Abstract Oxidative stress and inflammatory dysregulation play crucial roles in pathogenesis of acute lung injury (ALI), and their cyclic synergy drives excessive inflammatory responses and further exacerbates ALI. Therefore, new effective strategies to treat ALI are urgently needed. Herein, a novel synergistic selenium based chlorogenic acid nanoparticle was developed to disrupt the cyclic synergistic effect between oxidative stress and inflammatory response in ALI. The chlorogenic acid, a polyphenol commonly found in herb, had been effectively conjugated with human serum albumin and coated on selenium nanoparticles (Se NPs) to create CHSe NPs. The CHSe NPs exhibited superoxide dismutase(SOD) like and glutathione peroxidase(GPX) like activities, effectively scavenging various types of reactive oxygen species (ROS), and inhibited the inflammatory response of macrophages. Additionally, with excellent biosafety, CHSe NPs exhibited superior therapeutic effects in ALI mice models in vivo, surpassing the performance of the clinic drug dexamethasone. They remarkably reduced ROS levels, and elevated the SOD and GPX enzyme activities in lung tissue to exert antioxidant effects. In addition, the CHSe NPs modulated the immune microenvironment of ALI by reversing M1 macrophage polarization, downregulating the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-a), nitric oxide synthase (iNOS), and myeloperoxidase (MPO), and upregulating anti-inflammatory cytokine (IL-10) levels, thereby alleviating excessive inflammation and decreasing neutrophil infiltration. Further mechanistic research revealed that CHSe NPs directly acted on and modulated the expression of Mapk8ip1 and Itga2b, which were upstream proteins of MAPK signaling pathway and PI3K-Akt signaling pathway, therefore impeding the cyclic synergy between oxidative stress and inflammatory dysregulation. In summary, CHSe NPs synergistically exert antioxidant and anti-inflammatory effects by regulating the MAPK signaling pathway and PI3K-Akt signaling pathway, showing enormous potential in the treatment of ALI. Graphical Abstract [42]graphic file with name 12951_2025_3114_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03114-6. Keywords: Acute lung injury, CHSe NPs, ROS scavenging, Inflammatory modulation, Synergistic treatment Introduction Acute lung injury (ALI) is a severe disease caused by lung infection from pathogens such as bacteria, fungi, and viruses [[43]1]. Though respiratory support and anti-pathogen drugs are applied in clinical practice [[44]2], the mortality rate of sever ALI patients remains high, ranging from 30 to 40% [[45]3]. Therefore, there is an urgent need to develop new and effective strategies for treating ALI. The microenvironment of ALI is characterized by excessive generation of reactive oxygen species (ROS) and the activation of macrophages, which polarized from a resting state to pro-inflammatory M1 macrophages. Notably, during the progression of ALI, activated M1 macrophages overexpress ROS, including superoxide anions (O^2−), hydrogen peroxide (H[2]O[2]), hydroxyl radicals (•OH) and etc. Elevated ROS levels induce oxidative stress, disrupt lung oxidative homeostasis [[46]4], promote the pro-inflammatory M1 phenotype, and further recruitment of inflammatory neutrophils [[47]5, [48]6]. This induces the excessive synthesis and releases of various pro-inflammatory cytokines and harmful mediators, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) [[49]7, [50]8]. Those factors prompt the inflammatory imbalance, recruit inflammatory neutrophils [[51]9], release toxic mediators such as myeloperoxidase (MPO) [[52]10], which cause oxidative damage to lung tissue, and further exacerbate the inflammatory response in the lungs. Additionally, the stimulation of external pathogens activates relevant pro-inflammatory signaling pathways such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K-Akt) signaling pathways, initiating the pulmonary inflammatory response. Therefore, the cyclic synergistic effect between inflammatory response and elevated ROS levels that significantly exacerbating ALI [[53]11]. Selenium (Se), an essential nutrient and trace element, plays crucial roles in maintaining physiological activity [[54]12]. As the active center of natural antioxidant enzyme-glutathione peroxidase (GSH-Px) [[55]13], selenium enhances the activity of GSH-Px and ROS scavenging, thereby maintaining the redox balance of human body [[56]14]. Importantly, selenium exerts anti-inflammatory effects by remarkably enhancing the phagocytosis of macrophages and regulating the transform of pro-inflammatory M1 type to the anti-inflammatory M2 phenotype [[57]15]. There are four distinct valence states of selenium in nature: Se (-2), Se (0), Se (+ 4), and Se (+ 6) [[58]16]. These states have varying biological effects in vivo [[59]17, [60]18]. Among them, zero valent selenium nanoparticles (Se NPs) have lower toxicity and higher bioavailability [[61]19, [62]20], making them highly effective in the treatment of inflammatory diseases [[63]21], cancer [[64]22] and Alzheimer’s disease [[65]23]. Nevertheless, the instability and aggregation tendency of Se NPs limit their biological activity [[66]24]. To improve stability, biological activity and therapeutic effects, Se NPs are frequently modified by polysaccharides [[67]25], proteins [[68]26] or metal polyphenols [[69]27]. For instance, the chitosan coated selenium nano-enzymes can remove high levels of O^2− and •OH, and exhibit catalase (CAT) activity to inhibit the excessive H[2]O[2] production, demonstrating their significant antioxidant effects [[70]28]. Additionally, the albumin coated selenium nanoparticle can inhibit the expression of pro-inflammatory cytokines and alleviate the development of intestinal mucositis [[71]29]. However, the limited modifications of selenium nanoparticles restrict their broader applications. Natural phenolic compounds have gained widespread attention due to their extensive biological and pharmacological effects [[72]30].Chlorogenic Acid (ChA), a common dietary polyphenol in herb, {[73]32} exhibits various biological activities such as antioxidant, anti-inflammatory, and immune regulation [[74]32, [75]33]. It has shown therapeutic functions in treatment of non-alcoholic fatty liver [[76]34], neuroinflammation [[77]35], and colitis [[78]36] through regulating macrophage polarization. Moreover, it inhibits the infiltration of activated neutrophils and exerting antioxidant and anti-inflammatory effects in lipopolysaccharide (LPS) induced lung inflammation [[79]37] and exerts anti-inflammatory effects by inhibiting the expression of KAT2A and pro-inflammatory cytokines, including IL-1b, IL-6, and TNF-a, thereby ameliorating LPS induced ALI [[80]38]. Although ChA has multiple beneficial properties, its clinical application is limited by its poor hydrophilicity, weak stability, rapid metabolism (metabolizes in half an hour in vivo), and low bioavailability [[81]39]. To address these limitations, selenium nanoparticles are combined with ChA to achieve enhanced therapeutic efficacy. In this study, we successfully combined Se NPs with ChA by using human serum albumin (HSA) as a medium, creating CHSe NPs. These nanoparticles displayed superior therapeutic effect on ALI through impeding the cyclic synergistic effect of inflammatory responses and ROS. As depicted in Scheme [82]1, CHSe NPs inhibited the lung inflammatory in ALI mice by reversing M1 macrophage polarization, reducing overexpression of pro-inflammatory cytokines, and inhibiting neutrophil infiltration. Moreover, they exhibited GPX and SOD enzyme activities, which could scavenge various types of ROS, improve the oxidative stress environment, and maintain lung oxidative homeostasis. Mechanism studies revealed that CHSe NPs modulated the expression of key protein such as Mapk8ip1 and Itga2b to regulate the MAPK and PI3K-Akt signaling pathways, aggravating the pulmonary inflammatory microenvironment. Notably, the virtual calculations further predicted that ChA could directly bind to Mapk8ip1 at the sites E185, Y230 and Q253, while it interacts with Itga2b through five residues (V295, R422, S292, S172, L421). CHSe NPs presented several advantages: (i) overcoming the limitation of single and limited effects of HSe NPs, providing a new scheme for collaborative therapy; (ii) efficient delivery of ChA, enhancing its pharmacological effects and bioavailability; (iii) synergistic anti-inflammatory and antioxidant properties through the combination of Se NPs and ChA. In summary, our study demonstrated the successful synthesis of synergistic nanomedicines CHSe NPs, and further revealed their mechanism to impede the cyclic synergistic effect between ROS and inflammation, and providing a new strategy for treatment of ALI. Scheme 1. [83]Scheme 1 [84]Open in a new tab Schematic illustration of the synthesis pathway of CHSe NPs and its therapeutic mechanism in ALI Experimental section Materials Chlorogenic acid (ChA) was purchased from Chengdu Manchester Biotechnology Co., Ltd. (Chengdu, China). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), human serum albumin (HSA), sodium selenite (Na[2]SeO[3], 99%), L-ascorbic acid, dexamethasone (Dex), dihydroethylene ingot (DHE), and lipopolysaccharide (LPS, from Escherichia coli O111: B4) were obtained from Sigma-Aldrich (Beijing, China). Phosphate buffer solution (PBS), DPPH and ABTS free radical scavenging capacity assay kit, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity assay kit, superoxide anion and hydroxyl free radical scavenging capacity assay kit, thiazolyl blue tetrazolium bromide (MTT) assay kit, 4’,6-diamidino-2-phenylindole (DAPI), reactive oxygen species assay kit, sterile enzyme free water, malondialdehyde (MDA) content assay kit, the reduced glutathione (GSH) assay kit, red blood cell lysate, and 4% paraformaldehyde fixed solution were bought from Beijing Solarbio Technology Co., Ltd. (Beijing, China). DMEM basic culture medium, fetal bovine serum (FBS), and penicillin streptomycin (PS) solution were acquired from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). Lyso-Tracker Green and nitric oxide (NO) content assay kit were obtained from Beyotime (Shanghai, China). N-hydroxysuccinimide (NHS) was purchased from Tianjin Xiensi Technology Co., Ltd. (Tianjin, China). N, N-Dimethylformamide (DMF) was bought from Tianjin Kermel Chemical Reagent Co.,Ltd. (Tianjin, China). The borate buffer solution (pH 8.2) was obtained from Xiamen Haibiao Technology Co., Ltd. (Xiamen, China). HSA-cyanidin dye (Cy5) was purchased from Xian Ruixi Biological Technology Co., Ltd. (Xian, China). Flow cytometry antibodies were all bought from BioLegend (San Diego, USA). TRNzol universal reagent was purchased from Tiangen Co., Ltd. (Beijing, China). HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) and AceQ qPCR SYBR Green Master Mix were obtained from Vazyme (Beijing, China). Isoflurane was purchased from Tianjin Ringpu Bio-technology Co., Ltd. (Tianjin, China). The mouse interleukin-1b (IL-1b), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), and interleukin-10 (IL-10) enzyme-linked immunosorbent assay kits were all acquired from Ruixin Biotechnology Co., Ltd. (Guangzhou, China). Preparation of CHSe NPs 3 mL solution of ChA-HSA was stirred, and then 2 mL of 3 mg/mL Na[2]SeO[3] was added, followed by the injection of 200 µL of 120 mg/mL L-ascorbic acid solution. The mixed solution was stirred and reacted for 30 min. After the reaction, the solution was washed with PBS and ddH[2]O and filtrated through a 0.22 μm membrane. After centrifugation, the precipitates were dispersed in 1 mL of ddH[2]O to obtain CHSe NPs. Characterization of CHSe NPs The particle size, zeta potential, and stability of CHSe NPs were characterized using the Zetasizer Nano series (Malvern Instruments, UK). The morphology and elemental distribution of particles were detected by Transmission Electron Microscope (TEM) (JEM-F200, Japan). The characteristic absorption peak of CHSe NPs was tested by Ultraviolet-Visible Spectroscopy (UV2700, Japan). The molecular structure of CHSe NPs was determined by Raman Spectroscopy (Horiba LabRAM HR Evolution, Japan). The crystal structure of CHSe NPs was analyzed by X-ray Diffraction (XRD) (Rigaku SmartLab SE, Japan). The elements and valence states of CHSe NPs were examined by X-ray Photoelectron Spectroscopy (XPS) (Thermo Fisher Scientific, ESCALAB250Xi, USA). The functional groups of CHSe NPs were identified by Fourier transform infrared spectroscopy (FTIR) (Thermo Scientific Nicolet iS20, USA). The concentration of Se in CHSe NPs was measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Agilent 7700s, USA). ROS scavenging capacity The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) free radical scavenging capacity assay kit, 2,2ʹ-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS) free radical scavenging capacity assay kit, superoxide anion scavenging capacity assay kit, hydroxyl radical scavenging capacity assay kit as well as electron spin resonance (ESR) spectrometer (Bruker EMXPLUS, Germany), SOD activity assay kit and GPX activity assay kit were applied to probe the antioxidant ability of ChA, HSe NPs, and CHSe NPs in vitro. Endocytosis and metabolic capacity The RAW 264.7 were seeded in a confocal dish at a density of 1 × 10^5 cells/well and cultured overnight. The cells were stimulated with 1 µg/mL LPS for 24 h. After serum-starvation for 4 h (h), the medium was replaced with complete medium containing ChA (ChA: 488 ng/mL), HSe-Cy5 NPs (Se: 400 ng/mL), or CHSe-Cy5 NPs (ChA: 488 ng/mL, Se: 400 ng/mL). After culturing for different time points (2, 4, 6, and 8 h), the cells were fixed with 4% paraformaldehyde, stained with Hoechst (represent nuclei) and further observed under a confocal laser scanning microscope (Leica, Germany). Data were analyzed by Image J (National Institutes of Health, USA). To evaluate the intracellular lysosomal metabolic capacity of CHSe NPs, RAW264.7 cells were incubated with CHSe-Cy5 NPs (ChA: 488 ng/mL, Se: 400 ng/mL) for 8 h, and stained with a mixture of Hoechst and 50 nM Lyso-Tracker Green. The cells were observed under a confocal laser scanning microscope and data were analyzed by Image J. Intracellular ROS scavenging activity RAW 264.7 cells were cultured overnight in a confocal dish at a density of 1 × 10^5 cells/well. Then cells were divided into 5 groups: (1) Control group, untreated group; (2) LPS group, cells were treated with 1 µg/mL LPS; (3) LPS + HSe NPs group, cells were incubated with 1 µg/mL of LPS and HSe NPs (Se: 400 ng/mL); (4) LPS + ChA group, cells were incubated with 1 µg/mL of LPS and ChA (ChA: 488 ng/mL); (5) LPS + CHSe NPs group, cells were incubated with 1 µg/mL of LPS and CHSe NPs (Se: 400 ng/mL, ChA: 488 ng/mL). After 24 h of incubation, the cells were stained with the DCFH-DA ROS assay kit, and the nuclei were stained by Hoechst. The intracellular level of ROS in the five groups were examined by confocal scanning microscopy and data were analyzed by Image J. For flow cytometry experiment, RAW264.7 cells were cultured overnight in a 24 well plate(1 × 10^5 cells/well), and treated as described above. After the cells were collected, the intracellular ROS levels of 5 groups was quantitative assessed by flow cytometry (FACSVerse, BD, USA) and analyzed using FlowJo_v10.8.1 (Tree Star Inc, USA). Macrophage polarization RAW 264.7 cells were seeded in a confocal dish with a density of 1 × 10^5 cells/well, cultured overnight and treated as described in 2.6. After incubation for 24 h, cells from the five groups were collected, stained with anti-mouse PE-CD206 antibody, anti-mouse PerCP-CD11b antibody, and anti-mouse APC-F4/80 antibody, detected by flow cytometry and further analyzed using FlowJo_v10.8.1. Real-time fluorescence quantitative PCR (qRT-PCR) detection RAW264.7 cells were treated as described in Sect. 2.6. Aftertreatment, Then, total RNA from 5 groups were extracted by Trizol General Co., Ltd. (Tiangen, Beijing, China), and reverse transcription were performed by using The HiScript IIQ RT SuperMix for qPCR (+ gDNA wiper) kit. Then, the expressions of following genes were detected by qPCR SYBR Green Master Mix, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the internal reference. The specific primers for each gene were listed as follows Table. [85]1. Table 1. Primer sequence Gene name Primer sequence GAPDH Forward 5’ - GGCAAATTCAACGGCACAGTCAAG − 3’ Reverse 5’ - TCGCTCCTGGAAGATGGTGATGG − 3’ IL-1b Forward 5’ - AGCTTCAGGCAGGCAGTATC − 3’ Reverse 5’ - AAGGTCCACGGGAAAGACAC − 3’ IL-6 Forward 5’ - CTTCTTGGGACTGATGCTGGTGA − 3’ Reverse 5’ - TCTGTTGGGAGTGGTATCCTCTGTG − 3’ TNF-a Forward 5’ - CGCTCTTCTGTCTACTGAACTTCGG − 3’ Reverse 5’ - GTGGTTTGTGAGTGTGAGGGTCTG − 3’ iNOS Forward 5’ - TGCTTTGTGCGAAGTGTCAG − 3’ Reverse 5’ - CCCTTTGTGCTGGGAGTCAT − 3’ IL-10 Forward 5’ - GCTCTTACTGACTGGCATGAG − 3’ Reverse 5’ - CGCAGCTCTAGGAGCATGTG − 3’ [86]Open in a new tab Molecular docking detection The inflammation-related proteins structures (Mapk8ip1, Itga6, Ereg, Itga2b) were obtained from the Protein Data Bank (PDB, [87]http://www.rcsb.org) with PDB IDs 2G01, 7CEC, 8HGP, and 8T2U. Molecular docking analysis were conducted using Discovery Studio 2019. Initially, the X-ray crystal structures of the protein targets were preprocessed by adding hydrogen atoms, performing energy minimization using the CHARMm force field to optimize their conformations, and leaving all non-hydrogen atoms fixed. Then, the active sites within the protein cavities were identified. Subsequently, a docking protocol was applied to analyze the interactions between GA and the differential proteins by LibDock, ultimately selecting the predicted binding site with the lowest CDOCKER INTERACTION ENERGY. PyMOL software (Version 3.0, Schrödinger, LLC) was utilized for enhanced visualization and understanding of the results. Immunofluorescence staining RAW264.7 cells were processed and grouped as described in 2.6. Subsequently, the cells were fixed with 4% paraformaldehyde, washed with PBS, permeabilized by 0.1% Triton X-100, and blocked with 5% BSA for 1 h. Then cells were incubated with the primary antibodies MAPK8IP1 (ProteinTech Group, Chicago, IL, USA, dilution: 1:200) or ITGA2B (ProteinTech Group, Chicago, IL, USA, dilution: 1:200) for 1.5 h at room temperature and further incubated with secondary antibody Alexa Fluor 594-goat anti-rabbit IgG (AS039, dilution: 1:200) for 1 h. Fluorescent images were observed using a confocal laser scanning microscope. Establishment of acute lung injury mice model C57BL/6 mice (6 weeks old, male, 20–22 g) were purchased from Beijing Yaokang Biotechnology Co., Ltd. (Beijing, China). All experiments were performed in strict compliance with the permission of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences Animal Care and Use Committee (SYXK-2019–0002). The mice were randomly divided into six groups: (1) Control group (tracheal injection of PBS); (2) LPS group (tracheal injection of 2.5 mg/mL LPS); (3) HSe NPs group (tracheal injection of 2.5 mg/mL LPS and HSe NPs); (4) ChA group (tracheal injection of 2.5 mg/mL LPS and ChA); (5) CHSe NPs group (tracheal injection of 2.5 mg/mL LPS and CHSe NPs); (6) Dex group (tracheal injection of 2.5 mg/mL LPS and 2.5 mg/mL Dex). Mice were anesthetized with isoflurane. (Note: Se: 50 µg/mL, ChA: 61 µg/mL). After anesthesia, the mice were fixed on a surgical board. Then 40 µL of LPS was slowly injected into the tracheal cartilage connection to established an acute lung injury mice model. After 2 h, 40 µL of the drug was administered into the trachea according to the different treatment methods mentioned above. After 22 h of treatment, the mice were euthanized and the main tissues of the six groups of mice were harvested for subsequent correlation analysis. Detection of antioxidant indicators in vivo The lungs from the six groups were excised, minced, diluted with PBS containing protease inhibitors. The homogenate was subjected to repeated freeze-thaw and ultrasound fragmentation. Finally, the supernatants were collected after centrifuge at 5000 g for 10 min at 4 ℃ for further analysis of antioxidant indicators. The malondialdehyde (MDA) content, SOD activity, and reduced GSH content in each group of mice were analyzed according to the respective assay kit instructions. Collection and flow cytometry detection of immune cells in bronchoalveolar lavage fluid (BALF) The mice were euthanized 22 h post treatment and the lungs from the six groups were washed with 2 mL PBS to collect BALF. Then BALF was lysed by 2 mL of red blood cell lysate and further washed by PBS. The cells in BALF were stained with antibodies of M1 macrophages (PerCP-CD11b, APC-F4/80) or neutrophils (FITC-CD45, PE-CD11b, and APC-LY6G) and examined by flow cytometry. The experimental results were analyzed using FlowJo_v10.8.1. Immunohistochemical detection The lung tissue from six groups of mice was harvested, embedded, sliced, dewaxed and dehydrated, for further staining of iNOS or MPO. After antigen repair, slices were with 3% hydrogen peroxide to inhibit endogenous peroxidase and sealed with 10% goat serum. Then the lung slices were incubated with primary antibodies overnight at 4 ℃. Subsequently, the secondary antibodies (goat anti rabbit-HRP) were incubated with slice for 45 min, sealed, dried and observed under a microscope (Olympus, Japan) to investigate the expression levels of iNOS and MPO. Statistical analysis All data in this article were presented as mean ± standard deviation. The data were analyzed using one-way analysis of variance (ANOVA) (GraphPad Prism 8, GraphPad Softwar, San Diego, USA). The statistical significance was displayed throughout the text, charts, and legends using the following terms: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001, ^# represented the comparison between the LPS group and the control group ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, ^* indicated comparison between other experimental groups and LPS group; ns denoted no significant difference. Results and discussion Synthesis and characterization of CHSe NPs The synthesis of CHSe NPs were demonstrated in Scheme [88]1. HSA was conjugated with ChA via amide reaction to form ChA-HSA, which was subsequently coated on the surface of Se NPs through a redox reaction to create CHSe NPs. Initially, the successful synthesis of ChA-HSA was validated. In Fig. [89]S1, the results of ultraviolet-visible spectroscopy (UV-vis) indicated that ChA-HSA included the characteristic peaks of HSA (280 nm) and ChA (335 nm), confirming the successful synthesis of ChA-HSA. Next, matrix assisted laser dissociation ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to examine the coupling rate of ChA per HSA, and results showed that the coupling rate was 26 ChA per HSA (Fig. [90]S2). Additionally, changes of Zeta potential further confirmed the successful synthesis of ChA-HSA (Fig. [91]S3). Next, the synthesis of CHSe NPs was demonstrated. The transmission electron microscopy (TEM) image of CHSe NPs showed excellent spherical morphology with good dispersion (Fig. [92]1A). The insert figure further proved the successfully coated of ChA-HSA on the surface of Se NPs. Subsequently, the average hydrodynamic particle size of CHSe NPs was determined by dynamic light scattering (DLS). With good dispersion in water, the size of CHSe NPs was approximately to 35 nm (Fig. [93]1B and [94]S4). As shown in Fig. [95]1C, the zeta potential of Se NPs (-29.9 mV) increased to -24 mV after coated with ChA-HSA (-24.5 mV), which confirmed the successful construction of CHSe NPs. In addition, elemental mapping images indicated the presence of C (blue line), N (purple line), O (yellow line), and Se (red line) elements in CHSe NPs (Fig. [96]1D). Notably, the Se element was centrally located and other elements were distributed around it, confirming the successfully coated of ChA-HSA on Se NPs. Additionally, energy dispersive X-ray spectroscopy (EDX) revealed the characteristic energy peaks of C, N, O, and Se elements, further proving the successfully synthesis of CHSe NPs (Fig. [97]S5). Next, the UV-vis spectroscopy proved that the characteristic absorption peaks of CHSe NPs were in consistent with characteristic peaks of Se NPs and ChA-HSA, demonstrating the successful construction of CHSe NPs (Fig. [98]S6). The Raman spectroscopy (Fig. [99]S7) and X-ray diffraction (XRD, Fig. [100]S8) analysis further supported that. Moreover, X-ray photoelectron spectroscopy (XPS) analysis revealed the expected peaks corresponding to C1s, N1s, O1s and Se3d in CHSe NPs (Fig. [101]1E) as well as C1s, N1s, O1s in ChA-HSA (Fig. [102]1H), respectively. In Fig. [103]1F, the spectrum of C1s showed the peaks at 284.80 eV, 286.18 eV, and 288.30 eV, corresponding to C-C/C-H (66.48%), C-O (21.28%), and C = O (12.23%), respectively, indicating the present of ChA-HSA in CHSe NPs. In addition, the spectrum of Se3d displayed two characteristic peaks: Se3d5/2 and Se3d3/2 in CHSe NPs (Fig. [104]1G). Subsequently, the characteristic peaks of Se NPs (1541 and 1656 cm^− 1) and ChA-HSA (2849 and 2917 cm^− 1) were also showed in CHSe NPs (1532, 1641, 2850, and 2917 cm^− 1) by Fourier transform infrared spectroscopy (FTIR) (Fig. [105]1I). In brief, above results confirmed the successful synthesis of CHSe NPs. Finally, the stability of CHSe NPs over 30 days were monitored in ddH[2]O, PBS, and DMEM + FBS. The CHSe NPs showed excellent stability under three conditions (Fig. [106]1J). The polydispersity index (PDI) of CHSe NPs further proved that (Fig. [107]S9). In addition, the concentration of Se in HSe NPs (0.28 mg/mL) and CHSe NPs (0.41 mg/mL) were detected by ICP (Fig. [108]S10). In summary, well-dispersed CHSe NPs was successfully synthesized with excellent stability. Fig. 1. [109]Fig. 1 [110]Open in a new tab Characterization of CHSe NPs. (A) TEM image of CHSe NPs (scale bar = 30 nm). The insets showed higher-magnification images of CHSe NPs (scale bar = 30 nm). (B) Hydrodynamic diameter of CHSe NPs. (C) Zeta potential of ChA-HSA, Se NPs, and CHSe NPs. (D) Elemental mapping images of CHSe NP, and the dashed circles indicated location of nanoparticle. (E) The XPS survey spectrum of CHSe NPs. (F, G) High resolution XPS spectra of the C1s (F) and Se3d (G) in CHSe NPs. (H) XPS survey spectrum of ChA-HSA. (I) FTIR spectra of ChA-HSA, Se NPs, and CHSe NPs. (J) Stability analysis of CHSe NPs in ddH[2]O, PBS, and DMEM + FBS media during 30 days. The inset showed the color of CHSe NPs in different condition on day 30. Data were presented as mean ± SD. n = 3, data were presented as mean ± SD ROS scavenging capacity of CHSe NPs in vitro In view of the excessive ROS levels in ALI, the ROS scavenging ability of CHSe NPs was crucial in treatment of ALI. The antioxidant property of CHSe NPs was evaluated (Fig. [111]2A). Initially, the general free radical scavenging activity of CHSe NPs were assessed by 2,2-diphenyl-1-picrylhydrazine radical (DPPH•), a stable nitrogen-centered radical with a characteristic UV-vis absorption peak at 515 nm. In Fig. [112]2B, compared with the control group, the absorbance of DPPH• in ChA and HSe NPs groups were significantly decreased at 515 nm, and further declined in CHSe NPs group, indicating the superior DPPH• scavenging activity of CHSe NPs. The inset photos showed the scavenging of DPPH• in the four groups. Statistical data further demonstrated the dose-dependent DPPH• scavenging activity of CHSe NPs (Se: 5, 10, 20, 50, 100, 200, 400, and 800 µg/mL) (Fig. [113]2C, S14). Notably, when the concentration of CHSe NPs reached 400 µg/mL, the DPPH• scavenging rate was approaching saturation (98%). Similarly, the scavenging ability of CHSe NPs on 2,2-diazo-di (3-ethylbenzothiazole-6-sulfonic acid) diamine radical (ABTS^•+) was further evaluated. The characteristic absorption peak of ABTS^•+ at 405 nm and 734 nm was decreased in ChA, HSe NPs, and CHSe NPs groups than in control group, with the CHSe NPs group showed the lowest absorbance. The inset figures showed the scavenging of ABTS^•+ in the four groups. (Fig. [114]2D). The ABTS^•+ scavenging ability of CHSe NPs also displayed concentration dependence manner blow 50 µg/mL(Fig. [115]2E, S14 ). In brief, the CHSe NPs exhibited superior free radical scavenging activity than ChA and HSe NPs. Next, the scavenging ability of CHSe NPs on representative ROS types including superoxide anion (O[2]^•−) and hydroxyl radical (•OH) were evaluated to further explore their antioxidant capacity. As shown in Fig. [116]2F, compared with control group, the characteristic absorption peak of O[2]^•− was decreased in ChA and HSe NPs groups, and further reduced in CHSe NPs group, confirming their excellent O[2]^•− scavenging ability. The inset photos showed the scavenging of O[2]^•− in the four groups. Statistical data further proved the dose-dependent scavenging ability of CHSe NPs on O[2]^•− (Fig. [117]2G, S14). Furthermore, the •OH scavenging ability of CHSe NPs was also the strongest among the three treatment groups (Fig. [118]2H), and displayed dose-dependent scavenging ability on •OH (Fig. [119]2I, S11 and S14). Additionally, the scavenging abilities of CHSe NPs on O[2]^•− and •OH was further determined by electron spin resonance spectroscopy (ESR). As shown in Fig. [120]2J-M, the control group exhibited strong ESR signals, which gradually weakened in the three treatment groups, and CHSe NPs exhibited the greatest degree of signal reduction, indicating their efficiently scavenging abilities for O[2]^•− and •OH. Subsequently, the SOD and GPX enzyme-like activity of CHSe NPs were assessed. For the SOD-like activity, the absorbance spectrums of O[2]^•− in four groups were compared. Compared with the control group, the absorbance spectrum of O[2]^•− at 560 nm were decreased in ChA and CHSe NPs groups, with no significant change in the HSe NPs group, highlighting the dominant role of ChA in exerting SOD-like activity of CHSe NPs. The inset photos showed the inhibition of O[2]^•− in the four groups (Fig. [121]2N). Statistical data further proved the dose-dependent SOD enzyme-like ability of CHSe NPs (Fig. [122]2O, [123]S13 and [124]S14). In Fig. [125]2P, HSe NPs and CHSe NPs showed better GPX-like activity than ChA, indicating by the remarkable reduction of absorption peak at 412 nm than ChA. The inset photos depicted the inhibition of GSH in the four groups. Notably, the GPX-like activity of CHSe NPs displayed a dose-dependent manner (Fig. [126]2Q, [127]S14), and the inhibition rate approached saturation (72%) when the concentration of CHSe NPs reached 200 µg/mL (Fig. [128]S13). Summarily, CHSe NPs combined the advantages of ChA and HSe NPs in ROS scavenging and enzyme-like activities, thereby exerting the superior antioxidant capacity in vitro. Fig. 2. [129]Fig. 2 [130]Open in a new tab The antioxidant properties of CHSe NPs in vitro. (A) Schematic illustration depicted the antioxidant activity of CHSe NPs in vitro. (B) UV-vis absorbance spectra of DPPH• in control, ChA, HSe NPs, and CHSe NPs groups (the illustration showed the colors of different groups). (C) Statistical scavenging rate for DPPH• in various concentrations of CHSe NPs. (D) UV-vis absorbance spectra of ABTS^•+ for the four groups (the illustration showed the colors of four groups). (E) Statistical scavenging rate for ABTS^•+ under various concentrations of CHSe NPs. (F) UV-vis absorbance spectra of O[2]^•− in the four groups (the illustration showed the colors of the four groups). (G) Statistical scavenging rate for O[2]^•− under various concentrations of CHSe NPs. (H) Scavenging rate of •OH in the four groups. (I) Statistical scavenging rate for •OH by various concentrations of CHSe NPs. (J) ESR spectra of O[2]^•− in the four groups. (K) Corresponding quantitative ESR results for O[2]^•− in the four groups. (L) ESR spectra to characterize the •OH scavenging ability of four groups. (M) Corresponding quantitative ESR results for •OH in the four groups. (N) UV-vis absorbance spectra of O[2]^•− in the four groups (the illustration showed the colors of the four groups). (O) Statistical inhibition rate for O[2]^•− in various concentrations of CHSe NPs. (P) UV-vis absorbance spectra of GSH in the four groups (the illustration showed the colors of the four groups). (Q) Inhibition rate of GSH in various concentrations of CHSe NPs. n = 3, data were presented as the mean ± SD. **** P < 0.0001; * indicated a significant difference in comparison to the control group The intracellular metabolism and ROS scavenging ability of CHSe NPs The Mouse Monocyte-macrophage Leukemia Cells (RAW264.7) was selected as a model cell line to evaluate the intracellular role of CHSe NPs. To investigate the biosafety of CHSe NPs, different concentration of HSe NPs, ChA, and CHSe NPs were incubated with RAW264.7. All treatment groups (HSe NPs, ChA, and CHSe NPs groups) exhibited dose-dependent cytotoxicity on RAW264.7, and the cytotoxicity of CHSe NPs was lower than that of HSe NPs and higher than that of ChA (Fig. [131]3A). Notably, the survival rate of RAW264.7 cells remained above 80%, when the concentration of Se in CHSe NPs reached 400 ng/mL, demonstrating the superior biocompatibility of CHSe NPs. Therefore, this concentration was selected for subsequent cell experiments. Next, HSA was labeled with a cyanine dye (Cy5) to construct CHSe NPs-Cy5 for detecting their endocytosis ability. With the increasement of incubation time (2, 4, 6 and 8 h), more CHSe NPs were endocytosed by RAW264.7 (Fig. [132]3B, [133]S15). Notably, the CHSe NPs group displayed the highest red fluorescence than other three groups, indicating a greater uptake of CHSe NPs by RAW264.7 (Fig. [134]3C), which was further confirmed by statistical mean fluorescence of the four groups (Fig. [135]S16). Figure [136]3D presented the co-localization of CHSe NPs and lysosomes, suggesting that they could be metabolized via the lysosomal pathway. Fig. 3. [137]Fig. 3 [138]Open in a new tab Biosafety, endocytosis, metabolism and ROS-scavenging ability of CHSe NPs. (A) Viability of RAW264.7 cells after incubation with various concentrations of ChA, HSe NPs, and CHSe NPs. The x-axis represented various concentrations of Se and ChA. (B) Fluorescence images represented the endocytosis of CHSe NPs-Cy5 by RAW 264.7 cells in different time points (2, 4, 6 and 8 h), red fluorescence indicated CHSe NPs-Cy5, blue fluorescence from Hoechst33342 represented nuclei). Scale bar = 50 μm. (C) Cellular uptake efficiency of ChA, HSe NPs-Cy5 and CHSe NPs-Cy5 by RAW 264.7 cells (red fluorescence indicated HSe NPs-Cy5 or CHSe NPs-Cy5, blue fluorescence from Hoechst33342 represented nuclei). Scale bar = 50 μm. (D) Representative fluorescence images displayed the colocalization of CHSe NPs-Cy5(red fluorescence) with lysosomes(green fluorescence). Scale bar = 30 μm. (E) Fluorescence images of the intracellular ROS levels in the control, LPS, LPS + HSe NPs (HSe NPs), LPS + ChA (ChA), and LPS + CHSe NPs (CHSe NPs) groups (DCFH-DA, green fluorescence indicated ROS level, blue fluorescence from Hoechst33342 represented nuclei). Scale bar = 50 μm. (F, G) Representative flow cytometry images (F) and corresponding quantitative statistical data (G) showed the intracellular ROS levels in the five groups. n = 3, data were presented as the mean ± SD. ^####P < 0.0001, **** P < 0.0001; ^# indicated a significant difference in comparison to the control group, and * indicated a significant difference compared to the LPS group In view of the excellent ROS scavenging effect of CHSe NPs in vitro, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, green fluorescence) probe was utilized to assess their intracellular ROS scavenging ability. The LPS stimulated RAW 264.7 was employed as cellular model. The green fluorescence signal was remarkably elevated in LPS group than in control group, indicating the successfully established of ROS generation model (Fig. [139]3E). After treatment, the intensities of green fluorescence were significantly reduced in HSe NPs and ChA groups and further declined in CHSe NPs group (Fig. [140]3E and Fig. [141]S17), demonstrating the strongest ROS scavenging ability of CHSe NPs. The flowcytometry results confirmed that CHSe NPs group exhibited the lowest florescence intensity (represented ROS) among the five groups (Fig. [142]3F). Statistical data demonstrated that the ratio of ROS positive cells in LPS group (54.6%) was four times higher than that of control group (14.1%) and decreased to 46.9%, 41.2%, and 33.4% in HSe NPs, ChA, and CHSe NPs groups, respectively, proving the superior ROS scavenging ability of CHSe NPs than HSe NPs and ChA (Fig. [143]3G). Briefly, with favorable biosafety, CHSe NPs could be easily endocytosed by RAW 264.7, metabolized through the lysosome and displayed remarkable intracellular ROS scavenging ability. Intracellular anti-inflammatory abilities of CHSe NPs During pathogenesis of ALI, macrophages were activated and polarized into inflammatory M1 type, which caused severe lung injury and promoted the progression of ALI. Therefore, the anti-inflammatory effect of CHSe NPs on RAW264.7 was investigated. In Fig. [144]4A, compared with control group (3.88%), the frequency of M1 type macrophages significantly increased after LPS stimulation (54.4%), indicating the successful activation of RAW264.7. Notably, the proportion of M1 macrophages slightly decreased in HSe NPs group (52.7%), and further significantly reduced in ChA (45.5%) and CHSe NPs groups (39.8%) (Fig. [145]4A). The statistical data further confirmed the M1 type macrophages downregulatory ability of ChA and CHSe NPs but not HSe NPs, highlighting the dominant role of ChA in regulating M1 type macrophages (Fig. [146]4C). Simultaneously, the changes of anti-inflammatory M2 type macrophages in different treatment groups were also evaluated. In Fig. [147]4B, compared with control group (1.27%), the proportion of M2 macrophages was elevated in the LPS group (17.7%) and further increased in HSe NPs (20.3%) and CHSe NPs (25.8%) groups, but not in ChA group (11.0%). Statistical data proved the dominant regulatory role of HSe NPs in elevating portions of M2 type macrophages (Fig. [148]4D). The ratio of M1/M2 macrophage, an important indicator of inflammation development, was conducted in the five groups. Compared with control (2.8) and LPS group (3.1), the ratio of M1/M2 decreased in HSe NPs (2.5) and CHSe NPs (1.6) groups and elevated in ChA group (4.0) (Fig. [149]4E). Particularly, CHSe NPs combined the inhibitory effect of ChA on M1 type macrophage with the activated effect of HSe NPs on M2 type macrophage. Briefly, CHSe NPs reversed the polarization of inflammatory macrophages, thereby exerting anti-inflammatory effects. Fig. 4. [150]Fig. 4 [151]Open in a new tab Intracellular anti-inflammatory effect of CHSe NPs. (A, B) Representative flow cytometry images for M1-type (A) and M2-type (B) macrophages in the control, LPS, LPS + HSe NPs (HSe NPs), LPS + ChA (ChA), and LPS + CHSe NPs (CHSe NPs) groups. (C-E) Statistical data showed the percentages of M1-type (C), M2-type (D) macrophages as well as the ratio of M1/M2 macrophages (E) in the five groups. (F-J) mRNA expression of Il-1β (F), Il-6 (G), Tnf-a (H), Inos (I), and Il-10 (J) in the five groups. (K) The production levels of extracellular NO in the five groups. n = 3, data were presented as the mean ± SD. ^****P < 0.0001, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant; ^# indicated a significant difference in comparison to the control group, and * indicated a significant difference in comparison to the LPS group Given the excessive production of pro-inflammatory cytokines by overactivated macrophages in microenvironment of ALI, the anti-inflammatory effect of CHSe NPs was explored. The expression levels of pro-inflammatory cytokines were examined. After stimulation with LPS, the genes expression of IL-1b, IL-6, TNF-a, and iNOS were all remarkably elevated compared to the control group(Fig. [152]4F and I). In Fig. [153]4F, compared with LPS group, the expression of Il-1β gene was significantly decreased in HSe NPs and ChA groups and further remarkably reduced in CHSe NPs group, indicating their superior anti-inflammatory ability. Similarly, the gene expression levels of Il-6 (Fig. [154]4G), Tnf-a (Fig. [155]4H), and Inos (Fig. [156]4I) were also declined in HSe NPs and ChA groups and further downregulated in CHSe NPs group when compared with LPS group. Regarding the anti-inflammatory cytokines, the gene expression level of Il-10 was slightly increased in the LPS group and further significantly upregulated in HSe NPs, ChA, and CHSe NPs groups (Fig. [157]4J) than in control group. Notably, CHSe NPs displayed the optimal upregulation effect on expression of Il-10 among the three treatment groups. Since the overexpression of iNOS led to increased release of NO and promoted the development of inflammatory response, the regulatory effect of CHSe NPs on NO release were further investigated, and the standard curve was established (Fig. [158]S18). In Fig. [159]4K, compared with the LPS group, the release of NO was significantly reduced in HSe NPs and CHSe NPs groups, with no significant change in ChA group. In addition, HSe NPs(Fig. [160]S19) and CHSe NPs (Fig. [161]S20) but not ChA (Fig. [162]S21) exhibited a dose-dependent regulatory manner in downregulating expression of NO. Together, CHSe NPs reversed the polarization of M1 macrophages and regulated inflammatory responses, thereby exerting anti-inflammatory effects. Oxidative stress and inflammatory response regulatory mechanisms of CHSe NPs To further elucidated the oxidative stress and inflammatory regulatory mechanisms of CHSe NPs, the transcriptomic analysis was conducted. The Venn diagram showed that 19,459 genes were expressed among Control, LPS, HSe NPs, ChA, and CHSe NPs groups (Fig. [163]5A). The volcano map presented the distribution of 2197 differentially expressed genes (DEGs) between the LPS and CHSe NPs group, including 1424 upregulated genes (red dots) and 773 downregulated (green dots) genes (Fig. [164]5B). Additionally, a Venn plot in Fig. [165]5C showed a total of 24 DEGs shared among the three groups (LPS vs. CHSe NPs, Control vs. CHSe NPs, and Control vs. LPS), suggesting that these genes were essential genes in pathogenesis of ALI or they played critical role in the treatment of ALI. Subsequently, GO (Gene Ontology) or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted. The GO analysis showed that there were 2197 DEGs between the LPS group and the CHSe NPs group, categorized into three major parts: biological processes (BP, response to oxygen levels and apoptotic signaling pathway), cellular components (CC, cytoplasmic stress granule and endoplasmic reticulum membrane components), and molecular functions (MF, electron transfer and ATPase activity). Specifically, in BP, the CHSe NPs dominantly regulated inflammation related pathways [regulation of MAPK cascade (GO: 0043408), regulation of phosphatidylinositol 3-kinase signaling (GO: 0014066)] and oxidative stress-related pathways [regulation of oxidoreductase activity (GO: 0051341), response to oxidative stress (GO: 006979)]. The analysis of CC and MF suggested that CHSe NPs participated in the transcriptional translation process of genes and the regulation of kinases, such as transcription regulator activity (GO:0140110) and MAP kinase phosphatase activity (GO: 0033549) (Fig. [166]5D). To better elucidate the biological function of CHSe NPs, KEGG pathway enrichment analysis was utilized. Forty differential signaling pathways were identified between the LPS and CHSe NPs group, with 20 pathways related to inflammatory response and oxidative stress (Fig. [167]5E), including the MAPK signaling pathway (ko04010) and PI3K-Akt signaling pathway (ko04151) (Fig. [168]5E). Above findings further proved the regulating role of CHSe NPs on inflammatory response and oxidative stress pathways. Correspondingly, the gene heatmap of DEGs related to inflammatory responses and oxidative stress among the five groups were generated (Fig. [169]5F). Notably, the inflammatory response and oxidative stress related genes, including Mapk8ip1, Ereg, Itga2b Itga6 and etc., were upregulated after LPS stimulation. After treatment, expressions of those genes were reduced in HSe NP and ChA groups, and further remarkably declined in CHSe NPs group. In conclusion, CHSe NPs modulated the inflammatory microenvironment by synergistically regulating signaling pathways related to oxidative stress and inflammatory responses in macrophages. Fig. 5. [170]Fig. 5 [171]Open in a new tab Inflammatory response and oxidative stress regulatory mechanisms of CHSe NPs. (A) Venn diagram illustrated co-expressed genes in the control, LPS, LPS + HSe NPs (HSe NPs), LPS + ChA (ChA), and LPS + CHSe NPs (CHSe NPs) groups. (B) Volcano map depicted DEGs between the LPS and CHSe NPs groups (blue: genes that were not significantly different, green: downregulated genes, red: upregulated genes). (C) Venn diagram of DEGs in the three paired groups (control vs. LPS, control vs. CHSe NPs and LPS vs. CHSe NPs. (D, E) GO (D) and KEGG (E) enrichment analysis of the differential gene expression between LPS and CHSe NPs groups (BP: biological process, CC: cellular component, MF: molecular function). (F) The heatmap of differentially expressed gene related to oxidative stress and inflammatory response in the five groups. Differential gene screening criteria: pValue < 0.05 and | log[2]FoldChange | >1 Molecular docking explored the regulatory mechanism of inflammation Since ChA was exposed on the surface of CHSe NPs, the interactions between ChA and potential upstream target proteins of MAPK (Mapk8ip1 and Ereg) and PI3K-Akt signaling pathway (Itga2b and Itga6) were further explored based on the results of RAN-seq. The molecular docking analysis was performed using Discovery Studio 2019 software. The docking model of ChA and Mapk8ip1 revealed that the ChA situated in the interface between two subunits of Mapk8ip1, exquisitely establishing three crucial hydrogen bonds between E185 and the carboxyl group and hydroxyl group of ChA, Y230 and the hydroxyl group on the six-membered ring of ChA, Q253 and hydroxyl group on the phenyl ring of ChA (Fig. [172]6A and B). The DOCKER INTERACTION ENERGY value of ChA and Mapk8ip1 was − 35.4346 kJ/mol (Fig. [173]S22). Similarly, ChA interacted with Itga2b through five key residues (V295; R422; S 292; S172; L421) (Fig. [174]6C and D), with DOCKER INTERACTION ENERGY value of -47.628 kJ/mol (Fig. [175]S23). In addition, the molecular docking results of ChA with Ereg (Fig. [176]S24 and [177]S25) or Itga6 (Fig. [178]S26 and [179]S27) were also investigated. In brief, ChA could directly act on Mapk8ip1, Itga2b, Ereg, and Itga6, thereby regulating the MAPK and PI3K-Akt signaling pathway. Fig. 6. [180]Fig. 6 [181]Open in a new tab The CHSe NPs modulated inflammatory signaling pathways by inhibiting expression of Mapk8ip1 and Itga2b. (A) Molecular docking models of ChA with Mapk8ip1. (B) The key residues formed by hydrogen bonding between ChA and Mapk8ip1. (C) Molecular docking models of ChA with Itga2b. (D) The key residues formed by hydrogen bonding between ChA and Itga2b. (E, F) Fluorescence images presented the expression levels of Mapk8ip1 (E) and Itga2b (F) in RAW264.7 cells in the control, LPS, LPS + HSe NPs (HSe NPs), LPS + ChA (ChA), and LPS + CHSe NPs (CHSe NPs) groups (red fluorescence indicated Mapk8ip1 or Itga2b, blue fluorescence from DAPI indicated nuclei). Scale bar = 50 μm Subsequently, the regulatory effects of CHSe NPs on key proteins Mapk8ip1 and Itga2b were further validated through cell immunofluorescence staining. The RAW 264.7 cells were stimulated with LPS and further treated with HSe NPs, ChA, and CHSe NPs, respectively. Compared to the control group, the expression of Mapk8ip1 (represented by red fluorescence) was significantly increased in the LPS group and decreased in the HSe NPs and CHSe NPs groups, with ChA group showing a slight increase (Fig. [182]6E). Notably, the CHSe NPs group had the weakest red fluorescence intensity, indicating their most substantial inhibitory effect on Mapk8ip1 expression. In consistent with Mapk8ip1, CHSe NPs displayed most effective effect in inhibiting the expression of Itga2b among the three treatment groups (Fig. [183]6F). Summarily, CHSe NPs directly downregulated the expression of Mapk8ip1 and Itga2b, thereby modulating the MAPK signaling pathway and PI3K-Akt signaling pathway, and ultimately impeding the cyclic synergy of oxidative stress and excessive inflammatory response. Western blots were further utilized to confirm the above conclusion. As shown in Fig. [184]S28A, the expression of Mapk8ip1 and Itga2b were remarkably increased in LPS group when compared with control group in RAW264.7 cell. After treatment with HSe NPs, ChA, and CHSe NPs, the expression of Mapk8ip1 and Itga2b were reduced to varying degrees. It was worth noting that the expression of Mapk8ip1 and Itga2b in the CHSe NPs group were the lowest among three treatment groups. The statistical data of gray scales in five groups further proved that, indicating the excellent antioxidant and anti-inflammatory effects of CHSe NPs by inhibiting the expression levels of Mapk8ip1 and Itga2b (Fig. [185]S28B) Antioxidant capacity of CHSe NPs in vivo In view of the superior antioxidant and anti-inflammatory effects of CHSe NPs in vitro, these capabilities were further investigated in LPS induced ALI mouse model. Figure [186]7A presented the establishment of this model. LPS was injected into the lungs through tracheal administration, followed by different treatments after two hours. Twenty-two hour later, the mice were sacrificed, bronchoalveolar lavage fluid (BALF), major organs, and blood were collected for further analysis (Fig. [187]7A). The mice were divided into five groups: control, LPS, LPS + HSe NPs (HSe NPs), LPS + ChA (ChA), LPS + CHSe NPs (CHSe NPs) and LPS + dexamethasone (Dex) groups (dexamethasone: a drug approved by the China National Food and Drug Administration for the treatment of ALI, used as a positive control). The lung wet dry weight ratio (W/D), a typical indicator for monitoring initial pathological changes in pulmonary edema, was investigated. In Fig. [188]7B, compared with the control group, the W/D ratio of the LPS group was significantly increased, indicating the severe pulmonary edema, inflammatory cell infiltration and secretion of pro-inflammatory cytokines. The W/D value were significantly decreased in ChA and Dex groups, and further remarkably reduced in CHSe NPs group, while there was no change in the HSe NPs group, suggesting the strongest effect of CHSe NPs in mitigating pulmonary edema (Fig. [189]7B). Hematoxylin and eosin (H&E) staining further demonstrated that the LPS group displayed typical symptoms of ALI, including damage to alveolar epithelial cells and capillary endothelial cells, diffuse interstitial and alveolar edema, increased permeability, thickened alveolar walls, and pulmonary fibrosis. Those lung tissue damages were significantly mitigated in HSe NPs, ChA, CHSe NPs, and Dex groups, with CHSe NPs exhibiting optimum protective effect (Fig. [190]7C). Fig. 7. [191]Fig. 7 [192]Open in a new tab Antioxidant capacity of CHSe NPs in vivo. (A) Schematic illustration of ALI mouse model and treatment schedule. (B) Wet dry weight ratio (W/D) of lung in the control, LPS, LPS + HSe NPs (HSe NPs), LPS + ChA (ChA), LPS + CHSe NPs (CHSe NPs) and LPS + Dex (Dex) groups. (n = 6). (C) Representative H&E staining images of the lung tissue in the six group (The magnification of the upper row of image is 50x magnification and the lower row was 200 x, the arrow indicated the position of lower row in the above image). Scale bar = 100 μm. (D-F) MDA content (D), SOD enzyme activity (E), and GSH content (F) in lung tissue of the six groups. (G) Representative fluorescence images of ROS in lung tissue of the six groups (red fluorescence indicated ROS, blue fluorescence from DAPI indicated nuclei). Scale bar = 100 μm. Data were presented as the mean ± SD. ^##P < 0.01, ^####P < 0.0001, * P < 0.05, ** P < 0.01, **** P < 0.0001, and ns, not significant; ^# indicated a significant difference in comparison to the control group, and * indicated a significant difference in comparison to the LPS group In the microenvironment of ALI, excessive ROS generation led to oxidative stress and imbalanced physiologic homeostasis. The antioxidant capacity of CHSe NP was further probed in vivo. MDA, an important products of membrane lipid peroxidation, presented the degree of pulmonary oxidative damage and affected the activity of mitochondrial respiratory chain complexes and modulated key antioxidative enzymes. In lung tissue grinding fluid, the concentration of MDA was significantly increased in the LPS group, and remarkably reduced in HSe NPs, ChA, CHSe NPs, and Dex groups than that in control group. Notably, the CHSe NP showed the lowest MDA content, indicating their superior antioxidant effect (Fig. [193]7D). Subsequently, the activity of antioxidant enzymes SOD and GPX (represented by content of GSH) were measured in the six groups. In Fig. [194]7E and F, compared with control group, the SOD enzyme activity and GSH content were significantly decreased in LPS group and remarkably elevated in CHSe NPs group. Surprisingly, the SOD enzyme activity (Fig. [195]7E) and GSH content (Fig. [196]7F) were barely restored in HSe NPs, ChA, and Dex groups than that of LPS group. The above results confirmed the excellent anti-oxidative capacity of CHSe NP in vivo. Additionally, the ROS scavenging ability of CHSe NPs was validated in vivo by immunofluorescence technology. The dihydroethidium (DHE), a dye emitting red fluorescence, was selected as indicator of ROS. Compared with the control group, the red fluorescence intensity of the LPS group was significantly elevated, indicating increased ROS levels. Notably, the red fluorescence intensities were declined in four treatment groups and CHSe NPs group exhibited the weakest intensity, indicating their superior ROS scavenging effect (Fig. [197]7G). Statistical data further proved that (Fig. [198]S29). The biosafety of CHSe NPs was further examined by H&E and routine blood tests. In Fig. [199]S30, pathological changes of main tissues (liver, heart, spleen and kidney) were minimal in HSe NPs, ChA, CHSe NPs, and Dex groups than in control group. In addition, no significant changes among twenty blood parameters were observed in treatment groups than that of control group (Fig. [200]S31). The above results indicated the good biocompatibility of CHSe NPs in vivo. In summary, with excellent biosafety, CHSe NPs alleviated pathological lung tissue damage and exhibited superior antioxidant capacity in vivo. The anti-inflammatory ability of CHSe NPs in vivo Since the polarization of M1 macrophages, infiltration of neutrophils as well as imbalanced inflammatory cytokine secretion disturbed the immune homeostasis of lung tissue in ALI, the anti-inflammatory ability of CHSe NPs was determined. In Fig. [201]8A, compared with control group (4.06%), more macrophages in BALF were polarized to M1 type in LPS group (36.4%), suggesting the successfully established of ALI mouse model. The proportion of M1 macrophages were decreased in HSe NPs (29.3%), ChA (25.2%), and Dex (26.8%) groups, and markedly‌ reduced in CHSe NPs (19.2%) group than LPS group, highlighting their extraordinary regulatory ability on macrophage polarization. Statistical data further proved that (Fig. [202]8C). Similarly, compared with control group (0.34%), the frequency of neutrophils was significantly elevated in the LPS group (72.5%) and remarkably decreased in the HSe NPs (63.8%,), ChA (51.5%), CHSe NPs (48.6%), and Dex (52.6%) groups (Fig. [203]8B). Among treatment groups, CHSe NPs group exhibited lowest frequency of neutrophils, indicating the pronounced treatment effect of CHSe NPs than clinic drug Dex. Statistical data further confirmed the superior effect of CHSe NPs in inhibiting neutrophil infiltration (Fig. [204]8D). Fig. 8. [205]Fig. 8 [206]Open in a new tab The anti-inflammatory ability of CHSe NPs in vivo. (A, B). Representative flow cytometry images depicted M1 macrophages (A) and neutrophils (B) in BALF of the control, LPS, LPS + HSe NPs (HSe NPs), LPS + ChA (ChA), LPS + CHSe NPs (CHSe NPs) and LPS + Dex (Dex) groups. (C, D) Statistical frequency of M1 macrophage (C) and neutrophil (D) in BALF of the six groups (n = 3). (E-H) The serum levels of IL-1β (E), IL-6 (F), TNF-α (G), and IL-10 (H) in the six group (n = 3). (I) Representative immunohistochemical staining images of M1 type macrophage in lung tissue of the six groups (brown indicates iNOS and M1 macrophage, blue indicates nuclei). The magnification of upper row is a 50x and lower row were 200x (the arrow indicated the position of lower row in the above image). Scale bar = 100 μm. (J) Representative immunohistochemical staining images of neutrophile in lung tissue of the six groups (brown indicated MPO and neutrophils, blue indicates nuclei). The magnification of upper row is a 200x and lower row were 400x (the arrow indicated the position of lower row in the above image). Scale bar = 100 μm. Data are presented as the mean ± SD. ^###P < 0.001, ^####P < 0.0001, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, not significant; ^# indicated a significant difference in comparison to the control group, and * indicated a significant difference in comparison to the LPS group To evaluate regulatory impact of CHSe NPs on cytokine expression, the serum level of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were analyzed in the six groups. Compared with the control group, the expression levels of IL-1β (Fig. [207]8E), IL-6 (Fig. [208]8F), and TNF-α (Fig. [209]8G) were significantly increased in the LPS group. After treatment with ChA, CHSe NPs and Dex, the serum levels of IL-1β and IL-6 were significantly reduced. Notably, the CHSe NPs group had the lowest expression levels of IL-1β and IL-6. In contrast, HSe NPs showed minimal impact on the expression of these cytokines(Fig. [210]8E and F). Additionally, the expression levels of serum TNF-α was remarkably reduced in CHSe NPs group than in LPS group, with minimal changes observed in HSe NPs, ChA and Dex groups(Fig. [211]8G), indicating their excellent ability mitigating pro-inflammatory cytokines secretion. Furthermore, the expression of the classic anti-inflammatory cytokine IL-10 was examined in the six groups. Serum level of IL-10 was significantly reduced in the LPS group and were remarkably increased in the HSe NPs, ChA, CHSe NPs, and Dex groups than in control group, with the highest expression level of IL-10 in CHSe NPs group, highlighting their excellent anti-inflammatory effect (Fig. [212]8H). Briefly, CHSe NPs inhibited the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and enhanced the expression of anti-inflammatory cytokine (IL-10) in vivo. Next, immunohistochemical techniques were used to verify the infiltration of immune cells in situ lung tissue. The expression of iNOS, a biomarker for M1 macrophages, was detected to reveal the polarization phenomenon of M1 macrophages in lung tissue. Compared with the control group, the expression of M1 macrophages was significant elevated in LPS group (iNOS: brown, representing M1 macrophages). After treatment, the number of M1 macrophages were noticeable reduced in HSe NPs, ChA, CHSe NPs and Dex group, with the lowest number of M1 macrophages in CHSe NPs group, indicating their superior inhibitory effect on M1 macrophage polarization (Fig. [213]8I). In addition, Myeloperoxidase (MPO), a functional and activation marker of neutrophils, was assessed. In consistent with iNOS, the number of neutrophils (MPO: brown, representing neutrophils) was the lowest in the CHSe NPs group, confirming their superior effect on inhibiting the infiltration of inflammatory lung neutrophil (Fig. [214]8J). In brief, CHSe NPs inhibited M1 macrophage polarization and inflammatory pulmonary neutrophil infiltration, as well as regulated inflammatory related cytokines expression in vivo, therefor synergistically exert anti-inflammatory effects. Conclusions In conclusion, we successfully constructed ChA-HSA coated selenium nanoparticles to disrupt the cyclic synergistic effect between ROS and inflammatory response, exerting superior inflammatory microenvironment modulating therapeutic effect of ALI. The results showed that combined the enzyme like activities of SOD(derived from ChA) and GPX (derived from HSe NPs), CHSe NPs exerted the remarkable antioxidant effect to eliminate various types of ROS, and improved the oxidative stress environment in ALI. In addition, they modulated the inflammatory microenvironment of ALI mice, by reversing M1 macrophage polarization, inhibiting overexpression of pro-inflammatory cytokines (IL-1b, IL-6, TNF-a) and reducing neutrophil infiltration. Further mechanistic studies illustrated that CHSe NPs directly acted on and modulated the expression of Mapk8ip1 and Itga2b, which were upstream proteins of MAPK signaling pathway and PI3K-Akt signaling pathway, therefore impeding the cyclic synergy between oxidative stress and inflammatory dysregulation. It is worth noting that the therapeutic function of CHSe NPs was significantly better than antioxidant drug ChA and commonly used clinic drug Dex for treatment of ALI, by remarkably alleviating the severity of ALI and modulated anti-inflammatory and antioxidant indicators. Specially, compared with Dex, the immune cells modulation function of CHSe NPs was superior to Dex. Overall, this study demonstrated that the CHSe NPs synergistically exert antioxidant and anti-inflammatory effects by regulating the MAPK signaling pathway and PI3K-Akt signaling pathway, thereby treating ALI. Our study provided a new nano-therapy for the clinical treatment of ALI, as well as other ROS and inflammation related diseases. We believe that the synthesis of CHSe NPs offers several advantages, including mature chemical synthesis technologies for nano-selenium, straightforward synthesis methods, ease of operation, readily available raw materials, and low cost. These factors provide strong support for scaling up the production of CHSe NPs. However, the synthesis yield remains relatively not that high, therefore, further research was conducted to enhance the yield in future. Despite the lower toxicity of nano-selenium compared to elemental selenium, the long-term use safety of CHSe NPs still requires further exploration, which presents a challenge for clinical translation. In further, we aim to optimize the synthesis process to further enhance the safety of CHSe NPs for prolonged use and to facilitate their clinical application. Electronic supplementary material Below is the link to the electronic supplementary material. [215]Supplementary Material 1^ (4.2MB, docx) Acknowledgements