Abstract

Objective

   The exacerbation of extreme high-temperature events due to global
   climate change poses a significant challenge to public health,
   particularly impacting the central nervous system through heat stroke.
   This study aims to develop Poly(amidoamine) (PAMAM) nanoparticles
   loaded with curcumin (PAMAM@Cur) to enhance its therapeutic efficacy in
   hypothalamic neural damage in a heat stroke model and explore its
   potential mechanisms.

Methods

   Curcumin (Cur) was encapsulated into PAMAM nanoparticles through a
   hydrophobic interaction method, and various techniques were employed to
   characterize their physicochemical properties. A heat stroke mouse
   model was established to monitor body temperature and serum biochemical
   parameters, conduct behavioral assessments, histological examinations,
   and biochemical analyses. Transcriptomic and proteomic analyses were
   performed to investigate the therapeutic mechanisms of PAMAM@Cur,
   validated in an N2a cell model.

Results

   PAMAM@Cur demonstrated good stability, photostability, cell
   compatibility, significant blood–brain barrier (BBB) penetration
   capability, and effective accumulation in the brain. PAMAM@Cur markedly
   improved behavioral performance and neural cell structural integrity in
   heat stroke mice, alleviated inflammatory responses, with superior
   therapeutic effects compared to Cur or PAMAM alone. Multi-omics
   analysis revealed that PAMAM@Cur regulated antioxidant defense genes
   and iron death-related genes, particularly upregulating the PCBP2
   protein, stabilizing SLC7A11 and GPX4 mRNA, and reducing iron-dependent
   cell death.

Conclusion

   By enhancing the drug delivery properties of Cur and modulating
   molecular pathways relevant to disease treatment, PAMAM@Cur
   significantly enhances the therapeutic effects against hypothalamic
   neural damage induced by heat stroke, showcasing the potential of
   nanotechnology in improving traditional drug efficacy and providing new
   strategies for future clinical applications.

Significance

   This study highlights the outlook of nanotechnology in treating
   neurological disorders caused by heat stroke, offering a novel
   therapeutic approach with potential clinical applications.

Graphical abstract

   [28]graphic file with name 12951_2024_2771_Figa_HTML.jpg

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-024-02771-3.

   Keywords: Heat Stroke Treatment, PAMAM Nanoparticles, Curcumin,
   Hypothalamus Nerve Injury, Antioxidant Defense

Introduction

   Global climate change has had a serious impact on the frequent
   occurrence of extreme high-temperature events, exacerbating heat stress
   reactions, including heat stroke (HS), which has become an urgent issue
   in the field of global public health [[29]1, [30]2]. Heat shock (HS) is
   a condition triggered by high temperatures and a humid environment,
   characterized mainly by symptoms such as elevated body temperature and
   fluid loss, which can lead to organ dysfunction and death in severe
   cases [[31]3, [32]4]. With the increasing hot weather conditions due to
   climate change, people are more susceptible to heat stress, resulting
   in a rising incidence of HS [[33]5–[34]8]. Therefore, studying the
   mechanisms of HS and finding effective treatment methods are crucial to
   safeguarding people's health [[35]9, [36]10].

   The pathophysiology of HS involves the disruption of the body's heat
   balance regulation system, leading to elevated body temperature and
   further disarray in heat stress reactions, triggering systemic
   physiological and biochemical responses [[37]11, [38]12]. During an HS
   episode, the body faces severe heat stress, resulting in damage to
   vital organs such as the central nervous system, cardiovascular system,
   and kidneys [[39]13–[40]15]. The impact of the central nervous system
   on HS is crucial; heat injuries cause brain inflammation responses,
   neuronal damage, and disrupted nerve conduction, potentially leading to
   cerebral edema and neuronal apoptosis in severe cases [[41]16–[42]18].
   Thus, protecting the central nervous system and reducing inflammation
   responses are particularly important in the treatment of HS
   [[43]19–[44]21].

   With the advancement of nanotechnology, the application of
   nano-carriers in drug delivery is receiving increasing attention. As a
   novel nano-carrier, PAMAM@Cur encapsulating Cur shows promise in
   improving drug stability, bioavailability, and targeting ability,
   potentially enhancing the therapeutic efficacy in the treatment of HS.
   PAMAM nanoparticles exhibit excellent biocompatibility and cellular
   penetration ability, capable of crossing the BBB to reach the central
   nervous system, offering a new avenue for treating neuro-related
   diseases [[45]22–[46]24]. Cur, a natural active ingredient renowned for
   its anti-inflammatory, antioxidant, and anti-apoptotic properties, when
   combined with PAMAM nano-carriers, can further enhance its
   pharmacological effects and mitigate adverse reactions, thus opening up
   new possibilities for treating HS [[47]25].

   To investigate the therapeutic effects and mechanism of action of
   PAMAM@Cur on HS, we designed a series of systematic experimental
   protocols. Initially, employing a hydrophobic interaction method, we
   successfully encapsulated Cur into PAMAM nanoparticles and ensured the
   stability and biocompatibility of the nanoparticles through detailed
   physicochemical characterization [[48]26]. Subsequently, we established
   a mouse model of HS by exposing them to 42 °C for 1 h followed by
   recovery at 24 °C, monitoring body temperature changes, assessing serum
   biochemical indicators, and evaluating behavioral performance
   [[49]27–[50]29]. Furthermore, through histological examinations and
   molecular mechanism studies, we investigated the therapeutic effects of
   PAMAM@Cur on hypothalamus nerve injury. Additionally, through in vitro
   and in vivo experiments, we validated the drug delivery properties and
   cell compatibility of PAMAM@Cur. These comprehensive experimental
   designs will help elucidate the mechanisms and potential of PAMAM@Cur
   in the treatment of HS, providing a scientific basis for future
   clinical applications.

   This study aims to explore the therapeutic effects and underlying
   mechanisms of PAMAM@Cur on hypothalamus nerve injury induced by HS. Our
   research findings indicate that PAMAM@Cur effectively improves
   behavioral performance, and neural cell structural integrity, and
   reduces inflammation response in HS mice, demonstrating superior
   efficacy. By modulating the expression of antioxidant defense genes and
   ferroptosis-related genes, PAMAM@Cur exhibits significant therapeutic
   effects. This study is of vital significance in enhancing our
   understanding of the pathophysiological mechanisms of HS, developing
   novel therapeutic drugs, and advancing clinical treatment. It provides
   strong support for future strategies in treating HS.

Materials and methods

Ethical statement

   All mouse experiments conducted in this study have adhered to
   international and national ethical guidelines and standards regarding
   the use of experimental animals, including but not limited to the
   "Three Rs principle" (Replacement, Reduction, Refinement). The
   experimental procedures in this study were approved by the Animal
   Ethics Committee (Ethical approval number: DWLL20200111). We ensured
   that all experimental animals were treated with respect and in a humane
   manner throughout the research process. The housing and handling of all
   mice were carried out under conditions aimed at minimizing their pain
   and distress. Following the conclusion of the experiments, all mice
   were humanely euthanized.

Establishment of an HS mouse model

   The Animal Ethics Committee (DWLL20230101). Male C57BL/6 mice aged 9 to
   10 weeks (strain code: 219, Beijing Vital River Laboratory Animal
   Technology Co., Ltd.) were selected for the experiments. One week
   before the start of the experiment, the mice were acclimatized to a
   room temperature of 22 ± 2 °C, relative humidity of 50 ± 8%, with a
   12-h light–dark cycle, and provided ad libitum access to food and
   water. Subsequently, the mice were exposed to a whole-body heat (WBH)
   environment at 42 °C and 50–55% relative humidity for 1 h to simulate
   HS conditions. Following the WBH exposure, the mice were returned to a
   room temperature of 24 °C for recovery. The criteria for successful
   modeling included signs such as deep breathing, cyanosis of the mouth,
   nose, and limbs, abdomen closely attached to the treadmill surface, and
   a rectal temperature reaching 42.0 °C as the HS standard. Additionally,
   proper feeding and hydration were provided to the mice after 1 h of WBH
   exposure.

   To evaluate the HS mouse model, physiological and biochemical tests
   were conducted on the mice's blood. After the experiment, whole blood
   was collected from the mice and serum was separated. The blood samples
   were left at room temperature for 2 h, then centrifuged at 3000 rpm for
   15 min at 2–8 °C to obtain the upper layer of serum for biochemical
   testing. Biochemical parameters analyzed included alanine
   aminotransferase (ALT), aspartate aminotransferase (AST), alkaline
   phosphatase (ALP), creatinine (CREA), urea (UREA), creatine kinase
   (CK), among others. Finally, the physiological and biochemical status
   of the HS mice as well as multi-organ function were evaluated [[51]30].

Open field test (OFT)

   The experimental setup for the OFT comprises a square testing arena
   divided into four sections, each measuring 50 cm × 50 cm × 40 cm.
   Boundary lines are marked on the floor of the area. Mice (Control
   group: 10 mice, HS group: 8 mice) are placed into the arena and given a
   30-s habituation period. Each mouse undergoes a 10-min test in the
   arena, which is recorded by a video camera positioned 120 cm away from
   the testing area. Subsequently, the time spent by the mouse in the
   central area (defined as the time when all four limbs are
   simultaneously within the central square) and the distance traveled
   within this zone are analyzed. The testing environment is maintained
   under normal lighting conditions (800 lx) [[52]31].

Novel object recognition (NOR) Test

   Before the training session, mice are placed in the apparatus for 3 min
   to acclimate to the environment. Following acclimation, the mice
   (Control group: 10 mice, HS group: 8 mice) undergo a training session
   during which two identical objects are introduced into the apparatus
   for 5 min. The test session is conducted 2 h post-training, during
   which one of the familiar objects is replaced with a new object of a
   different shape but similar properties. Mice are allowed to explore
   these two objects for 5 min. The time spent by the mice exploring each
   object is recorded using the Any-maze video tracking system, and the
   recognition index and frequency of interactions with the new object are
   calculated. The recognition index is defined as the ratio of time spent
   by the mice on the new object to the total test duration [[53]32].

Immunofluorescent staining of C-Fos and NeuN

   After the completion of behavioral experiments, the mice in each group
   were euthanized, and their brain tissues were immediately fixed in a 4%
   paraformaldehyde solution (P0099, Beyotime, Shanghai, China). The fixed
   tissues were dehydrated, embedded, and cut into 5 μm thick consecutive
   sections. The sections were subjected to antigen retrieval and washed
   three times with PBS. Subsequently, the sections were treated with a
   0.3% hydrogen peroxide solution (National Medicinal Code 10,011,208,
   National Medicinal Group Chemical Reagents Co., Ltd.) for 30 min to
   block endogenous peroxidase activity. Following this, the sections were
   incubated in a PBS solution containing 5% goat serum (C0265, Beyotime,
   Shanghai, China) to block nonspecific binding sites for 1 h. The
   sections were then separately incubated overnight at 4 °C with primary
   antibodies against C-Fos (ab208942, 1:200, Abcam, UK) and NeuN
   (ab177487, 1:300, Abcam, UK). Subsequently, the sections were incubated
   for 1 h at room temperature in the dark with a secondary antibody, goat
   anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150077, 1:200, Abcam, UK). In
   the final step, the sections were counterstained with DAPI (D9542,
   Sigma-Aldrich), mounted with an antifade reagent, and observed under a
   confocal microscope (Leica, STELLARIS 5, Germany) [[54]30].

Preparation of PAMAM@Cur

   In the first step, the fourth generation (G4) PAMAM dendrimer polymer
   (412,449, Sigma-Aldrich) was fluorescently labeled with Cy5.5 (170C0,
   Lumiprobe). The labeled dendrimer polymer was dialyzed overnight in a
   0.9% sodium chloride solution and further dialyzed in water. The
   Cy5.5-labeled dendrimer polymer was freeze-dried for 48 h and stored at
   -20 °C until further use. Subsequently, 1 mL of Cur (CAS No: 458–37-7,
   Sigma-Aldrich) dissolved in methanol (1 mg/mL) was added dropwise to
   the PAMAM solution in 1 mL of methanol (1 mg/mL) and 2 mL of PBS. The
   mixture solution was vigorously stirred for 2 days to encapsulate Cur
   within the PAMAM cavities. Methanol was removed using a rotary
   evaporator, leaving behind a PBS suspension. The PAMAM@Cur mixture was
   centrifuged (5000 rpm, 15 min at 4 °C) to remove the precipitate of
   non-complexed free Cur insoluble in PBS. The precipitate was collected
   and dissolved in 1 mL of methanol, and the amount of unloaded drug was
   determined by UV–visible spectroscopy, with the supernatant stored
   light-protected at 4 °C [[55]33].

Enzyme-linked immunosorbent assay (ELISA)

   The levels of TNF-α, IL-1β, and IL-6 in the hypothalamic lysates of the
   mice groups were determined using the Mouse TNF-α ELISA Kit (MTA00B,
   R&D Systems, USA), Mouse IL-6 ELISA Kit (M6000B, R&D Systems, USA), and
   Mouse IL-1β ELISA Kit (MLB00C, R&D Systems, USA). The experimental
   procedures were conducted according to the instructions provided in the
   respective kits [[56]30].

Physical and chemical characterization of nanoparticles

   The Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire,
   England) was utilized to measure the hydrodynamic size and Zeta
   potential of PAMAM-Cur formulation and PAMAM at a 20:1 N/P ratio before
   and after Cur loading using dynamic light scattering (DLS). The results
   were evaluated using the standard deviation (SD) ± method. To ascertain
   the encapsulation of Cur in the system (PAMAM@Cur), ultraviolet–visible
   absorption spectroscopy measurements were performed on free Cur, PAMAM,
   and PAMAM@Cur [[57]33].

Cur Release from PAMAM@Cur

   To determine the release of Cur from PAMAM@Cur complexes, a phosphate
   buffer solution (PBS, pH 7.4) was used as the medium, and the
   experiment was conducted using a dialysis bag method. Initially,
   1 mg/mL of Cur-containing PAMAM@Cur was dissolved in 5 mL of PBS,
   followed by placing this solution into a dialysis bag with a molecular
   weight cutoff of 10 kDa. Subsequently, the dialysis bag was oscillated
   at 200 rpm in a water bath shaker at 37 °C with 50 mL of external PBS
   as the medium, allowing free Cur molecules to diffuse into the external
   medium. At specific time points of incubation (for 3 days), 500 μL of
   samples were withdrawn for analysis, and an equal volume of fresh PBS
   was immediately replenished to maintain system constancy. The released
   amount of Cur was quantified using high-performance liquid
   chromatography (HPLC) analysis. The procedure involved extracting Cur
   from the samples with chloroform (in a 1:1 volume ratio), followed by
   its dissolution in 50 μL of methanol. This solution was then injected
   into a reverse-phase HPLC system equipped with a Cecil 1100 series UV
   detector for analysis, with the mobile phase comprising a mixture of
   75% acetonitrile and 25% water with 5% acetic acid, a flow rate of
   1 mL/min, and a detection wavelength of 430 nm. The amount of released
   Cur was calculated by comparing it against the experimentally
   determined standard curve (linear range of 0.1–50 μg/mL) to ascertain
   the effective payload release [[58]33].

Photostability profiles of Cur and PAMAM@Cur

   Free Cur or PAMAM@Cur (at a concentration of 1 mg/mL) was dissolved in
   4 mL of PBS and exposed to direct light at various time intervals from
   1 to 72 h. This procedure aimed to compare the photostability of Cur
   enclosed in PAMAM dendrimer with free Cur by measuring the degradation
   by-products and reduction in absorption peaks of Cur and PAMAM-Cur.
   Sample measurements were performed using HPLC to calculate the
   remaining amount in each sample within 72 h [[59]33].

Cell viability staining

   Live and dead cells were identified using Calcein AM (C3099, Thermo
   Fisher, USA) and Propidium Iodide (PI) (DN1005-010, inibio, USA). Cells
   were incubated with Calcein AM (1 μM) at 37 ℃ for 30 min, followed by
   three washes with PBS. Subsequently, PI (1 μM) was added and incubated
   for 10 min, followed by three washes with PBS. Images were captured
   using a confocal microscope (880, Carl Zeiss AG, Germany), with each
   image representing a different field of view. The experiments were
   conducted with three independent samples. Cell viability was calculated
   using ImageJ software (V1.8.0) [[60]34].

CCK-8

   Cells were treated according to the instructions of the CCK-8 kit
   (C0041, Beyotime, Shanghai, China). Cell viability was assessed using
   the CCK-8 assay at 48 h post-culturing. For each measurement, 10 μL of
   CCK-8 detection solution was added, and the cells were then incubated
   in a CO[2] incubator for 4 h. Subsequently, the absorbance at 450 nm
   was measured using a microplate reader to determine cell viability
   [[61]35].

Animal grouping

   The animal experiment was divided into the following groups: HS group
   (HS model group), HS + PAMAM group (HS model with intracarotid
   injection of PAMAM nanoparticles), HS + Cur group (HS model with
   intracarotid injection of 20 mg/kg Cur), and HS + PAMAM@Cur group (HS
   model with intracarotid injection of PAMAM@Cur nanoparticles containing
   20 mg/kg Cur).

In Vitro BBB permeability

   An in vitro BBB model was established using mouse brain endothelial
   bEnd.3 cells (CL-0598, Wuhan Punuosi Life Science and Technology Co.,
   Ltd.). The bEnd.3 cells were cultured on transwell membranes with a
   pore size of 0.4 μm, positioned above Na2 cells. When the cells formed
   a confluent monolayer, this model was considered a reasonable
   approximation of the BBB in vitro. An endothelial electrical resistance
   of at least 200 Ω•cm^2 indicated the stability of the cell layer for
   subsequent experiments. Samples from the lower chamber were collected
   at regular intervals, and their fluorescence intensity was measured
   using a fluorescence spectrophotometer to evaluate the efficiency of
   PAMAM@Cur crossing the BBB. The bottom chamber was observed using a
   confocal microscope (Leica, STELLARIS 5, Germany), and ImageJ software
   was used to analyze the obtained images [[62]36].

In Vivo BBB permeability

   PAMAM@Cur and Cur were intracarotidly injected into male C57BL/6 mice
   (9–10 weeks old) in groups of six. Brain fluorescence images were
   obtained at 2, 4, 8, 12, and 24 h post-injection using an in vivo
   imaging system (IVIS) (Perkin Elmer). Additionally, brain, heart,
   liver, spleen, lung, and kidney samples were collected for ex vivo
   imaging 48 h post-administration. The recorded images were analyzed
   using Living Image 4.3.1 software [[63]36].

Biochemical parameter analysis

   The metabolic parameters in the serum of each group of mice were
   analyzed using the ALT activity assay kit (E1010, Sigma-Aldrich, USA),
   AST activity assay kit (E1020, Sigma-Aldrich, USA), blood urea nitrogen
   (BUN) assay kit (60–1100, BioAssay Systems, USA), and creatinine assay
   kit (CR200, Randox Laboratories Ltd, UK). Specific procedures were
   performed according to the instructions provided with the respective
   assay kits.

Hematoxylin and Eosin (H&E) staining

   Hypothalamus tissues of the mice in each group were collected, fixed in
   10% neutral formalin, embedded in paraffin for sectioning, and
   deparaffinized in xylene. The sections were then stained with
   hematoxylin (C0107, Beyotime, Shanghai, China), rinsed with distilled
   water, immersed in 95% ethanol, stained with eosin (C0109, Beyotime,
   Shanghai, China), dehydrated with a gradient of ethanol, cleared in
   xylene, air-dried, fixed with neutral resin, and observed under an
   optical microscope [[64]37].

Detection of neuronal apoptosis

   Hypothalamus slices were first incubated with 10% normal donkey serum
   and then labeled with a series of antibodies: rabbit anti-mouse
   neuronal nuclear protein NeuN (ab104224, 1:200, Abcam, UK) and rabbit
   anti-cleaved caspase-3 (PA5-114,687, 1:100, Invitrogen, USA).
   Subsequently, the slices were incubated with fluorescently labeled
   secondary antibodies, including goat anti-mouse IgG H&L (Alexa Fluor®
   568) (ab175473, 1:200, Abcam, UK) and goat anti-rabbit IgG H&L (Alexa
   Fluor® 488) (ab150077, Abcam, UK). Next, nuclear counterstaining was
   performed with DAPI (D9542, Sigma-Aldrich) to enhance nuclear
   visualization. In addition, selected sections were stained using the
   terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
   (TUNEL) assay kit (#630,108, Clontech, Palo Alto, CA, USA). Images were
   observed with a confocal microscope (Leica, STELLARIS 5, Germany), and
   neuronal apoptosis was quantitatively analyzed using ImageJ software
   [[65]38].

qRT-PCR

   RNA was extracted from tissues and cells using Trizol (Cat# 16,096,020,
   Thermo Fisher Scientific, USA). The concentration and purity of RNA
   were determined using the NanoDrop One/OneC spectrophotometer (Thermo
   Fisher Scientific) with an A260/A280 ratio of 2.0 and a concentration
   greater than 5 μg/μL. The cDNA first-strand synthesis kit (Cat# D7168L,
   Beyotime, Shanghai) was used for RNA reverse transcription. RT-qPCR
   experiments were performed using the RT-qPCR kit (Cat# Q511-02, Vazyme
   Biotech, Nanjing) following the manufacturer's instructions. For each
   reaction, 2 μL of cDNA template, 0.2 μL of each forward and reverse
   primer, and 10 μL of RT-qPCR Mix were mixed and brought to a final
   volume of 20 μL with RNase-free water. PCR amplification was carried
   out using the Bio-Rad CFX96 real-time PCR system with the following
   cycling conditions: initial denaturation at 95 ℃ for 30 s, followed by
   denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 30 s, and
   extension at 72 ℃ for 30 s, repeated for 40 cycles, with a melt curve
   analysis from 65 ℃ to 95 ℃. The primer sequences were designed and
   provided by Shanghai Sangon Biotech and are listed in Table S1. The
   relative expression of the target gene in the experimental group
   compared to the control group was calculated using the 2^−ΔΔCt method
   with GAPDH as the reference gene [[66]39]. The formula used was
   ΔΔCt = ΔCt(exp)—ΔCt(control), where ΔCt = Ct(target gene)—Ct(reference
   gene), and the experiment was repeated three times.

Western blot

   Cell extracts for total protein were prepared using RIPA lysis buffer
   (Cat# P0013B, Beyotime, Shanghai) containing 1% PMSF. Nuclear proteins
   were extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Kit
   (Cat# 78,833, ThermoFisher Scientific). The total protein concentration
   of each sample was determined using the BCA assay kit (Cat# P0011,
   Beyotime, Shanghai). SDS-PAGE gels ranging from 8 to 12% were prepared
   based on the target protein band size, and equal amounts of protein
   samples were loaded into each lane for electrophoresis using a
   micropipette. Proteins separated on the gel were transferred to a PVDF
   membrane (Cat# 1,620,177, BIO-RAD, USA) and then blocked with 5%
   non-fat milk at room temperature for 1 h. The membrane was probed with
   primary antibodies: Anti-BNDF (ab108319, 1:1000), Anti-HSP70 (ab2787,
   1:1000), Anti-HIF-1α (ab308433, 1:1000), Anti-IL-1β (ab283818, 1:1000),
   Anti-TNF-α (ab183218, 1:1000), Anti-PCBP2 (ab200835, 1:1000),
   Anti-SLC7A11 (xCT) (ab307601, 1:1000), Anti-GPX4 (ab125066, 1:1000),
   Anti-CytoC (ab133504, 1:1000), Anti-Bax (ab32503, 1:1000), Anti-Bcl-2
   (ab182858, 1:1000), and anti-GAPDH (ab8245, 1:2000), and then incubated
   overnight at 4 ℃. The membrane was washed three times for 5 min each
   with 1 × TBST at room temperature, followed by incubation with
   HRP-conjugated secondary antibodies: goat anti-rabbit IgG (ab6721,
   1:2000) or goat anti-mouse IgG (ab6728, 1:2000) for 1 h at room
   temperature, all antibodies were purchased from Abcam, UK. After three
   washes with 1 × TBST buffer at room temperature, the membrane was
   incubated with ECL reagent (Cat# 1,705,062, Bio-Rad, USA), and the
   bands were visualized using the Image Quant LAS 4000C gel imaging
   system (GE, USA). The protein of interest was normalized to GAPDH, and
   the relative protein expression levels were analyzed using ImageJ
   software (V1.8.0.112) by comparing the grayscale values of the target
   band to the reference band, to detect the protein expression levels
   [[67]40]. Each experiment was performed in triplicate.

High-throughput transcriptome sequencing

   RNA was extracted from the hypothalamus tissues of different groups of
   mice (HS, n = 3; HS + PAMAM@Cur, n = 3) using Trizol reagent (Thermo
   Fisher Scientific, catalog no. 16096020, USA). The RNA concentration,
   purity, and integrity were assessed using the Qubit®2.0 Fluorometer®
   (Thermo Fisher Scientific, catalog no. [68]Q33238, USA) with the Qubit®
   RNA Analysis Kit (Shanghai Bogoo Biotechnology Co., Ltd., catalog no.
   HKR2106-01, Shanghai, China), Nanophotometer (IMPLEN, USA) and
   Bioanalyzer 2100 system with the RNA Nano 6000 Assay Kit (Agilent,
   catalog no. 5067–1511, USA), ensuring a RNA concentration greater than
   100 ng/μL and a 260/280 ratio between 1.8 and 2.1. A total of 3 μg of
   RNA from each sample was used as input material for RNA library
   preparation. Following the manufacturer's instructions, the NEBNext®
   UltraTM RNA Library Prep Kit (New England Biolabs, catalog no. E7435L,
   Beijing, China) suitable for the Illumina® platform was used to
   generate cDNA libraries, which were then evaluated for quality on the
   Agilent Bioanalyzer 2100 system. The indexed samples were clustered
   using the TruSeq PE Cluster Kit v3 cBot HS (Illumina, catalog no.
   PE-401–3001, USA) on the cBot cluster generation system. After cluster
   generation, library preparation was sequenced on the Illumina HiSeq 550
   platform, producing 125 bp/150 bp paired-end reads [[69]41, [70]42].

   The quality of the paired-end reads from the raw sequencing data was
   assessed using FastQC software v0.11.8. Preprocessing of the raw data
   was performed using Cutadapt software version 1.18 to remove Illumina
   sequencing adapters and poly(A) tail sequences. Reads with N content
   exceeding 5% were filtered out using a perl script. Reads with 70% base
   quality over 20 were extracted using FASTX Toolkit software version
   0.0.13. The BBMap software was used for repairing paired-end sequences.
   Finally, the filtered high-quality read fragments were mapped to the
   mouse genome using HISAT2 software (version 0.7.12) [[71]43, [72]44].

Proteomic analysis

   In proteomic analysis, the precipitate was first sonicated on ice in a
   lysis buffer containing 4% SDS, 0.1 M dithiothreitol (DTT), and 20 mM
   Tris–HCl. Subsequently, the supernatant was collected by centrifuging
   the sample at 14,000 g for 10 min at 4 °C. Ice-cold acetone (5 volumes)
   was added to each sample and allowed to precipitate overnight at
   -20 °C. The resulting protein precipitate was resuspended and
   quantified for concentration using the BCA Protein Assay Kit (cat. no.
   23225, Thermo Fisher Scientific, USA) following the manufacturer's
   instructions. Next, FASP technology was employed to perform trypsin
   digestion of 100 μg of each sample on a 30 kD filter (Millipore,
   Billerica, USA) according to established protocols. Following peptide
   collection, drying was carried out using a freeze dryer (Labconco,
   Kansas City, Missouri, USA). Eight-plex iTRAQ reagents (Applied
   Biosystem Inc., CA, USA) were used to label 20 μg of each sample from a
   total of 4 samples per group, after which the labeled samples were
   combined. The combined samples were then separated using the Pierce
   High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher
   Scientific, USA). The eluate from each fraction was
   vacuum-concentrated, reconstituted in 8 μL of 0.1% formic acid, and
   subsequently analyzed by nanoLC-orbitrap MS, following established
   protocols. Three replicates were conducted for each sample. The
   collected MS/MS spectra were converted to MGF files and searched
   against the SwissProt mouse database using Mascot Daemon. A filtering
   criterion was set at a 1% false discovery rate to minimize the
   possibility of erroneous peptide identification. Finally, significantly
   differentially expressed proteins were determined with a standard of
   P < 0.05 and |log2FC|> 1.00 [[73]30].

Analysis of differentially expressed genes (DEGs)

   DEGs were identified using the "limma" package in R with a threshold of
   |log2FC|> 2 and a P-value < 0.05. The differential gene expression
   volcano plots were generated using the "ggplot2" package in R [[74]45,
   [75]46].

GO and KEGG enrichment analysis

   The DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of
   Genes and Genomes (KEGG) enrichment analysis using the
   "clusterProfiler," "org.Hs.eg.db," "enrichplot," and "ggplot2" packages
   in R. Enrichment plots for the biological process (BP), cellular
   component (CC), and molecular function (MF) categories in GO were
   generated alongside a bubble plot for KEGG enrichment analysis results
   [[76]47].

Protein–protein interaction (PPI) analysis

   Proteins encoded by the relevant DEGs were analyzed for PPI using the
   String website, filtering out non-interacting proteins to construct the
   PPI network [[77]48]. The PPI network was imported into Cytoscape
   software for further analysis using the MCODE plugin. By leveraging the
   network's topological characteristics, the MCODE plugin identifies
   highly connected subgraphs within the network, which often represent
   protein complexes or closely interacting protein groups involved in
   specific biological processes [[78]49].

Determination of Fe^2+ content

   The level of Fe^2+ was determined using the iron ion detection assay
   kit (ab83366, Abcam) following the manufacturer's instructions.
   Briefly, cellular or tissue samples were weighed, homogenized in the
   iron ion detection assay kit buffer on ice, and rinsed with cold PBS.
   Subsequently, the homogenate was centrifuged at 16,000 g for 10 min at
   4 °C; 5 μL of the assay kit buffer was added to each supernatant. The
   mixture was then incubated at 37 °C for 30 min, and the absorbance was
   measured at 593 nm using a microplate reader to determine the optical
   density (OD) [[79]50].

GSH content analysis

   The glutathione (GSH) content in cells/tissues was measured using a GSH
   fluorescence assay kit (EIAGSHF, Thermo Scientific, USA). Samples or
   standards were mixed with the assay reagent and incubated at room
   temperature for 15 min. The fluorescence product was then read at
   510 nm in a fluorescence microplate reader with an excitation
   wavelength of 390 nm. Initially, free GSH was measured after 15 min,
   followed by the addition of the reaction mixture to convert all
   oxidized glutathione (GSSG) to free GSH, which reacted with excess
   assay reagent to generate a signal related to the total GSH content.
   The total concentration of generated GSH in the samples was calculated
   based on the signal produced. [[80]51].

ELISA detection of MDA and SOD levels

   The levels of Malondialdehyde (MDA) and Superoxide Dismutase (SOD) were
   assessed using the MDA Colorimetric Assay Kit (EEA015, Invitrogen, USA)
   and the SOD Colorimetric Activity Assay Kit (EIASODC, Invitrogen, USA)
   following the specific protocols outlined in the respective assay kit
   manuals [[81]52].

Measurement of mitochondrial membrane potential

   The changes in mitochondrial membrane potential were evaluated using
   the mitochondrial staining assay kit with the cationic fluorescent dye
   JC-1 ([82]M34152, Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells
   were seeded in a 96-well black plate at a density of 5 × 10^3 cells per
   well. After 48 h of treatment, the cells were co-incubated with the
   JC-1 dye for 20 min. To image JC-1 monomers, a live-cell imaging system
   (BD Pathway™ Bioimager System, BD Biosciences) was utilized with an
   excitation wavelength of 490 nm and emission wavelength of 530 nm; for
   J-aggregates, the excitation wavelength was set at 525 nm and emission
   wavelength at 590 nm [[83]53].

Detection of intracellular reactive oxygen species (ROS)

   Hypothalamic tissue samples from various groups of mice were treated
   with 20 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, C2938,
   Invitrogen, USA) and then incubated for 1 h. Following the completion
   of the incubation, the tissue samples were washed three times with PBS
   to remove any unabsorbed DCFH-DA dye. Subsequently, the samples were
   exposed to 850 nm light for 20 min to activate the fluorescence of DCF.
   Prior to imaging, the samples were stained with 1.0 μM Hoechst 33,342
   (62,249, Thermo Fisher Scientific) for 10 min to label the cell nuclei.
   Fluorescence signals generated by 2,7-dichlorofluorescein (DCF) were
   monitored using a 488 nm excitation laser, while Hoechst 33,342 was
   excited at 405 nm. All imaging procedures were performed using a
   confocal microscope (Leica, STELLARIS 5, Germany) [[84]54].

Establishment of HS cell model

   In vitro experiments were conducted using N2a cells (CL-0168, Wuhan
   Punose Life Technology Co., Ltd), cultured in RPMI-1640 medium
   (11,875,093) supplemented with 1% glutamine (25,030,081), 10% fetal
   bovine serum (10099141C), and 1% penicillin/streptomycin (10,378,016),
   and incubated at 37 °C in a humidified atmosphere containing 5% CO[2].
   The aforementioned reagents were procured from ThermoFisher [[85]55].
   For HS treatment, the cells were subjected to a 2-h incubation at 43 °C
   followed by a 6-h recovery period at 37 °C [[86]56].

Cell infection and grouping

   N2a cells were manipulated through lentivirus infection for either
   overexpression or silencing, with lentivirus packaging services
   provided by Sangon Biotech (Shanghai), China. The pHAGE-puro series
   plasmid, along with the helper plasmids pSPAX2 and pMD2.G, and the
   pSuper-retro-puro series plasmid with the helper plasmids gag/pol and
   VSVG were co-transfected into 293 T cells (CL-0005, Wuhan Punose Life
   Technology Co., Ltd). After 48 h of cell culture, the supernatant was
   collected, filtered through a 0.45 μm filter, centrifuged, and the
   viral supernatant was collected. Subsequently, the supernatant was
   concentrated by centrifugation after 72 h, the two batches of virus
   were mixed, and the titer was determined. When the cells were in the
   logarithmic growth phase, they were trypsinized and seeded into 6-well
   plates at 1 × 10^5 cells per well. Following 24 h of routine culture,
   when the cell confluency reached approximately 75%, the culture medium
   containing an appropriate amount of lentivirus (MOI = 10, working titer
   around 5 × 10^6 TU/mL) and 5 μg/mL polybrene (Merck, TR-1003, USA) was
   added to infect the cells. After 4 h of infection, an equal amount of
   medium was added to dilute the polybrene, and 24 h post-infection, the
   medium was replaced with a fresh culture medium. To establish stable
   cell lines, 4 μg/mL puromycin (Sangon Biotech, A100339, Shanghai,
   China) was used for resistance selection to obtain stable cell lines.
   The sequences for silencing lentivirus are listed in Table S2.

   Cell grouping: Control group (control group), HS group (HS model
   group), HS + PAMAM@Cur (cells treated with PAMAM@Cur before HS
   induction), HS + PAMAM@Cur + sh-PCBP2 (cells transfected with sh-PCBP2
   lentivirus and treated with PAMAM@Cur before HS induction),
   HS + PAMAM@Cur + sh-PCBP2 + oe-SLC7A11 (cells transfected with
   sh-PCBP2 + oe-SLC7A11 lentivirus and treated with PAMAM@Cur before HS
   induction), HS + PAMAM@Cur + sh-PCBP2 + oe-GPX4 (cells transfected with
   sh-PCBP2 + oe-GPX4 lentivirus and treated with PAMAM@Cur before HS
   induction).

RIP-qPCR

   Initially, rinse the cells once with cold PBS, then add 2 mL of cold
   PBS to a 10 cm culture dish for cell collection. Extract the cell
   nuclei from these cells and proceed with sonication. Bind 2 μg of PCBP2
   antibody (ab184962, Abcam) or corresponding rabbit IgG control
   (ab172730, Abcam) to Protein A/G magnetic beads (88,802, Thermo Fisher
   Scientific), and incubate the mixture at 4 °C for 4 h. Subsequently,
   wash the beads three times and then incubate them with pre-cleared
   nuclear extract in RIP buffer (150 mM KCl, 25 mM Tris (pH 7.4), 5 mM
   EDTA, 0.5 mM DTT, 0.5% NP-40, 1 × protease inhibitor, 10 U/mL RNase
   inhibitor) at 4 °C overnight. Following incubation, wash the beads
   three times with RIP buffer, resuspend the beads in 80 μL of PBS, and
   perform DNA digestion at 37 °C for 30 min, followed by treatment with 2
   μL of proteinase K (25,530,031, 20 mg/mL, Thermo Fisher) at 55 °C for
   30 min. Finally, extract RNA from the Input and IP samples using the
   TRIzol method and analyze the levels of SLC7A11 and GPX4 by
   quantitative PCR [[87]57].

Experiment on RNA half-life

   Cells were treated with streptomycin D (129,935, Millipore) at a
   concentration of 5 mg/ml. After incubation for 0, 0.5, 1.5, and 2 h,
   cells were collected, and RNA was extracted for RT-qPCR analysis. The
   rate of RNA degradation was estimated using the formula Nt/N0 = e^−kt,
   where t represents the time of transcriptional inhibition, and Nt and
   N0 denote the RNA expression levels at time t and time 0, respectively.
   The RNA half-life (t[1/2]) was calculated from the degradation rate as
   follows: t[1/2] = ln[2]/k [[88]58].

Statistical analysis

   Our study utilized R language version 4.2.1, with R language
   compilation performed through the integrated development environment
   RStudio, version 2022.12.0–353. Data was processed using GraphPad Prism
   8.0, and continuous data were presented as mean ± standard deviation
   (Mean ± SD). Unpaired t-tests were used for comparisons between two
   groups, while one-way analysis of variance was applied for comparisons
   among multiple groups. The homogeneity of variance was tested using the
   Levene test. In cases of homogeneity, Dunnett’s T3 and LSD-t tests were
   used for pairwise comparisons. When variance was not homogeneous,
   Dunnett’s T3 test was employed. A p-value < 0.05 indicated statistical
   significance for differences between the two groups [[89]59].

Results

Successful establishment of a HS mouse model

   As extreme high-temperature events become more frequent and intense due
   to global climate change, HS, a severe heat stress response triggered
   by high-temperature environments, is on the rise annually, becoming a
   significant public health concern. HS not only results in the failure
   of the body's temperature regulation mechanism but also triggers
   systemic inflammatory responses and multi-organ dysfunction, with
   notable damage to the central nervous system, particularly the
   hypothalamus. The hypothalamus plays a crucial role in maintaining
   temperature stability, water-electrolyte balance, and various endocrine
   functions [[90]60, [91]61]. Therefore, this study initially established
   an HS mouse model to investigate the effects of high-temperature
   exposure (Fig. [92]1A). In the model group, mice were exposed to a
   high-temperature climate chamber at 42 °C for 1 h and then allowed to
   recover at room temperature of 24 °C. The rectal temperature of HS
   model group mice gradually increased, with 8 mice reaching 42 °C within
   100 min (Fig. [93]1B). These 8 mice were selected as the HS group for
   subsequent experiments. Subsequently, the change in body weight before
   and after modeling was measured for each mouse. It was observed that
   the body weight of the HS group mice decreased, likely due to
   dehydration from sweating (Fig. [94]1C). Blood samples were collected
   from mice in each group, and serum was isolated for biochemical
   analysis, which revealed that compared to the control group, markers
   such as ALT, AST, ALP, CREA, UREA, and CK showed significant increases
   (Fig 1D-I), confirming the successful establishment of the HS mouse
   model.

Fig. 1.

   [95]Fig. 1
   [96]Open in a new tab

   Construction of an HS mouse model. Note: A Schematic diagram of the
   construction process of the HS mouse model (mice exposed to
   high-temperature conditions in a climatic chamber, allowed to recover
   at room temperature, and blood collected for biochemical analysis); B
   Monitoring of rectal temperature in mice from each group; C Comparative
   graph of body weight changes before and after modeling in mice from
   each group; D-I Evaluation of changes in serum levels of ALT, AST, ALP,
   CREA, UREA, and CK in mice from each group. * indicates P < 0.05
   compared to the Control group, ** indicates P < 0.01, *** indicates
   P < 0.001, ns indicates no significant change. The Control group
   comprised 10 mice, while the HS group had 8 mice

HS induces hypothalamic nerve injury in mice

   Following the successful establishment of an HS mouse model, we
   conducted behavioral tests to observe the effects of HS on the brain
   (Fig. [97]2A). In the OFT, mice in the HS group exhibited a significant
   reduction in the time spent in the central zone compared to the control
   group, indicating a potential decrease in cognitive abilities and
   induction of anxiety-like behavior (Fig. [98]2B-C). In the NOR test,
   mice in the HS group showed a notable decrease in the recognition index
   and the number of entries to the new object compared to the control
   group (Fig. [99]2D-E). These results suggest that HS leads to increased
   stress and anxiety-like behavior in mice, accompanied by brain damage.

Fig. 2.

   [100]Fig. 2
   [101]Open in a new tab

   Impact of HS on hypothalamic nerve injury in mice. Note: A Evaluation
   process of the HS mouse model, including behavioral tests, histological
   analysis, and biochemical assays; B Tracing path diagram of OFT; C
   Comparison of total distance traveled and time spent in the central
   zone in the OFT; D Tracing path diagram of NOR test; E Comparison of
   exploratory behavior towards a novel object in the NOR test; F
   Immunofluorescence staining showing c-Fos protein expression in the
   hypothalamus (scale bar = 50 μm); G Immunofluorescence staining showing
   NeuN-positive cells in the hypothalamus (scale bar = 25 μm); H-J ELISA
   detection of TNF-α, IL-1β, and IL-6 in hypothalamic lysates of mice
   from each group; K Western blot analysis of HSP70, Bax, cyto C, and
   Bcl2 protein expression in the hypothalamus of mice from each group. *
   indicates P < 0.05 compared to the Control group, ** indicates
   P < 0.01, *** indicates P < 0.001. The Control group consisted of 10
   mice, while the HS group had 8 mice

   Based on the aforementioned observations and experimental findings,
   further research focused on elucidating how HS specifically affects the
   structure and function of the hypothalamus. Through immunofluorescence
   analysis, we found an increase in c-Fos expression in the hypothalamus
   of HS mice, indicating stress response and activation of hypothalamic
   neurons in response to high-temperature exposure (Fig. [102]2F).
   Furthermore, the decrease in NeuN-positive cells suggests damage to
   hypothalamic neurons due to HS (Fig. [103]2G). Examination of
   inflammatory markers TNF-α, IL-1β, and IL-6 levels in hypothalamic
   lysates revealed a 1.4-fold increase in TNF-α, a 1.2-fold increase in
   IL-1β, and a 1.3-fold increase in IL-6 levels post HS, further
   confirming the inflammatory response triggered by HS (Fig. [104]2H-J).

   To ascertain the heat stress-related biological responses in the
   hypothalamus, Western blot analysis was performed to assess the
   expression changes of heat shock protein (HSP70), apoptosis-related
   proteins Bax and Bcl-2, and mitochondrial functional protein cytochrome
   C. The results indicated an increase in the expression of HSP70, Bax,
   and cytochrome C, along with a decrease in Bcl-2 expression in the
   hypothalamus post-HS, highlighting the stress response, apoptosis, and
   mitochondrial dysfunction caused by HS (Fig. [105]2K).

   In conclusion, this study successfully established an HS mouse model
   and uncovered that HS leads to hypothalamic neuronal damage, heightened
   inflammatory response, and apoptotic reactions.

Preparation and characterization of PAMAM nanoparticles loaded with Cur

   After identifying the specific impacts of HS-induced hypothalamus
   damage, this study proceeded to evaluate potential intervention
   strategies. Considering the known anti-inflammatory and antioxidant
   benefits of Cur, as well as its demonstrated protective effects in
   other neural injury models (such as Alzheimer's disease, Parkinson's,
   ischemic brain injury, spinal cord injury) [[106]62–[107]65],
   supporting its potential use as a therapeutic strategy for neurological
   disorders, including hypothalamus damage caused by HS. However, due to
   the limited solubility and bioavailability of Cur, this study employed
   PAMAM nanoparticles as carriers, aiming to enhance its therapeutic
   efficiency by improving its physicochemical properties. The excellent
   biocompatibility and surface modifiability of PAMAM nanoparticles are
   expected to enhance the stability and efficacy of Cur, facilitating its
   targeted delivery to the hypothalamus to more effectively alleviate
   neural damage induced by HS.

   Initially, Cur was encapsulated into the cavities of PAMAM (4th
   generation) dendrimer via hydrophobic interactions in a 1:1 mass ratio,
   forming PAMAM@Cur (Fig. [108]3A). UV–visible absorption spectroscopy
   revealed a significant absorption peak at 420 nm and a shoulder peak at
   about 370 nm, indicating enhanced solubility and stability of Cur
   within the dendritic structure, resulting in a redshift in absorbance
   (Fig. [109]3B). DLS results showed average hydrodynamic diameters of
   24.5 nm for PAMAM and 90.2 nm for PAMAM@Cur (Fig. [110]3C), with zeta
   potentials of + 6.6 mV and + 2.1 mV, respectively (Fig. [111]3D). TEM
   observation depicted spherical Cur-DA nanoparticles with an average
   diameter of 83.5 ± 18.6 nm, consistent with the DLS results
   (Fig. [112]3E). In PBS, an in vitro release curve of Cur from PAMAM@Cur
   at physiological pH 7.4 over 24 h showed approximately 48% release
   within 24 h (Fig. [113]3F). Further evaluation of the photostability of
   PAMAM@Cur revealed that the absorption of free Cur dissolved in PBS
   decreased gradually from 100 to 15% after 72 h, while PAMAM@Cur only
   decreased by 20%, indicating higher photostability (Fig. [114]3G).

Fig. 3.

   [115]Fig. 3
   [116]Open in a new tab

   Preparation and characterization of PAMAM nanoparticles loaded with
   Cur. Note: A Schematic illustration of Cur encapsulated into the
   cavities of PAMAM (4th generation) dendrimers through hydrophobic
   interactions to form PAMAM@Cur complexes; B UV–Vis absorption spectra
   showing characteristic absorption peaks of encapsulated Cur and
   potential changes in solubility and stability; C DLS data indicating
   the average hydrodynamic diameters of PAMAM and PAMAM@Cur
   nanoparticles; D Zeta potential measurement of PAMAM and PAMAM@Cur
   nanoparticles; E TEM images displaying the spherical morphology and
   average diameter of nanoparticles (scale bar = 100 nm); F In vitro
   release curve of Cur from PAMAM@Cur in PBS over 24 h; G Comparative
   evaluation of photo-stability of free Cur and PAMAM@Cur in PBS over
   72 h; H CCK-8 assay showing the cytotoxicity of different
   concentrations of Cur and PAMAM@Cur after 24 h of treatment on Na2
   cells; I Live/dead cell staining images after treatment with 50 mg/mL
   Cur and PAMAM@Cur for 24 h, demonstrating cell viability (scale
   bar = 25 μm). Cell experiments were repeated three times. * indicates
   P < 0.05 compared to the PAMAM group, ** indicates P < 0.01; #
   indicates P < 0.05 compared to the Cur group, ### indicates P < 0.001

   Furthermore, we evaluated the cytotoxicity of PAMAM@Cur in Na2 cells
   using the CCK-8 assay and live/dead cell staining. The results
   demonstrated that after 24 h of cultivation with varying concentrations
   of Cur and PAMAM@Cur, cell viability in the Cur group decreased as the
   concentration increased to 20 mg/mL, reaching around 80% at 100 mg/mL.
   In contrast, in the PAMAM@Cur group, possibly due to its good
   sustained-release properties, cell viability remained above 90%
   throughout (Fig. [117]3H). Treatment with a concentration of 50 mg/mL
   for 24 h and observation using live/dead cell staining showed that the
   cell survival rate was approximately 90% in the Cur group and around
   96% in the PAMAM@Cur group (F[118]ig. [119]3I), indicating excellent
   cell compatibility of PAMAM@Cur.

   In summary, by utilizing PAMAM nanoparticles, we improved the
   physicochemical properties of Cur. The in vitro experimental results
   demonstrated that PAMAM@Cur exhibits good cell compatibility and
   enhanced photostability. This supports the potential of PAMAM@Cur in
   the treatment of hypothalamus damage induced by HS.

The ability of PAMAM@Cur to penetrate the BBB both in Vitro and in Vivo

   To investigate the role of PAMAM@Cur in hypothalamic damage induced by
   HS, we initially validated the penetration ability of PAMAM@Cur through
   the BBB both in vitro and in vivo (Fig. [120]4A). In the in vitro
   experiments, a monolayer model of brain microvascular endothelial cells
   (bEnd.3 cells) mimicking the BBB was established to assess the
   penetration ability of PAMAM@Cur. Cy5.5 fluorescent-labeled PAMAM@Cur
   was introduced into the upper chamber of the model, samples from the
   lower chamber were collected at timed intervals, and the fluorescence
   intensity was measured using a fluorescence spectrophotometer to
   evaluate the efficiency of PAMAM@Cur crossing the BBB. The results
   indicated that compared to the standalone Cur group, the fluorescence
   intensity in the lower chamber of the in vitro BBB model significantly
   increased with PAMAM@Cur, demonstrating successful penetration of the
   simulated BBB (Fig. [121]4B). Immunofluorescence staining of Na2 cells
   also confirmed the successful penetration of PAMAM@Cur through the
   simulated BBB (Fig. [122]4C).

Fig. 4.

   [123]Fig. 4
   [124]Open in a new tab

   Evaluation of the ability of PAMAM@Cur to penetrate the BBB in Vitro
   and in Vivo. Note: A Schematic representation of the in vitro and in
   vivo experimental procedures for investigating the penetration of
   PAMAM@Cur across the BBB; B Statistical analysis of the fluorescence
   intensity of Cy5.5-labeled PAMAM@Cur in an in vitro BBB model; C
   Assessment of the effectiveness of PAMAM@Cur crossing the simulated BBB
   using immunofluorescence staining technique (scale bar = 25 μm); D-E
   Time-dependent fluorescence images of Cy5.5-labeled PAMAM@Cur
   distribution in the mouse brain recorded by IVIS imaging. Each group
   consisted of 6 mice. * indicates P < 0.05 compared to the Cur group, **
   indicates P < 0.01, *** indicates P < 0.001

   In the in vivo experiments, C57BL/6 mice were intravenously injected
   with Cy5.5-labeled PAMAM@Cur via the carotid artery, and the
   fluorescence signal in the brain was observed and recorded at different
   time points using an IVIS to evaluate the BBB penetration ability and
   distribution of PAMAM@Cur in the brain. The results showed a
   significant enhancement of the fluorescence signal in the brains of
   mice injected with PAMAM@Cur compared to the Cur group, particularly
   within 2 to 24 h post-injection, with the fluorescence signal
   concentrating in specific brain regions and gradually diminishing
   thereafter (Fig. [125]4D-E). Additionally, H&E staining of mouse heart,
   liver, spleen, lungs, and kidneys revealed no notable morphological
   changes in the treated tissues (Figure S1A), indicating that PAMAM@Cur
   nanoparticles did not induce significant tissue damage. Furthermore,
   the biochemical parameters such as ALT, AST, BUN, and CR in the serum
   of treated mice remained within normal levels, showing no significant
   differences compared to the normal control group (Figure S1B-E),
   demonstrating excellent biocompatibility and safety.

   These series of experiments demonstrate that PAMAM@Cur effectively
   crosses a simulated BBB in vitro and accumulates in the brain in vivo,
   providing experimental evidence for the potential of PAMAM@Cur as a
   drug delivery system for treating brain disorders.

PAMAM@Cur improves hypothalamic damage caused by HS

   Following the confirmation of PAMAM@Cur's effective penetration of the
   BBB, our study aimed to assess the therapeutic effects of PAMAM@Cur on
   HS-induced hypothalamic damage. Experimental animals were divided into
   four groups: HS group, HS + PAMAM group, HS + Cur group, and
   HS + PAMAM@Cur group. Results from the NOR test revealed that compared
   to the HS and HS + PAMAM groups, mice in the HS + PAMAM@Cur group
   exhibited significantly enhanced ability to recognize a new object,
   suggesting a marked improvement in cognitive function. In contrast,
   although mice in the HS + Cur group showed behavioral improvements
   compared to the HS model group, indicating some degree of improvement,
   the limited ability of Cur to penetrate the BBB resulted in an inferior
   outcome compared to the HS + PAMAM@Cur group (Fig. [126]5A-C). In the
   OFT, mice in the HS + PAMAM@Cur group displayed a significant increase
   in exploration activity in the central area, reflecting a marked
   reduction in anxiety behavior. While the HS + Cur group also showed an
   increase in central area activity, the extent of improvement was not as
   significant as that in the HS + PAMAM@Cur group (Fig. [127]5D-F).

Fig. 5.

   [128]Fig. 5
   [129]Open in a new tab

   The therapeutic effect of PAMAM@Cur on hypothalamic damage induced by
   HS. Note: A Tracing path diagram of NOR test; B-C Comparison of
   exploratory behavior towards a novel object in the NOR test; D Tracing
   path diagram of OFT; E–F Comparison of total distance traveled and time
   spent in the central zone in the OFT; G Histopathological changes
   observed in hypothalamic tissue sections stained with H&E (scale
   bar = 50 μm); H Immunofluorescence double staining of neuronal-specific
   nuclear antigen (NeuN) and apoptosis-executing protein
   (cleaved-caspase-3) in hypothalamic tissue sections (scale
   bar = 25 μm); I Immunofluorescence double staining of NeuN and TUNEL in
   hypothalamic tissue sections (scale bar = 100, 50, 25 μm); J-K Western
   blot analysis of the protein expression of BDNF, HSP70, HIF-1α, IL-1β,
   and TNF-α in the hypothalamus of mice from each group. * indicates
   P < 0.05 compared to the HS group, ** indicates P < 0.01; # indicates
   P < 0.05 compared to the HS + Cur group, ## indicates P < 0.01. Each
   group comprised 6 mice

   Observation of hypothalamic tissue slices stained with H&E revealed
   that treatment with PAMAM@Cur significantly improved the structural
   integrity of nerve cells, and reduced inflammation and cell death,
   surpassing the outcomes of the HS group and HS + PAMAM group. Although
   the HS + Cur group showed neuroprotective effects and reduced
   inflammation, limitations in drug delivery efficiency resulted in
   inferior outcomes compared to the HS + PAMAM@Cur group (Fig. [130]5G).
   Further support for these findings was provided by immunofluorescence
   labeling, showing a significant decrease in c-Fos expression and a
   significant increase in NeuN-positive neurons in the HS + PAMAM@Cur
   group compared to the HS group, indicating a reduction in neural stress
   response and an increase in neuronal survival rate (Figure S2A-B).

   To investigate the role of HS + PAMAM@Cur in HS-induced hypothalamic
   neuronal apoptosis, we conducted immunofluorescent double staining on
   hypothalamic slices. The results revealed that compared to the HS group
   and HS + PAMAM group, the HS + Cur and HS + PAMAM@Cur groups exhibited
   significantly reduced NeuN + cleaved-caspase3 + and
   NeuN + TUNEL + positive cells, indicating a significant reduction in
   HS-induced hypothalamic neuronal apoptosis with both PAMAM@Cur and Cur
   treatments, with the efficacy of PAMAM@Cur still superior to Cur
   (Fig. 5H-I). Additionally, Western blot analysis unveiled a significant
   increase in BDNF, HSP70, and HIF-1α expression, along with a notable
   decrease in IL-1β and TNF-α expression in the HS + PAMAM@Cur group,
   reflecting significant neuroprotection and anti-inflammatory effects
   (Fig. [131]5J-K).

   In conclusion, PAMAM@Cur, by enhancing the BBB penetration capability
   and bioavailability of Cur, significantly ameliorated hypothalamic
   nerve injury in the HS model. This improvement encompasses enhanced
   cognitive function, reduced anxiety behavior, protection of neural cell
   structure, alleviation of neural stress response, and mitigation of
   inflammatory reactions. In contrast, while standalone Cur treatment
   exhibited some therapeutic effects, its limited BBB penetration
   capability resulted in outcomes inferior to the significant effects of
   PAMAM@Cur.

Multi-omics analysis reveals the therapeutic mechanism of PAMAM@Cur in
treating HS by regulating antioxidant defense genes and ferroptosis-related
genes

   To delve deeper into the molecular mechanisms of PAMAM@Cur in treating
   hypothalamic nerve injury caused by HS, we systematically analyzed the
   transcriptome and proteome of hypothalamic tissues from mice in the HS
   group and the HS + PAMAM@Cur group. Transcriptomic data revealed that
   after PAMAM@Cur treatment, the expression levels of 47 genes were
   upregulated, while 72 genes were downregulated (Fig. [132]6A). Heatmap
   analysis further demonstrated a consistent regulatory pattern of these
   genes' expression in the HS + PAMAM@Cur group, highlighting the
   specific gene expression changes induced by the treatment
   (Fig. [133]6B). KEGG pathway enrichment analysis indicated that these
   DEGs underwent significant changes in biological pathways closely
   related to neuronal damage and repair, such as ferroptosis, oxidative
   stress response, and central nervous system myelination (Fig. [134]6C).
   Furthermore, GO analysis emphasized the roles of these genes in
   biological processes such as oxidative stress response, and neuronal
   development, as well as in molecular functions related to structural
   composition and receptor activity (Fig. [135]6D). By conducting protein
   interaction analysis using the STRING database and the MCODE plugin in
   Cytoscape software on 119 DEGs, we found that Cluster 1 mainly
   comprised genes associated with neuronal signal transduction and
   protein stability (Fig. [136]6E), while Cluster 2 focused on genes
   closely linked to ferroptosis, further confirming the crucial role of
   ferroptosis in hypothalamic nerve injury induced by HS (Fig. [137]6F).

Fig. 6.

   [138]Fig. 6
   [139]Open in a new tab

   Analysis of transcriptome sequencing results. Note: A Volcano plot of
   gene expression in transcriptome sequencing, where the x-axis (logFC)
   represents the logarithm of fold change in gene expression, and the
   y-axis (-log10(p-value)) represents the statistical significance. The
   colors of data points indicate the direction of gene expression
   changes: red for upregulated genes, green for downregulated genes, and
   black for genes with insignificant changes; B Heatmap illustrating
   differential gene expression, where each row represents a gene and each
   column represents a sample. The color intensity reflects the level of
   gene expression: blue for decreased expression and yellow for increased
   expression; C Display of KEGG enrichment analysis results for DEGs,
   depicting gene-pathway associations through lines. Line colors
   represent various diseases or biological processes, while the thickness
   of lines and the size of circles next to genes indicate the extent of
   gene expression changes; D Results of GO enrichment analysis for DEGs,
   showcasing the biological functions (BP: Biological Process), cellular
   components (CC: Cellular Component), and molecular functions (MF:
   Molecular Function) affected by gene expression changes. Each point
   represents an enriched GO term, where point size denotes the number of
   genes (Count), color depth (q-value) signifies statistical
   significance, and the x-axis (GeneRatio) indicates the proportion of
   significant genes in a specific GO term; E–F Analysis of DEGs in the
   PPI network from the STRING database using the MCODE plugin in
   Cytoscape software. The clusters in these graphs represent highly
   interactive protein groups, where proteins in each cluster may
   collectively participate in the same biological process. HS n = 3,
   HS + PAMAM@Cur n = 3

   Principal component analysis of the proteomic data revealed a
   significant overall difference in protein profiles between the HS group
   and the HS + PAMAM@Cur group (Fig. [140]7A), with consistent expression
   patterns of proteins of different molecular weights across the two
   groups (Fig. [141]7B). Differential protein analysis identified 18
   upregulated and 11 downregulated proteins (Table S3). This was further
   corroborated through heatmap analysis, demonstrating that PAMAM@Cur
   significantly influenced the expression levels of key proteins,
   including GPX4 and SLC7A11, which play crucial roles in antioxidant
   defense, iron metabolism, and ferroptosis processes (Fig. [142]7C). The
   enrichment results of KEGG pathways for differential proteins showed
   significant enrichment of multiple biological pathways related to
   ferroptosis, particularly the ferroptosis pathway itself, further
   confirming our findings from transcriptomic analysis and protein
   expression heatmaps (Fig. [143]7D). GO enrichment analysis revealed
   significant enrichment of biological processes closely related to
   oxidative stress defense, electron transport chain, and GSH metabolism
   (Fig. [144]7E). The significance of these biological processes
   emphasizes the potential role of PAMAM@Cur in promoting redox balance
   and antioxidant stress response.

Fig. 7.

   [145]Fig. 7
   [146]Open in a new tab

   Proteomic analysis results. Note: A Principal component analysis
   illustrating the changes in protein profiles between the HS group and
   the HS + PAMAM@Cur group; B Box plot presenting reliable protein
   expression in the HS and HS + PAMAM@Cur groups; C Heatmap displaying
   differential protein expression, where each row represents a protein
   and each column represents a sample. Color variations indicate relative
   changes in protein expression levels, with red denoting increased
   expression and blue denoting decreased expression; D Enrichment
   analysis of KEGG pathways for differential proteins, where each point
   in the graph represents a specific KEGG pathway. The color of points
   reflects the significance of enrichment (p-value), with smaller
   p-values represented by a redder color, indicating greater statistical
   significance. The size of points indicates the number of differentially
   enriched proteins in that pathway. A higher enrichment score on the
   x-axis suggests more significant enrichment; E GO enrichment analysis
   results for differential proteins, encompassing biological processes
   (in green), cellular components (in orange), and molecular functions
   (in blue). The bar lengths in the graph indicate the significance of
   enrichment for each GO term, with higher -log10 p-values signifying
   greater statistical significance; F-G Analysis results of interactions
   between differential proteins using the MCODE plugin in Cytoscape
   software based on the STRING protein interaction data. The clusters in
   these graphs represent highly interacting protein groups, where
   proteins within each cluster may jointly participate in the same
   biological processes. HS n = 3, HS + PAMAM@Cur n = 3

   Furthermore, analysis using the MCODE module in Cytoscape unveiled that
   Cluster 1 was associated with signal transduction and protein stability
   (Fig. [147]7F), while Cluster 2 pointed to key genes involved in
   metabolic processes (Fig. [148]7G), aligning with the previous
   analytical results. Notably, the identification of Pcbp2, Slc7a11, and
   GPX4 not only confirmed findings from transcriptomic and proteomic data
   (Figure S3A-C) but also underscored their potential importance in
   regulating oxidative stress response and ferroptosis.

   Overall, integrating the multi-omics data, our study indicates that
   PAMAM@Cur may reduce ferroptosis and promote the repair of hypothalamic
   nerve injury induced by HS by modulating genes associated with
   antioxidant defense and neuroprotection.

PAMAM@Cur ameliorates hypothalamic nerve injury induced by HS through
mitigation of ferroptosis

   Through multi-omics analysis, we have identified that PAMAM@Cur may
   improve hypothalamic nerve injury induced by HS by modulating
   ferroptosis. To further confirm the role of iron accumulation and
   oxidative stress in hypothalamic damage caused by HS, Prussian Blue
   staining and assessment of oxidative stress markers (SOD activity, MDA,
   and GSH levels) were conducted. The results revealed that compared to
   the Control group, the HS group exhibited elevated levels of Fe^2+
   accumulation and MDA, along with decreased SOD and GSH levels. In
   contrast to both the HS and HS + PAMAM groups, the HS + PAMAM@Cur group
   demonstrated significant reductions in Fe^2+ accumulation and MDA
   levels, along with increased SOD activity and GSH content, indicating
   the effective inhibition of ferroptosis and oxidative stress by
   PAMAM@Cur (Fig. [149]8A-E). Additionally, Western blot analysis of the
   expression levels of PCBP2, SLC7A11, and GPX4 in the hypothalamus of
   mice across different groups revealed a significant increase in the
   protein expression levels of these markers in the HS + PAMAM@Cur group
   compared to the HS and HS + PAMAM groups, while the HS + Cur group
   showed only modest changes (Fig. [150]8F).

Fig. 8.

   [151]Fig. 8
   [152]Open in a new tab

   The impact of PAMAM@Cur on ferroptosis regulation in hypothalamus nerve
   injury induced by HS. Note: A Prussian blue staining observing the iron
   ion deposition in the hypothalamus of mice (scale bar = 50 μm); B
   Detection of Fe^2+ content in the hypothalamus tissue of mice using an
   assay kit; C-E Measurement of SOD activity, MDA levels, and GSH content
   in the hypothalamus tissue using assay kits; F Expression levels of
   PCBP2, SLC7A11, and GPX4 proteins in the hypothalamus of mice in
   different experimental groups assessed by Western blot; G-H Evaluation
   of mitochondrial membrane potential changes using JC-1 fluorescent
   staining (scale bar = 25 μm); -JI Detection of intracellular ROS levels
   using DCFDA fluorescent probe (scale bar = 25 μm).* indicates P < 0.05
   compared to the HS group, ** indicates P < 0.01; # indicates P < 0.05
   compared to the HS + Cur group, ## indicates P < 0.01. Six mice per
   group

   Mitochondrial dysfunction, a critical link in the ferroptosis pathway,
   is one of the direct causes of cell death induced by iron overload
   [[153]66]. Therefore, measurements of mitochondrial membrane potential
   and ROS levels were performed to assess mitochondrial health and
   function directly. Evaluation of mitochondrial membrane potential using
   JC-1 dye and detection of intracellular ROS levels with the DCFDA
   fluorescent probe revealed that compared to the Control group, mice in
   the HS group exhibited decreased mitochondrial membrane potential and
   significantly increased ROS production, indicating compromised
   mitochondrial function and exacerbated oxidative stress under HS
   conditions. However, in the HS + PAMAM@Cur group, the decrease in
   mitochondrial membrane potential was less pronounced, and ROS
   production was significantly reduced. These changes, in comparison to
   the HS group and HS + PAMAM group, suggest that PAMAM@Cur exerts a
   protective effect on mitochondrial function, effectively alleviating
   HS-induced mitochondrial damage and oxidative stress (Fig. [154]8G-J).

   Through a comprehensive analysis combining iron levels, oxidative
   stress markers, and mitochondrial function, we further confirmed that
   PAMAM@Cur mitigates hypothalamic nerve injury induced by HS by reducing
   ferroptosis.

PAMAM@Cur upregulates PCBP2 to stabilize SLC7A11 and GPX4 and reduce
ferroptosis

   In a previous proteomic analysis, we observed a significant
   upregulation of PCBP2 expression following treatment with PAMAM@Cur, a
   finding that captured our attention. PCBP2, an RNA-binding protein, has
   the potential to regulate the expression of specific proteins through
   interactions with mRNA [[155]67]. Based on this hypothesis, we focused
   on the potential targets of PCBP2, namely SLC7A11 and GPX4, both
   closely associated with the process of ferroptosis. To investigate the
   relationship between PCBP2 and these proteins, we conducted RIP
   experiments, confirming a direct interaction between PCBP2 and the mRNA
   of SLC7A11 and GPX4 (Fig. [156]9A). Furthermore, data from
   Starbase-CLIP supported this finding, demonstrating that PCBP2 can bind
   to both mRNAs (Figure S4A-B). Following PCBP2 knockdown and
   overexpression in the Na2 cell model, the most efficient sh-PCBP2-1
   sequence was selected for further studies (Figure S4C-D). Subsequent to
   the PCBP2 knockdown, mRNA levels of SLC7A11 and GPX4 significantly
   decreased (Fig. [157]9B). Treatment with actinomycin D revealed that in
   cells where PCBP2 was silenced, the half-life of SLC7A11 and GPX4 mRNA
   significantly shortened, indicating an impact on mRNA stability
   (Fig. [158]9C-D); conversely, in cells with PCBP2 overexpression, the
   half-life of these mRNAs extended significantly, suggesting that
   overexpression of PCBP2 enhanced the stability of SLC7A11 and GPX4 mRNA
   (Figure S4E-F). These experiments indicate that PCBP2 may enhance the
   stability of SLC7A11 and GPX4 mRNA by directly binding to them, thereby
   enhancing cellular regulation of iron ions and reducing ferroptotic
   cell death.

Fig. 9.

   [159]Fig. 9
   [160]Open in a new tab

   The influence of PAMAM@Cur on regulating PCBP2 stability to stabilize
   SLC7A11 and GPX4 in HS-induced ferroptosis. Note: A RIP experiment
   detecting the interaction between PCBP2 and SLC7A11 and GPX4 mRNA; B
   Detection of SLC7A11 and GPX4 mRNA levels after PCBP2 knockout; C-D
   Assessment of the effect of PCBP2 knockdown on the mRNA half-life of
   SLC7A11 and GPX4 following actinomycin D treatment; E Examination of
   the protein expression levels of PCBP2, SLC7A11, and GPX4 in cells from
   different experimental groups; F-I Utilization of assay kits to
   evaluate intracellular Fe^2+ accumulation, as well as changes in SOD,
   MDA, and GSH levels; J Evaluation of cell viability through CCK-8
   experiments. * indicates P < 0.05 compared to the Control group, **
   indicates P < 0.01; # indicates P < 0.05 compared to the HS group, ##
   indicates P < 0.01; & indicates P < 0.05 compared to the HS + PAMAM@Cur
   group, && indicates P < 0.01; @ indicates P < 0.05 compared to the
   HS + PAMAM@Cur + sh-PCBP2 group, @@ indicates P < 0.01. Cell
   experiments were repeated three times

   To determine whether PAMAM@Cur can stabilize SLC7A11 expression through
   PCBP2 and consequently reduce ferroptosis, we overexpressed SLC7A11 and
   GPX4 in PCBP2-knockdown N2a cells to test if the effects of ferroptosis
   could be reversed, as demonstrated in Figure S4G-H. Initially, the
   expression of relevant proteins in each group of cells was assessed.
   The results showed that compared to the Control group, the expression
   of PCBP2, SLC7A11, and GPX4 decreased in the HS group; in contrast, in
   the HS + PAMAM@Cur group, the expression of these proteins was
   upregulated compared to the HS group. When PCBP2 was silenced, the
   expression of PCBP2, SLC7A11, and GPX4 decreased again. Worth noting,
   through overexpression of SLC7A11 or GPX4
   (HS + PAMAM@Cur + sh-PCBP2 + oe-SLC7A11 and
   HS + PAMAM@Cur + sh-PCBP2 + oe-GPX4 groups), the levels of these
   proteins could be partially restored (Fig. [161]9E).

   Further assessment of intracellular Fe^2+ accumulation, SOD, MDA, and
   GSH levels using an iron ion detection kit was conducted to evaluate
   the status of ferroptosis and oxidative stress. The results revealed
   that in comparison to the Control group, the HS group exhibited
   significant increases in Fe^2+ accumulation and MDA levels, accompanied
   by notable decreases in SOD and GSH levels, indicating exacerbated
   oxidative stress and ferroptosis. Relative to the HS group, the
   HS + PAMAM@Cur group displayed considerable improvement in the levels
   of these oxidative stress markers, reflecting the protective effect of
   the treatment. However, in the PCBP2 knockdown HS + PAMAM@Cur group,
   significant increases in Fe^2+ accumulation and MDA levels were
   observed, while SOD and GSH levels decreased significantly, suggesting
   that inhibition of PCBP2 may attenuate the protective effect of
   PAMAM@Cur. Notably, in cells overexpressing SLC7A11 and GPX4, there was
   a reduction in Fe^2+ accumulation, decreased MDA levels, and increased
   SOD and GSH levels, indicating that overexpression of SLC7A11 and GPX4
   could reverse the adverse effects caused by PCBP2 silencing
   (Fig. 9F-I). These findings suggest that PAMAM@Cur may enhance cellular
   resistance to ferroptosis by upregulating PCBP2, thereby stabilizing
   the mRNA of SLC7A11 and GPX4.

   Subsequently, cell viability was assessed using the CCK-8 assay. The
   results revealed that compared to the Control group, the HS group
   exhibited a significant decrease in cell viability. Conversely, the
   HS + PAMAM@Cur group demonstrated increased cell viability compared to
   the HS group, while cell viability decreased in the
   HS + PAMAM@Cur + sh-PCBP2 group. Cell viability was restored or
   enhanced in the HS + PAMAM@Cur + sh-PCBP2 + oe-SLC7A11 and
   HS + PAMAM@Cur + sh-PCBP2 + oe-GPX4 groups, indicating a positive
   impact of SLC7A11 and GPX4 overexpression on reducing ferroptosis and
   improving cell survival (Fig. [162]9J).

   In conclusion, our study illustrates that PAMAM@Cur can mitigate
   HS-induced ferroptosis by upregulating the PCBP2 protein, thereby
   stabilizing and enhancing the levels of SLC7A11 mRNA effectively.

Discussion

   Previous research has brought significant attention to the application
   of nanotechnology in drug delivery systems [[163]68–[164]70]. However,
   studies on nano drug delivery systems for the treatment of HS, a severe
   heat stress reaction, remain limited. This study introduces a novel
   PAMAM nanoparticles loaded with Cur (PAMAM@Cur) and successfully
   demonstrates its significant neuroprotective effects in an HS model.
   Compared to traditional therapeutics, PAMAM@Cur nanoparticles exhibit
   improved bioavailability and stability of Cur, showcasing the potential
   to enhance therapeutic outcomes in HS treatment. The ability of
   PAMAM@Cur to effectively cross the BBB and target the hypothalamus
   makes it a promising alternative to existing treatments that often face
   limitations in drug delivery and efficacy.

   Traditional Cur, a natural compound with various biological activities
   such as anti-inflammatory and antioxidant properties [[165]71], faces
   limitations in drug delivery and low bioavailability [[166]72,
   [167]73]. Using PAMAM nanoparticles as carriers, this study
   encapsulates Cur within the nanoparticles, boosting its stability and
   bioavailability, thereby enhancing its efficacy in HS treatment.
   Compared to studies solely focusing on Cur, PAMAM@Cur exhibits a more
   significant therapeutic effect, showcasing the advantages of
   nanotechnology in drug delivery [[168]33]. Furthermore, the use of
   green nanomaterials in the formulation of PAMAM@Cur underscores the
   sustainability and biocompatibility of the approach, addressing
   environmental concerns associated with synthetic nanomaterials. Green
   nanomaterials, derived from natural sources, minimize toxicity and
   adverse effects, making PAMAM@Cur a safer and more eco-friendly option
   for clinical applications.

   This study significantly improved the bioavailability and stability of
   Cur by encapsulating it in PAMAM nanoparticles. This innovation not
   only demonstrated excellent efficacy in treating hypothalamic neural
   damage induced by heat shock but also showcased the broad prospects of
   nanotechnology in drug delivery. The PAMAM@Cur nanoparticles, with
   their remarkable Blood–brain barrier penetration capability and
   excellent cell compatibility, offer a novel and effective approach for
   neuroprotection and anti-inflammatory therapy. Furthermore, the
   development of this nanodelivery system provides insights for the
   delivery of other drugs that have difficulty crossing the Blood–brain
   barrier, thereby expanding the application of nanotechnology in the
   treatment of neurodegenerative diseases, brain injuries, and other
   neurological disorders. By utilizing green nanomaterials, this study
   contributes to the development of sustainable therapeutic strategies,
   aligning with global efforts to reduce the environmental footprint of
   medical interventions.

   Research on HS predominantly focuses on aspects like temperature
   regulation, inflammatory responses, and neural damage [[169]3, [170]74,
   [171]75]. This study, through behavioral assessments, histological
   examinations, and biochemical analyses, reveals the therapeutic effects
   of PAMAM@Cur on hypothalamus nerve injury in an HS mouse model. The
   results demonstrate improvements in behavior and reduction of
   inflammation, offering a comprehensive therapeutic approach for HS
   treatment. In comparison to previous studies concentrating on singular
   aspects, this research comprehensively explores the treatment
   mechanisms of HS, highlighting the potential of PAMAM@Cur as a robust
   alternative to traditional therapeutics.

   Moreover, in cellular biology experiments, this study further validates
   the cell compatibility and penetration capability of PAMAM@Cur in a BBB
   model. These experimental findings provide deeper support for the
   effectiveness of PAMAM@Cur in treating HS. Compared to the interaction
   mechanisms between traditional drugs and cells, nano-drug delivery
   systems exhibit superior cellular penetration and bioavailability,
   laying a solid foundation for their clinical applications.

   Multi-omics analysis plays a crucial role in elucidating disease
   treatment mechanisms. This study delves deep into the potential
   mechanisms of PAMAM@Cur in treating hypothalamus injury induced by HS
   through transcriptomic and proteomic analyses. The results demonstrate
   that PAMAM@Cur exerts its effects by regulating antioxidant defense
   genes and ferroptosis-related genes, playing a significant role in
   alleviating neural damage caused by HS. In comparison to past studies
   focusing solely on surface phenomena, this research delves more
   profoundly into the molecular-level regulatory effects of the drug
   [[172]76–[173]78].

   In summary, this study successfully developed PAMAM@Cur nanoparticles
   and validated their efficacy and mechanism in treating hypothalamus
   nerve injury induced by HS through a series of in vivo and in vitro
   experiments (Fig. [174]10). By loading Cur into PAMAM nanoparticles,
   the bioavailability and stability of Cur were enhanced, allowing it to
   effectively penetrate the BBB and directly act on the damaged
   hypothalamus region. The research findings indicate that PAMAM@Cur not
   only alleviates neural cell damage and reduces inflammation caused by
   HS but also, by regulating antioxidant defense genes and
   ferroptosis-related genes, particularly through the upregulation of
   PCBP2 protein, stabilizing SLC7A11 and GPX4 mRNA, enhancing the cell's
   regulation of iron ions, and reducing iron-dependent cell death. These
   discoveries offer new strategies for the treatment of HS and related
   neural injuries, showcasing the immense potential of nanotechnology in
   enhancing the efficacy of traditional drugs.

Fig. 10.

   [175]Fig. 10
   [176]Open in a new tab

   Mechanistic investigation of PAMAM nanoparticles loaded with Cur
   regulating PCBP2-mediated cell ferroptosis in the treatment of
   hypothalamus nerve injury induced by HS

   The scientific and clinical significance of this study lies in
   proposing an innovative treatment strategy for HS, a global public
   health concern, by utilizing PAMAM nanoparticles loaded with Cur to
   treat hypothalamus nerve injury induced by HS. Detailed investigations
   into nanoparticle preparation and drug delivery characteristics confirm
   the significant efficacy of PAMAM@Cur in the HS model, not only
   improving behavioral performance and neural cell structure but also
   reducing inflammation, paving the way for novel directions in HS
   treatment. The results of this study hold important scientific
   significance and hold promising potential for clinical applications by
   providing insights into the mechanisms of neural damage induced by HS
   and exploring new drug treatment approaches.

   Nevertheless, this study has certain limitations. For instance,
   potential biases and constraints in experimental design need further
   validation in terms of the reproducibility and reliability of the
   experimental results. Additionally, more in-depth research and
   exploration are required to investigate the stability,
   biocompatibility, and feasibility of the clinical application of
   PAMAM@Cur in long-term treatment.

   Looking ahead, building upon the outcomes of this study, future
   research could explore the potential application of PAMAM@Cur in a
   broader range of disease models, expanding its possibilities in
   treating other neurological disorders. By integrating high-throughput
   technologies such as transcriptomics and proteomics, and delving deeper
   into the therapeutic mechanisms, it is promising to provide new
   insights and strategies for the development of nanomedicine in the
   field of neural injury treatment, advancing the application and
   development of nanotechnology in clinical medicine. Therefore, despite
   the challenges and outstanding issues, this study brings hope and
   enlightenment for future research and clinical therapy in related
   fields.

Supplementary Information

   [177]Additional file1 (JPG 2678 KB)^ (2.6MB, jpg)
   [178]Additional file2 (JPG 2189 KB)^ (2.1MB, jpg)
   [179]Additional file3 (JPG 631 KB)^ (630.9KB, jpg)
   [180]Additional file4 (JPG 1306 KB)^ (1.3MB, jpg)
   [181]Additional file5 (DOCX 18 KB)^ (17.8KB, docx)

Acknowledgements