Abstract

Background

   The phenomenon of population aging presents a significant global
   challenge, with the aging population in China steadily increasing. As
   individuals progress in age, there is a gradual deterioration of human
   organs and systems, as well as a decline in the immune system, referred
   to as immunosenescence. Atractylodis macrocephalae rhizoma (BZ) has
   been historically used in China for its medicinal properties, including
   gastrointestinal improvement, immunomodulation, anti-aging, antioxidant
   effects, and anti-tumor effects. Nevertheless, there remains a gap in
   understanding the pharmacological and molecular mechanisms underlying
   its anti-immunosenescence effects.

Methods

   This study employed UPLC-ESI-MS and network pharmacology to create a
   network map of BZ ultrafine powder (BZU) and its aging targets.
   Enrichment analysis was then used to identify the primary mechanistic
   pathways underlying BZU’s anti-immunosenescence effects. The primary
   components of BZU were quantitatively analyzed using high-performance
   liquid chromatography (HPLC). Naturally aging rats were used to examine
   the effects of different oral doses (0.25, 0.5, and 1 g/kg) of BZU over
   5 weeks on aging performance, peripheral blood immunophenotyping and
   cell count, and splenic lymphocyte proliferation rate. To validate the
   findings of network pharmacology, quantitative RT-PCR, Western
   blotting, and immunofluorescence analyses were conducted.

Results

   Our analyses demonstrated that BZU improved various indicators of aging
   in naturally aging rats, such as increasing the number of voluntary
   activities, enhance grip strength and fatigue resistance, increasing
   the microcirculatory blood flow and improving hematological levels. The
   BZU administration enhanced T and B lymphocyte proliferation and
   significantly improved the lymphocyte-to-T cell subpopulation ratio. It
   can elevate serum IL-2 and IL-4 levels while reducing IL-6, IFN-γ and
   TNF-α levels in naturally aging rats. Finally, it increased CD3 protein
   expression in the spleen while decreasing protein levels of PI3K,
   p-AKT, IKKα/β, and NF-κB. It also decreased the mRNA expression of
   Pik3cg, Akt1, Pdk1 and Nfκb1.

Conclusion

   These findings suggest that BZU may enhance lymphocyte proliferation by
   inhibiting the PI3K/Akt/NF-κB signaling pathway, correcting immune cell
   imbalances, reducing inflammatory responses, and ultimately enhancing
   immune function and potentially delaying aging.

   Keywords: Atractylodis macrocephalae rhizoma, aged, PI3K/Akt/NF-κB,
   network pharmacology, immunosenescence

Graphical Abstract

   [39]graphic file with name FPHAR_fphar-2025-1550357_wc_abs.jpg

1 Introduction

   The increasing number and proportion of older individuals represent a
   significant global demographic challenge. China’s rapidly aging
   population has emerged as a significant social issue ([40]Wang et al.,
   2021).The decline in functions related to aging, including reduced
   immunity, neurasthenia, back and leg pain, and memory loss,
   significantly affects human health and quality of life ([41]Qiang,
   2009). Therefore, improving immunity and delaying aging is an urgent
   problem.

   Aging is a universal phenomenon experienced by all individuals,
   characterized by the gradual decline of vital essence substances,
   deterioration of internal organ functions, and structural changes in
   the body according to Chinese medicine theory ([42]Wanwan and Fusheng,
   2019; [43]Mohsen et al., 2020). A report published over 2,000 years ago
   in the Yellow Emperor’s Classic of Internal Medicine suggested a
   reduction in spleen and stomach qi (vital energy) would lead to a
   shorter lifespan ([44]Shiyu, 2017). Therefore, according to Chinese
   medicine theory, “strengthening the spleen” can slow down the aging
   process. In Chinese medicine, the “spleen” refers to functions beyond
   the anatomical organ, involving the spleen, pancreas, and
   gastrointestinal tract. It is connected to digestion, endocrine,
   nervous, blood, lymphatic systems, and exocrine glands, all crucial for
   immunity ([45]Feifei et al., 2018). And in modern medicine, there is
   also a close relationship between immunity and aging. Aging leads to
   immunosenescence, causing immune function alterations such as
   deficiency, autoimmunity, and inflammation imbalance ([46]Matthew et
   al., 2021).

   The aging of the immune system affects both the innate and adaptive
   immune systems. Natural killer (NK) cells constitute an important
   effector cell population in the innate immune system. Adaptive immune
   cells are broadly categorized into T and B lymphocytes. They are
   distributed throughout the body and, upon antigenic stimulation,
   activate, divide, proliferate, and initiate specific immune responses.
   With increasing age, T, B, and NK lymphocytes exhibit signs of
   senescence, such as disrupted subpopulation cell ratios and
   dysfunctional cellular responses, leading to impairments in immune
   response and immune function ([47]Rebeca et al., 2011). In particular,
   T cells have been the main focus of immunosenescence. ([48]Maria and
   Guido, 2021). A major division has also been made between T cells that
   are helpers (Th) and those that are cytotoxic (Tc). Th and Tc cells can
   be categorized as naïve, memory, or effector T cells, based on their
   differentiation stage ([49]Snell et al., 2016; [50]Monica et al.,
   2022). As the body ages, the thymus atrophies, leading to impairments
   in the development, differentiation, and maturation of T cells. This
   manifests as a decrease in the number of naive T cells exported from
   the thymus to the periphery, accompanied by an increase in memory T
   cells. These cellular changes can reduce the immune response level of
   elderly individuals to new antigens. The decline of CD8^+T cells during
   senescence was more pronounced than that of CD4^+T cells. The lack of
   CD28 expression in CD4^+ and CD8^+T cells among the elderly is a
   significant change associated with aging and senescence-related
   functional processes in human T cells. TH cells can differentiate into
   subpopulations of helper T cells 1 and 2 (Th1, Th2), Treg cells, and so
   on under different activation conditions. Th1 and Th2 cells interact
   with and regulate each other. The increased senescence of peripheral
   blood T cells leads to higher levels of pro-inflammatory cytokines
   (IFN-γ, TNF-α, IL-6) and lower levels of anti-inflammatory cytokines
   (IL-2, IL-4, IL-10) ([51]Cornelia M et al., 2013). CD4^+CD25^+Foxp3^+
   Treg cells are the most characteristic Treg cells, capable of
   inhibiting the proliferation and activation of effector T cells,
   reducing cytokine secretion, and thereby regulating immune responses to
   exert immunosuppressive effects. With increasing age, the number of
   Treg cells increases, and its ability to suppress lymphocyte function
   by inhibiting lymphocyte activation receptors also enhances ([52]Sanjay
   et al., 2013). T cells rely on multiple downstream signaling and
   metabolic pathways to maintain homeostasis and respond appropriately to
   TCR and cytokine stimulation during activation. Aging disrupts these
   pathways, most notably by hyperactivating basal signaling pathways in
   resting T cells, such as an increased basal activation of the PI3K/AKT
   signaling pathway. NF-κB is a signaling pathway involved in
   inflammatory responses, and the hyperactivation of the PI3K/AKT pathway
   can activate nuclear NF-κB, leading to inflammatory reactions that
   contribute to aging ([53]Carrie et al., 2013).

   Atractylodes Macrocephalae Rhizoma (BZ) is the dry rhizome of the
   Compositae plant Atractylodes macrocephala Koidz, which has the effect
   of invigorate the spleen and Qi, reduce dampness and moisture. BZ, a
   traditional and precious Chinese herbal medicine in China, has been
   included in the “List of Items that Can Be Used in Health Foods” by the
   Ministry of Health, and is expected to be further developed as a food
   and medicinal substance. BZ can regulate the spleen and stomach,
   replenish qi and nourish blood, qi and blood transport sufficient, and
   then enhance the function of the internal organs. It makes the body
   strong and improves immunity, thus playing a role in prolonging life.
   To date, there have been many studies on the anti-aging effects of BZ,
   such as oxidative stress, telomerase, and genetic doctrines, while the
   functional importance of immunosenescence has been less investigated
   ([54]Hua et al., 2022). Experimental research has shown that BZ
   significantly improved learning memory ability, and BZ enhanced the
   antioxidant capacity of D-galactose-induced senescence model mice
   ([55]Chunmei et al., 2021). Some studies have found a decrease in
   plasma malondialdehyde levels, and the rise in superoxide dismutase and
   total antioxidant activity in mice treated with BZ volatile oil
   suggests that BZ volatile oil possesses notable antioxidant properties
   ([56]Yi et al., 2024). Many studies have shown that BZ has the ability
   to promote the metabolism of organism substances, such as the
   regulation of body sugar and lipid metabolism, and can prevent and
   control lipid metabolism disorders, atherosclerosis, and other diseases
   in the elderly ([57]Qige et al., 2022; [58]Yi et al., 2024). The
   studies mentioned above thoroughly show that BZ can contribute to
   delaying the aging process.

   In studies, BZ has been proven to enhance and strengthen the body’s
   immune system. Xiang et al. found that BZ polysaccharides enhance the
   splenic index, counteract cyclophosphamide-induced immunosuppression in
   mice, and restore normal immune function ([59]Xuelian et al., 2020). BZ
   polysaccharide may also mitigate the immunosuppressive effects of
   cyclophosphamide in geese by preserving the equilibrium between humoral
   and cellular immunity ([60]Wanyan et al., 2018). Further studies
   revealed that glycoproteins extracted from BZ are significant
   immunologically active constituents of BZ extracts, capable of
   augmenting the induction of protective immune responses in mice
   ([61]Kyoung et al., 2018). Research has shown that BZ and its active
   ingredients have an immune enhancing effect.

   This paper initially identified the components in BZU using UPLC-ESI-MS
   analysis, followed by establishing component and disease target network
   maps through network pharmacology. Subsequently, Gene Ontology (GO) and
   Kyoto Encyclopedia of Genomes (KEGG) pathway enrichment analyses were
   conducted to identify the primary mechanistic pathways through which
   BZU delays aging. The main components of BZU, including Atractylodes I,
   II, III, and Atractylone, were determined by HPLC. We used naturally
   aging rats as the old rat model; BZU was administered by gavage for
   five consecutive weeks at different doses. Subsequently, the impact of
   BZU on systemic aging manifestations, peripheral blood immunophenotype,
   and cell counts in aging rats was assessed, along with the
   proliferation rate of splenic lymphocytes. The study further confirmed
   the mechanistic pathway through which BZU delays aging by enhancing
   immunity through immunohistochemistry, Western blotting, and PCR
   techniques.

2 Materials and methods

2.1 Data preparation

2.1.1 BZ active ingredient database establishment

   UPLC-ESI-MS analysis was performed using a ZenoTOF 7600 mass
   spectrometer (AB Sciex, California, USA) coupled with a UPLC system
   (Shimadzu, Rydalmere, Australia).

   Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH
   C18 column (2.1 mm × 50 mm, 1.7 μm) from Milford, MA, USA. Flow rate
   was 0.3 mL/min, and column temperature was 25°C. Acetonitrile and 0.5%
   formic acid were used as mobile phases A and B, respectively. A
   gradient elution in the following manner was applied: 0–8 min, 90–52%
   B; 8–12 min, 52–50% B; 12–16 min, 50–30% B; 16–18 min, 30–20% B; 18–26
   min, 20% B; 26–27 min, 20–90% B; 27–30 min, 90% B, with a 5 μL
   injection volume.

   The positive ion mode was used in the mass spectrometer. The MS
   parameters were capillary voltage at 3.0 kV (positive ion mode), ion
   source gas 1 (air) at 55 psi, ion source gas 2 (air) at 55 psi, and
   curtain gas (N[2]) at 35 psi. The ion spray temperature was 550°C, and
   the voltage was 5500V. The collision energy was set at 35 V with a
   spread of 15 V. Analysis of the data was carried out using Analyst TF
   1.7 and PeakView 2.1 software (SCIEX).

   The experimental data obtained were compared and integrated with the
   chemical composition of BZ in databases (TCMSP, TCMID, BATMAN-TCM, and
   Massbank) and literature (PubMed, Embase, CNKI, and Wanfang Database)
   ([62]Xiaoying et al., 2023; [63]Yafei et al., 2023). The active
   compound targets were collected from the TCMSP and SymMap databases.
   Obtaining the compound structural formula of other components in the
   PubChem database ([64]https://pubchem.ncbi.nlm.nih.gov/) and importing
   it into SwissTargetPrediction
   ([65]http://www.swisstargetprediction.ch/index.php) for coupling. The
   prediction targets were obtained with a probability >0 as the screening
   conditions.

2.1.2 Collection of drug targets for aging

   The genes of targets related to “senescence” or “aging” or “anti-aging”
   were screened via Durgbank ([66]http://www. drugbank. ca/), Malacards
   ([67]https://www.malacards.org/), TTD ([68]http://db.idrblab.net/ttd/),
   and OMIM ([69]https://www.ncbi.nlm.nih.gov/omim). After removing the
   duplicate genes, the common targets of the above keywords are collected
   as candidates. The BZ and aging targets, including their full names,
   abbreviations, and gene IDs, were obtained from the Uniprot database
   ([70]https://www.uniprot.org/).

2.2 Drug-compound-target interaction network construction

   We utilized Cytoscape 3.9.1 ([71]https://cytoscape.org/) to construct a
   drug-target-disease map, enabling the analysis of BZ’s active
   ingredients and their association with aging. Potential target genes
   for BZ therapy in aging were identified using the Venny 2.1
   intersection tool
   ([72]https://bioinfogp.cnb.csic.es/tools/venny/index.html).

2.3 Construction of a PPI protein interaction network

   In this study, intersection targets were imported into the STRING
   database ([73]https://string-db.org/), selecting ‘multiple proteins’
   and limiting the species to ‘Homo sapiens’. The interrelationship of
   these potential target genes is imported into Cytoscape 3.9.1, a PPI
   network is constructed, and core targets are screened out through the
   calculation for degrees of freedom.

2.4 Functional enrichment analysis

   Utilize the R packages “org.Hs.eg.db” and “clusterProfiler” to convert
   gene symbols to ENTREZID and conduct GO and KEGG enrichment analyses
   for visualization. The analysis results were filtered to include only
   those with a p-value <0.05 and then sorted by p-value and count value
   for detailed examination.

2.5 Preparation and quality control of BZU

2.5.1 Preparation of BZU

   Raw herbs were purchased from Zhejiang Intel Pharmaceutical Co., Ltd.
   (Zhejiang, China). A voucher specimen was made and deposited at the
   herbarium with voucher specimen number 2010018. The ultrafine powder of
   BZ was processed by Jinan Dawei Machinery Co., Ltd. and passed through
   a 300-mesh sieve to obtain drugs with a particle size of <48 μm. A
   distilled water solution was prepared by dissolving the ultrafine
   powder in distilled water at 4°C.

2.5.2 Scanning electron microscope observation of BZU

   Take an appropriate amount of tissue BZ ultrafine powder and coat it
   with gold powder on the cross-section surface. Powder morphology was
   checked using digital light microscopy and a Hitachi SU8010 field
   emission scanning electron microscope (SEM).

2.5.3 Determination of main components in BZU

   The total polysaccharide content of the water extract was determined
   using the phenol-sulfuric acid method, as outlined in the 2020 edition
   of the Chinese Pharmacopoeia ([74]Qiuling et al., 2023). HPLC was used
   to quantify the major components of BZU, such as atractylenolide I, II,
   III, and atractylone. Analyses of the content of the samples were
   conducted using an Agilent 2600 series HPLC system (1200, Agilent,
   Germany) equipped with a diode-array detector (DAD). An Ultimate LP-C18
   column (4.6 × 250 mm, 5 μm) was kept at 25°C. The mobile phase
   comprised 0.01 mol L^−1 water amine acetate (A) and acetonitrile (B)
   with a flow rate of 1.0 mL/min. The gradient program was: 0–20 min,
   50%–60%B; 20–30 min, 60%–70%B; 30–35 min, 70–100%B; 35–40 min, 100%B;
   40–45 min, 100–50%B. The injection volume was 10 μL. The detection
   wavelengths were 0–23 min, 222 nm; 23–30 min, 278 nm; and 30–45 min,
   222 nm, respectively.

2.6 Animals and experimental treatment

   The research was approved by a university ethics committee (Zhejiang
   Chinese Medical University Reference ZSLL-2016-118). Adult (4 months)
   and aged (14–15 months) male SD rats were sourced from Weitong Lihua
   Limited Company (Zhejiang, China). Rats were maintained under standard
   conditions (temperature 25°C ± 2°C, humidity 60% ± 5%, 12/12 h
   day/night cycle) with ad libitum access to food and water. The dosing
   regimen for BZU was determined based on the dosing amount of BZ
   specified in the Chinese Pharmacopoeia. Young male SD rats were
   assigned to the young control group (AC, n = 6). Aged male SD rats were
   randomly divided into four groups (n = 6 per group): old model group
   (AM), low-dose BZU group (BZU-L, 0.25 g/kg), medium-dose BZU group
   (BZU-M, 0.5 g/kg), high-dose BZU group (BZU-H, 1.0 g/kg). The
   administration was done by oral gavage once a day in the morning for
   five consecutive weeks.

2.7 Behavioral experiment

2.7.1 Autonomic activity test

   After 3 weeks of administration, the rats were placed in the autonomous
   activity instrument (YLS-1A, Jinan Yiyan Technology Development Co.,
   China) in a quiet environment for 5 min after adaptation, and the
   number of autonomous activities of the rats within 5 min was recorded.

2.7.2 Body temperature test

   The body temperature of the rats was measured using rectal probe
   thermometry (inserting a thermometer into the rectal lumen of the rats)
   at 3 weeks after administration.

2.7.3 Grip strength test

   After 3 weeks of administration, rat forelimb strength was measured
   using a Rodent Grip Strength Meter (YLS-13A, Jinan Yiyan Technology
   Development Co., China), which records peak force when grip is lost.

2.7.4 Antifatigue ability test

   Evaluation of fatigue resistance in rats by exhaustive swimming time.
   After 3 weeks of administration, the rats, each loaded with 5% of their
   body weight in lead skin 1 cm from the tail root, were placed in a
   swimming tank with 30 cm deep water at 24–26°C. The time counting was
   started immediately after placing the rats into water, and the end time
   was recorded when the rat’s head was submerged in water for 5 s without
   surfacing, and the rats were fished out.

2.7.5 Fecal moisture test

   After 3 weeks of administration, freshly excreted stool was collected
   in sterile tubes, weighed, dried at 60°C for 24 h, and reweighed. Fecal
   water content (%) was determined using the formula: [(fecal wet weight
   - fecal dry weight)/fecal wet weight] × 100.

2.7.6 Microcirculatory blood flow intensity test

   After 4 weeks of administration, the rats were anesthetized with 4%
   isoflurane via a face mask, and the tail microcirculatory blood flow
   intensity was measured using a speckle contrast imager (MoorFLPI, Moor
   Instruments Ltd., Devon, UK).

2.8 Immunophenotyping of peripheral blood cells and hematological parameters

2.8.1 Peripheral blood cell count

   A 2 mL whole blood sample in an EDTA-treated tube was analyzed for
   white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and
   blood platelet (PLT) using the routine hematology laboratory method
   (XT-2000i Automated Hematology Analyzer, Sysmex, Japan).

2.8.2 Immunophenotyping of peripheral blood cell

   Peripheral blood immune cell subsets were analyzed via flow cytometry
   as detailed below. We incubated whole blood with surface antibodies for
   30 min at 4°C, as detailed in [75]Table 1. To lyse erythrocytes, 1 mL
   of lysis buffer (BD Pharm Lyse, BD, USA) was added and incubated for
   10 min in darkness. Cells were centrifuged at 1100 rpm for 5 min, the
   supernatant discarded, and then washed with 1 mL of PBS followed by
   centrifugation at 1100 rpm for 10 min. Cells were washed, centrifuged,
   resuspended in PBS, and analyzed.

TABLE 1.

   Antibodies used for flowcytometry phenotyping.
   Antigen Flourochrome Antibody                  Clone Supplier
   CD3     FITC         Mouse BALB/c IgM, κ       1F4   BD Pharminge
   CD4     BV510        Mouse BALB/c IgG2a, κ     OX-35 BD Pharminge
   CD8a    Alexa 647    Mouse BALB/c IgG1, κ      OX-8  BD Pharminge
   CD44    BV421        Mouse BALB/c IgG2a, κ     OX-49 BD Pharminge
   CD62L   BB700        Armenian Hamster IgG2, λ1 HRL1  BD Pharminge
   CD45RA  APC-Cy7      Mouse BALB/c IgG1, κ      OX-33 BD Pharminge
   CD28    PE           Mouse BALB/c IgG1, κ      JJ319 BD Pharminge
   CD161A  R718         Mouse IgG1, κ             10/78 BD Pharminge
   [76]Open in a new tab

   Lymphocyte regions were identified based on their forward scatter (FSC)
   and side scatter (SSC) characteristics. Non-adhered cells were excluded
   by FSC-W and FSC-H measurements. The panels define the proportion and
   total number of major groups as follows: NK cell panel (CD3, CD161a); B
   cell subset panel (CD3, CD45RA); T cell subset panel (CD3); T helper
   cells (Th, CD3, CD4); T cytotoxic cell (Tc, CD3, CD8A); naïve, memory,
   or effector Th and Tc cells panel (CD3, CD4, CD8, CD44, CD62L).
   Moreover, the expression of CD28 was checked in the subpopulations CD4
   and CD8. The expression of Treg cells (CD4^+CD25^+Foxp3^+) was
   detected.

   Peripheral blood lymphocyte and T cell subset immunophenotyping was
   conducted using multiparameter flow cytometry on a BD LSRFortessa (BD
   Biosciences). Results were analyzed in FlowJo version 10.6.2 software.

2.8.3 Hematological parameters

   Blood was obtained from the abdominal aorta after the rats were
   anesthetized with ulatan. Plasma viscosity and whole blood viscosity at
   high, medium, and low shear rates were measured using the capillary
   method with an FASCO-3010D automatic blood rheology analyzer
   (FASCO-3010D, Chongqing, China).

2.9 Splenic lymphocyte proliferation assay

   After the rats were executed, the spleens were removed under sterile
   conditions. The harvested spleens were promptly placed in 15 mL of
   ice-cold PBS. Splenocyte single-cell suspensions were prepared by
   pressing spleens through a 70 μm cell strainer. Red blood cells were
   lysed using a lysing solution (BD, New York, USA). After two washes
   with Hank’s solution, the single-cell suspensions were prepared in
   500 μL of RPMI 1640 with 10% FBS and 1% penicillin-streptomycin (Gibco,
   New York, USA). The cells were quantified and adjusted to a working
   concentration of 1 × 10^7 cells/mL.

   CCK8 assays were conducted to evaluate cell proliferation ability. The
   100 μL cell suspension was inoculated into a 96-well plate, and splenic
   T and B lymphocytes were induced to proliferate by the addition of
   mitogens ConA and LPS, respectively ([77]Kai et al., 2023). The culture
   was cultured at 37°C in a 5% CO[2] humidified atmosphere for 72 h. Each
   well was added with 10 μL CCK8, incubated at 37°C for 2 h, and the OD
   value at 450 nm was detected by enzymoleter. The proliferation rate was
   calculated relative to that of the control cells.

2.10 Hematoxylin-eosin (HE) staining

   The spleen tissue samples were fixed in 10% formaldehyde for over 24 h
   and subsequently placed in running water overnight. An automated tissue
   dehydrator (Tissue-TekVIP5Jr, Japan) was used for 8 h to remove excess
   moisture. The specimen was embedded in paraffin wax and cut into
   4 mm-thick sections by a microtome (NanoZoomer S60, Hamamatsu, Japan).
   After dewaxing and rehydration, the sections were stained with HE for
   morphological analysis.

2.11 Immunohistochemical staining

   The spleen paraffin sections were dewaxed using xylene and rehydrated
   with graded alcohol. Antigens were microwaved in sodium citrate
   solution (P0083, Beyotime, Shanghai, China) for 15 min and then blocked
   with 3% hydrogen peroxide (P0260, Beyotime, Shanghai, China) for
   15 min. Following a 20-min block using blocking buffer (P0260,
   Beyotime, Shanghai, China), the sections were incubated overnight at
   4°C with anti-CD3 antibody (1:500, 17617-1-AP, Proteintech, Wuhan,
   China), anti-IKKα/β antibody (1:100, YT2302, ImmunoWay, California,
   USA), and anti-PDK1 antibody (1:150, ab202468, Abcam, Cambridge, UK).
   Finally, sections were washed in PBS, developed with DAB solution (19:1
   mixture of solution A and B; ZLI-9019, Origene), and counterstained
   with Hematoxylin. Sections were then observed under a NanoZoomer S60
   microscope (Hamamatsu, Japan).

2.12 Cytokine measurements

   After the last administration, the rats were fasted for 12 h, and blood
   was collected from the abdominal aorta after anesthesia, and serum was
   separated (centrifugation at 3,000 rpm for 15 min). Serum levels of
   IL-2, IL-6, IL-4, IL-10, TNF-α, and IFN-γ were measured using ELISA
   kits (MultiSciences, Zhejiang, China) following the manufacturer’s
   instructions.

2.13 Western blot analysis

   Spleen tissues of rats were powdered in liquid nitrogen, and then the
   cracking liquid with 1% PMSF was added (Beyotime, Shanghai, China).
   After lysis for a minimum of 30 min and centrifugation at 12,000 rpm
   for 10 min at 4°C, protein concentration was measured using a BCA kit
   (Beyotime, Shanghai, China).

   SDS-PAGE gels were prepared using the SDS-PAGE reagent kit (8%). The
   samples were prerun at 80 V for 30 min, and the samples were
   fractionated at 120 V for 90 min. The protein was transferred to
   0.22 μm PVDF membranes (EMD Millipore) at 200 mA for 120 min. Membranes
   were blocked for 60 min in 5% nonfat dry milk blocking buffer, then
   washed in PBST. The membranes were incubated overnight with primary
   antibodies: anti-PI3K (1:1000, ET1608-70, Huabio, Zhejiang, China),
   anti-Akt (1:1000, ET1609-47, Huabio, Zhejiang, China), anti-p-Akt
   (1:500, AF0016, Affinity Biosciences, Texas, USA), anti-CD3 (1:500,
   17617-1-AP, Proteintech, Wuhan, China), anti-NF-κB (1:1000, ab76302,
   Abcam, Cambridge, UK), and anti-β-actin (1:10000, EM21002, Huabio,
   Zhejiang, China).

   Excess primary antibody was washed out, followed by a 2 h incubation
   with secondary antibody at room temperature and four 5 min washes with
   PBST. The membranes were incubated with enhanced chemiluminescence
   reagents (BL520A, Biosharp, Anhui, China) for 1 min at room temperature
   and then developed in a dark room.

2.14 Gene expression analysis by quantitative RT-PCR

   Frozen spleen tissue was retrieved from the −80°C freezer and ground
   into a fine powder using liquid nitrogen. Total RNA was extracted using
   the SteadyPure Quick RNA Extraction Kit (AG21023, Accurate
   Biotechnology (Hunan) Co., Ltd., ChangSha, China). Reverse
   transcription was conducted with the MonScript™ RTIII Super Mix with
   dsDNase (Two-Step) kit (MR05201, Monad, Suzhou, China). Gene expression
   levels were measured using the ABI StepOne Plus instrument and the
   MonAmp™ SYBR ^® Green qPCR Mix (High ROX) kit (MQ10301S, Monad), with
   calculations based on the 2^−ΔΔCT method. The primer sequences of genes
   are listed in [78]Table 2.

TABLE 2.

   Gene primer sequence list.
   Rat gene Sequence (5′to 3′)                Product size
   Pik3cg-F CAC​CTC​CGC​AAC​CAA​TCC​TGA​C     130
   Pik3cg-R AGC​TCG​AAC​TCT​GTC​TCC​TTC​TGG
   Pdk1-F   CTT​AGA​GGG​CTA​CGG​GAC​GGA​TG    117
   Pdk1-R   TCG​TGG​TTG​GTT​CTG​TAA​TGC​TTC​C
   Akt1-F   CAC​AGG​TCG​CTA​CTA​TGC​CAT​GAA​G 95
   Akt1-R   GCA​GGA​CAC​GGT​TCT​CAG​TAA​GC
   Nfκb1-F  TGT​GGT​GGA​GGA​CTT​GCT​GAG​G     138
   Nfκb1-R  AGT​GCT​GCC​TTG​CTG​TTC​TTG​AG
   Actin-F  CAC​GAT​GGA​GGG​GCC​GGA​CTC​ATC   240
   Actin-R  TAA​AGA​CCT​CTA​TGC​CAA​CAC​AGT
   [79]Open in a new tab

2.15 Statistical analysis

   All statistical analyses were conducted using SPSS 26.0, and the data
   were presented as mean ± standard deviation. A one-way ANOVA was
   conducted to assess the statistical significance of group differences.
   When the homogeneity of variance was satisfied, the least significant
   difference t-test was applied. Tambane’s T^2 method was also employed
   for analysis. p < 0.05 or p < 0.01 indicate statistical significance.

3 Results

3.1 Identification of active ingredients and target selection

   The chemical constituents in BZ were analyzed using UPLC-EIS-MS, and a
   total of 69 active compounds were identified. We list 30 of these major
   and more abundant components in [80]Table 3.

TABLE 3.

   Identification of compounds in BZ samples by UPLC-EIS-MS.
   NO. tR/min Molecular formula Mass Theoretical mass Experimental mass
   Adduct MS^2 Identified constituents
   1 0.4 C[6]H[9]N[3]O[2] 155.06948 156.07675 156.07472 +H 110.0727,
   83.0596, 56.0483 L-histidine
   2 0.47 C[5]H[9]NO[2] 115.06333 116.07061 116.06902 +H 116.0709,
   70.0707, 43.0536 L-proline
   3 0.83 C[9]H[11]NO[2] 165.07898 166.08626 166.08437 +H 131.0481,
   120.0845, 103.0543 L-phenylalanine
   4 1.56 C[16]H[18]O[9] 354.09508 355.10236 355.09978 +H 145.0287,
   163.0436 Chlorogenic acid
   5 3.58 C[10]H[8]O[4] 192.04226 193.04954 193.04754 +H 178.0256,
   137.0594, 133.0260 Scopoletol
   6 3.7 C[10]H[10]O[4] 194.05791 195.06519 195.06314 +H 177.0525,
   145.0269, 117.0338 Ferulic Acid
   7 4.5 C[28]H[32]O[15] 608.17412 609.1814 609.17821 +H 463.1194,
   301.0702 Neodiosmin
   8 4.68 C[13]H[10]O 182.07317 183.08044 183.07601 +H 155.0446, 127.0156,
   98.9852 Atractylodin
   9 7.08 C[20]H[36]O[7] 388.2461 389.25338 389.25195 +H 371.2419,
   232.1680 8,9-epoxy atracolactone
   10 8.41 C[16]H[20]O 228.15142 229.15869 229.15639 +H 121.0626, 91.0548,
   77.0375 furan sesquiterpene
   11 9.4 C[15]H[20]O[3] 248.14124 249.14852 249.14636 +H 175.0747,
   163.0780, 135.0435 Atractylenolide Ⅲ
   12 10.23 C[16]H[22]O[2] 246.16198 247.16926 247.16697 +H 215.1407,
   187.1465, 159.1142 Methyl-atractylenolide II
   13 10.34 C[17]H[24]O[3] 276.17254 277.17982 277.17748 +H 259.1674,
   235.1757, 217.1562 atractylenolide Ⅳ
   14 11.43 C[15]H[20]O[2] 232.14633 233.15361 233.15151 +H 187.1497,
   151.0785 Atractylenolide Ⅱ
   15 11.93 C[14]H[12]O 196.08882 197.09609 197.09404 +H 128.0643,
   115.0524, 91.0541 4,6-Dimethyl dibenzofuran
   16 13.85 C[15]H[18]O[2] 230.13068 231.13796 231.13599 +H 185.1317,
   157.1004, 145.0999 Atractylenolide Ⅰ
   17 14.86 C[15]H[24] 204.1878 205.19508 205.19266 +H 163.1465, 149.0221,
   135.1153 beta-Selinene
   18 15.01 C[15]H[22]O 218.16707 219.17434 219.17216 +H 123.0811, 95.0856
   alpha-Cyperone
   19 15.6 C[17]H[22]O[3] 274.15689 275.16417 275.16158 +H 215.1404,
   197.1303, 145.0985 3β-acetoxy atractylon
   20 16.82 C[6]H[6]O[3] 126.03169 127.03897 127.03703 +H 98.9581,
   85.0653, 81.0677 5-Hydroxymethylfurfural
   21 16.97 C[17]H[24]O[2] 260.17763 261.18491 261.18222 +H 219.1732,
   173.1306, 145.1013 2-(3,7-Dimethyl-octa-2,6-dienyl)-4-methoxy-phenol
   22 18.72 C[15]H[22] 202.17215 203.17943 203.17717 +H 147.1186,
   133.1025, 119.0851 Atractylenolide Ⅵ
   23 18.73 C[6]H[14]N[2]O[2] 146.10553 147.1128 147.11441 +H 105.0676,
   91.0532, 41.0368 L-Lysine
   24 18.92 C[15]H[20]O 216.15142 217.15869 217.15622 +H 199.1426,
   147.1156, 143.0840 Atractylone
   25 19.93 C[16]H[30]O 238.22967 239.23694 239.23426 +H 203.1762,
   137.1319, 109.0946 Muscone
   26 20.04 C[10]H[16]O[2] 168.11503 169.12231 169.12036 +H 151.1100,
   133.0997, 123.1159 Trans-geranic acid
   27 21.63 C[16]H[32]O[2] 256.24023 257.24751 257.24465 +H 187.1668,
   173.1512, 145.1219 Plamitic acid
   28 21.92 C[19]H[32]O[2] 292.24023 293.24751 293.24446 +H 243.2104,
   237.1812, 183.1332 Methyl Linolenate
   29 24.43 C[19]H[34]O[2] 294.25588 295.26316 295.26008 +H 263.2350,
   245.2248, 189.1616 Methyl octadeca-9,12-dienoate
   30 26.58 C[29]H[48]O 412.37052 413.37779 413.37435 +H 343.0096,
   231.0760, 217.1596 Stigmasterol
   [81]Open in a new tab

   These components mainly contain sesquiterpenes and lactones,
   flavonoids, phenylpropanoids, alkynes, polysaccharides, and other
   components. Among them, sesquiterpenes are the main active ingredients
   of BZ, mainly including atractylenolide Ⅰ to Ⅶ, atractylenone, and
   β-eudesmol, etc. The organic acids include chlorogenic acid, ferulic
   acid, and palmitic acid. The coumarins include 7-hydroxymethylcoumarin
   and scopoletin. The amino acids include arginine, L-histidine,
   L-proline, and L-valine. The secondary mass spectra of atractylenolide
   I, II, III, and atractylone are presented in [82]Figure 1. We have
   provided the CAS number, SMILE number, and molecule name of all the
   ingredients in BZ, as well as the Uniprot numbers and abbreviations of
   the corresponding targets of the ingredients ([83]Supplementary Table
   S1). A total of 1124 targets corresponding to active ingredients in BZ
   were collected and predicted. Through the database search of DurgBank,
   Malacards, TTD, and OMIM, 303 predicted targets about aging were found
   after deduplication ([84]Supplementary Table S2).

FIGURE 1.

   [85]FIGURE 1
   [86]Open in a new tab

   The positive mode detected the mass spectrum of the biomarker. (A) A
   total ion flow chromatogram of BZU. (B) Second-order mass spectrum
   (MS^2) of the atractylenolide III. (C) MS^2 of the atractylenolide II.
   (D) MS^2 of the atractylenolide I. (E) MS^2 of the Atractylone.

3.2 Network construction and PPI protein interaction network

   We import the targets of BZ components and the predicted targets of
   aging into Cytoscape and Venny for network analysis to obtain the
   BZ-aging-potential targets gene network ([87]Figure 2). The
   BZ-aging-potential target gene network comprised 37 nodes and 532 lines
   ([88]Table 4). Further analysis identified the top 12 targets with
   significant roles in the network (degree >20): Protein kinase B (AKT1),
   Poly[ADP-ribose] synthase 1 (PPARG), Tumor necrosis factor receptor
   superfamily member 10A (TNF), Caspase-3 (CASP3), Hypoxia-inducible
   factor 1-alpha (HIF1A), Bcl-2-like protein 11 (BCL2), Estrogen receptor
   (ESR1), Transcription factor AP-1 (JUN), Acute-phase response factor
   (STAT3), Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and
   dual-specificity protein phosphatase PTEN (PTEN), Proto-oncogene c-Fos
   (FOS), and PPAR-gamma (PARP1).

FIGURE 2.

   [89]FIGURE 2
   [90]Open in a new tab

   Potential target analysis and PPI network construction. (A) The Venny
   results of potential target genes of BZ therapy for aging. (B)
   Protein-protein interaction network diagram (The corresponding colors
   in the legend are calculated according to the degrees of freedom). (C)
   Drug-compound–target interaction network diagram (The purple nodes
   represent the compounds in BZ, the orange nodes represent key targets,
   and the green nodes represent other targets.).

TABLE 4.

   37 potential target genes of BZ therapy for aging.
   No. Symbol Target Uniprot No. Symbol Target Uniprot
   1 ADRB2 Beta-2 adrenoceptor [91]P07550 20 SOD1 Superoxide dismutase
   [Cu-Zn] [92]P00441
   2 PARP1 Poly [ADP-ribose] synthase 1 [93]P09874 21 CASP3 Caspase-3
   [94]P42574
   3 CTSK Cathepsin K [95]P43235 22 CDKN2AIP CDKN2AIP N-terminal-like
   protein [96]Q96HQ2
   4 SIGMAR1 Sigma1-receptor [97]Q99720 23 CDKN1B Cyclin-dependent kinase
   inhibitor 1B [98]P46527
   5 TERT Telomerase reverse transcriptase [99]O14746 24 HIF1A
   Hypoxia-inducible factor 1-alpha [100]Q16665
   6 ADORA3 Adenosine receptor A3 [101]P0DMS8 25 PPARG PPAR-gamma
   [102]P37231
   7 ADORA1 Adenosine receptor A1 [103]P30542 26 FOS Proto-oncogene c-Fos
   [104]P01100
   8 ADORA2A Adenosine receptor A2a [105]P29274 27 STAT3 Acute-phase
   response factor [106]P40763
   9 ESR1 Estrogen receptor [107]P03372 28 TNF Tumor necrosis factor
   receptor superfamily member 10A [108]O00220
   10 PPARA PPAR-alpha [109]Q07869 29 XDH Xanthine oxidoreductase
   [110]P47989
   11 HSD11B1 11-beta-HSD1 [111]P28845 30 NOX4 NADPH oxidase 4 [112]Q9NPH5
   12 AKT1 Protein kinase B [113]P31749 31 PDGFRB Platelet-derived growth
   factor receptor beta [114]P09619
   13 TOP1 DNA topoisomerase 1 [115]P11387 32 IGF1R Insulin-like growth
   factor 1 receptor [116]P08069
   14 MAPK14 Mitogen-activated protein kinase 14 [117]Q16539 33 BRAF
   Serine/threonine-protein kinase B-raf [118]P15056
   15 MAPK1 Mitogen-activated protein kinase 13 [119]O15264 34 PNP Purine
   nucleoside phosphorylase [120]P00491
   16 ME2 NAD-dependent malic enzyme, mitochondrial [121]P23368 35 APP
   Amyloid-beta precursor protein [122]P05067
   17 BCL2 Bcl-2-like protein 11 [123]O43521 36 NAMPT Nicotinamide
   phosphoribosyltransferase [124]P43490
   18 PTEN Phosphatase and tensin homolog [125]P60484 37 SIRT2
   NAD-dependent protein deacetylase sirtuin-2 [126]Q8IXJ6
   19 JUN Transcription factor AP-1 [127]P05412
   [128]Open in a new tab

3.3 GO and KEGG pathway enrichment analysis

   We imputed 37 targets into the R language, enriched and analyzed the GO
   and KEGG pathways. Annotation of GO function enrichment is based on
   three aspects: biological process (BP), molecular function (MF), and
   cellular component (CC) ([129]Supplementary Table S3). GO enrichment
   analysis identified 1990 biological process functions (p < 0.05),
   encompassing aging, cell aging, responses to oxygen and decreased
   oxygen levels, radiation, hypoxia, regulation of DNA metabolic
   processes, and oxidative stress. Scatterplots were generated for the
   top 20 enriched gene biological function catalogs and the top five most
   significant biological function enriched targets ([130]Figure 3). GO
   analyses identified 120 enriched molecular function GO terms, such as
   catalytic activity on DNA, NAD+ binding, DNA-binding transcription
   factor binding, damaged DNA binding, sequence-specific single-stranded
   DNA binding, and RNA polymerase II-specific DNA-binding transcription
   factor binding. 52 GO terms in the cellular component were enriched,
   including chromosome, telomeric region, chromosomal region, nuclear
   chromosome, telomeric region, nuclear periphery, DNA repair complex,
   etc. The most significant genes in these pathways involved were TP53,
   JUN, HIF1A, STAT3, SIRT1, CDKN2A, FOXO3, etc.

FIGURE 3.

   [131]FIGURE 3
   [132]Open in a new tab

   GO enrichment analysis of potential genes. (A) Biological process
   analysis. (B) Molecular function analysis. (C) Cell component analysis.

   To gain insight into gene regulatory networks in BZ therapy for aging,
   a total of 102 KEGG signaling pathways were enriched
   ([133]Supplementary Table S4). The top 20 highly enriched KEGG pathways
   are presented ([134]Figures 4A–C). The KEGG analysis identified
   pathways including Cellular senescence, MAPK, PI3K-Akt, FoxO, mTOR, and
   T cell receptor signaling, etc. [135]Figure 4D highlights the primary
   targets in the T cell receptor signaling pathway.

FIGURE 4.

   [136]FIGURE 4
   [137]Open in a new tab

   KEGG pathway analysis. (A) Interaction network of pathways. (B)
   Target-pathway network. (C) Top 20 genes of pathway enrichment. (D) The
   T cell receptor signaling pathway of potential target genes of BZ in
   aging. Arrows indicate upstream and downstream relationships between
   genes. The red is a BZ target gene in the network.

3.4 Quantitative analysis of components in BZU

   As can be noticed in the SEM micrograph ([138]Figure 5A), the BZU is
   incomplete with large fragmentation and uniformity in particle size.
   The surfaces of the particles are rough, and the powders agglomerate
   with each other.

FIGURE 5.

   [139]FIGURE 5
   [140]Open in a new tab

   BZU content determination and behavioral results. (A) The SEM
   micrograph images of BZU. (B) HPLC chromatogram of the content of the
   main components of BZU (Peaks 1,2,3,4 are atractylenolide III, II, I
   and atractylone respectively.). Behavioral experiment: (C) the times of
   autonomous activity, (D) the body temperature, (E) the grasp strength,
   (F) the exhaustive swimming time, (G) the moisture content of fecal,
   (H) the microcirculatory blood flow and (I) densitometry of
   microcirculatory blood flow in the rat tail. (J) Changes of
   hemorheology indexes. Peripheral blood measurements for (K) WBC count,
   (L) RBC count, (M) HGB count and (N) PLT count. Statistical
   significance of differences between groups is depicted on the graphs.
   Data were shown as mean ± SD (Each group n = 6). AC, Young control
   group; AM, Old model group; BZU-L, low-dose BZU group; BZU-M,
   medium-dose BZU group; BZU-H, high-dose BZU group. ^△ p < 0.05, ^△△ p <
   0.01. vs. control; *p < 0.05, **p < 0.01. vs. model.

   The phenol-sulfuric acid method determined the total polysaccharide
   content of BZU to be 4.76%. In addition, the contents of
   atractylenolide I, II, III, and atractylone in BZU, determined by HPLC,
   were 0.2962, 0.3970, 0.2316, and 1.7089 mg/g, respectively. HPLC
   chromatograms of the BZU are depicted in [141]Figure 5B.

3.5 Effects of BZU on general signs

   As depicted in [142]Figures 5C–I, compared to the AC group, the times
   of autonomous activity, the body temperature, the grasping strength,
   the times of exhaustive swimming, and the microcirculatory blood flow
   of the AM group were significantly decreased (p < 0.05, p < 0.01).
   After 3 weeks of administration, all BZU groups exhibited a significant
   increase in autonomous activity, and the BZU-L group showed a
   significant rise in body temperature compared to the AM group (p <
   0.05, p < 0.01) ([143]Figures 5C,D). All BZU groups exhibited a
   significant increase in grasp strength compared to the AM group (p <
   0.05, p < 0.01) ([144]Figure 5E). The BZU-L and BZU-H groups showed a
   significant rise in exhaustive swimming time compared to the AM group
   (p < 0.05, p < 0.01) ([145]Figure 5F). However, no significant
   differences were observed in fecal water content (p > 0.05, p < 0.01)
   ([146]Figure 5G). Microcirculatory blood flow significantly increased
   in the BZU-M and BZU-H groups compared to the AM group (p < 0.05, p <
   0.01) ([147]Figure 5H). As [148]Figure 5I shows, at a glance, the
   change in intensity of microcirculatory blood flow in the tail of each
   group of rats.

3.6 Effect of BZU on hematological indices

   [149]Figure 5J illustrates that both whole blood and plasma viscosity
   were significantly elevated in the AM group compared to the AC group,
   with plasma viscosity showing a notable difference (p < 0.05).
   Post-intervention, whole blood viscosity at low shear rates was
   significantly reduced in both the BZU-L and BZU-M groups compared to
   the AM group (p < 0.05, p < 0.01).

   The AM group exhibited a significant decrease in WBC levels compared to
   the AC group (p < 0.05), while the levels of RBC, HGB, and PLT showed
   an increasing trend without reaching statistical significance (p >
   0.05). After 3 weeks of administration, the BZU-M and BZU-L groups
   exhibited a significant increase in WBC levels compared to the AM group
   ([150]Figure 5K). The RBC, HGB, and PLT levels showed a downward trend,
   but no significant differences were found (p > 0.05) ([151]Figures
   5L–N).

3.7 Effect of BZU on peripheral blood immunophenotyping ratios

   The logic diagram of flow cytometry gating of peripheral blood immune
   cells is shown in [152]Figure 6A. [153]Figures 6B,C show that compared
   with the AC group, the proportion of lymphocytes and T cells in the AM
   group significantly decreased (P < 0.05). There was a trend toward a
   decrease in the percentage of B and NK cells in the AM group, but there
   was no significant difference (p > 0.05) ([154]Figures 6D,E). While the
   proportion of Treg cells in the AM group was significantly higher (p <
   0.05) ([155]Figures 6F,G). After 3 weeks of oral gavage administration
   of BZU, the lymphocyte percentage significantly increased in the BZU-M
   group compared to the AM group (p < 0.01). T, B, and NK lymphocyte
   levels increased in the treatment group, but the change was not
   statistically significant (p > 0.05). The proportion of Treg cells in
   the BZU-M and BZU-H groups was significantly reduced.

FIGURE 6.

   [156]FIGURE 6
   [157]Open in a new tab

   Phenotype analysis of lymphocyte subpopulations by flow cytometry. (A)
   Flow cytogram circle gate logic diagram. (B) Lymphopenia, (C) T cells
   (CD3^+), (D) B cells (CD3^−CD45RA^+), (E) NK cells (CD3^−CD161a^+), (F)
   Treg cells (CD4^+CD25^+Foxp3^+) and (G) Flow densitogram of Treg cells.
   Statistical significance of differences between groups is depicted on
   the graphs. Data were shown as mean ± SD (Each group n = 6). AC, Young
   control group; AM, Old model group; BZU-L, low-dose BZU group; BZU-M,
   medium-dose BZU group; BZU-H, high-dose BZU group. ^△ p < 0.05, ^△△ p <
   0.01. vs. control; *p < 0.05, **p < 0.01. vs. model.

   [158]Figure 7 shows that compared with the AC group, the proportion of
   Tc cells in the AM group significantly increased, while the proportion
   of Th cells significantly decreased (p < 0.05). In addition, there was
   a significant difference in the proportion of naïve T, memory T and
   effector T cells between the two groups. The proportions of memory of
   Th and Tc cells in the AM group were significantly increased, while the
   proportions of naïve of Th and Tc cells, and effector Tc cells were
   significantly decreased (p < 0.05, p < 0.01). The AM group showed a
   significant decrease in the proportion of CD8^+CD28^+ subgroups, while
   there was no statistically significant difference in the proportion of
   CD4^+CD28^+ subgroups (p < 0.05).

FIGURE 7.

   [159]FIGURE 7
   [160]Open in a new tab

   Phenotype analysis of T cell sub-populations by flow cytometry. (A) Tc
   cells (CD3^+CD8^+), (B) Th cells (CD3^+CD4^+), (C) Histogram of CD4/CD8
   cells, (D) naïve Tc cells (CD3^+CD8^+CD44^−CD62^+), (E) effector Tc
   cells (CD3^+CD8^+CD44^+CD62^−), (F) memory Tc cells
   (CD3^+CD8^+CD44^+CD62^+), (G) naïve Th cells (CD3^+CD4^+CD44^−CD62^+),
   (H) effector Th cells (CD3^+CD4^+CD44^+CD62^−), (I) memory Th cells
   (CD3^+CD4^+CD44^+CD62^+), (J) Contour map of T-cell subpopulations, (K)
   CD8^+CD28^+, (L) CD4^+CD28^+. Statistical significance of differences
   between groups is depicted on the graphs. Data were shown as mean ± SD
   (Each group n = 6). AC, Young control group; AM, Old model group;
   BZU-L, low-dose BZU group; BZU-M, medium-dose BZU group; BZU-H,
   high-dose BZU group. ^△ p < 0.05, ^△△ p < 0.01. vs. control; *p < 0.05,
   **p < 0.01. vs. model.

   The BZU-H group significantly increased the percentage of Th and naïve
   Tc cells and decreased the percentage of Tc and memory Th cells (p <
   0.05, p < 0.01) ([161]Figures 7A–I). The BZU-M group significantly
   increased the percentage of effector Tc cells and naïve Th cells and
   decreased the percentage of memory Tc cells (p < 0.05, p < 0.01). The
   BZU-L group significantly increased the percentage of naïve Tc cells (p
   < 0.01). As shown in [162]Figure 7J, a contour plot of the T-cell
   subpopulations, the distribution of cell proportions can be visualized.
   The CD8^+CD28^+ subsets were significantly elevated in the BZU-M and
   BZU-H groups (p < 0.05, p < 0.01) ([163]Figure 7K). BZU had no
   significant effect on CD4^+CD28^+ cells ([164]Figure 7L).

3.8 Effect of BZU on the proliferation rate of spleen lymphocytes

   Compared with the AC group, the proliferation ability of T cells and B
   cells in the spleen of rats in the AM group was significantly reduced,
   as shown in [165]Figures 8A,B (p < 0.05). The BZU-H group exhibited a
   significantly higher proliferation rate of splenic T and B cells in
   rats compared to the AM group (p < 0.05, p < 0.01).

FIGURE 8.

   [166]FIGURE 8
   [167]Open in a new tab

   Lymphocyte proliferation rate, immunohistochemical expression and serum
   levels of inflammatory factors in the spleen. (A) T-lymphocyte
   proliferation rate. (B) B-lymphocyte proliferation rate. The content of
   (C) IL-2, (D) IL-4, (E) IL-6, (F) IL-10, (G) IFN-γ and (H) TNF-α in
   serums. Data were shown as mean ± SD (Each group n = 6). (I)
   Histopathology observations of spleen tissue (10×, 20×). (J)
   Immunohistochemistry plot of PDK1, CD3 and IKKα/β (20×). The
   immunohistochemical quantitative statistics of (K) PDK1, (L) CD3, and
   (M) IKKα/β. Data were shown as mean ± SD (Each group n = 3).
   Statistical significance of differences between groups is depicted on
   the graphs. AC: Young control group; AM: Old model group; BZU-L:
   low-dose BZU group; BZU-M, medium-dose BZU group; BZU-H, high-dose BZU
   group. ^△ p < 0.05, ^△△ p < 0.01. vs. control; *p < 0.05, **p < 0.01.
   vs. model.

3.9 Changes of serum IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ levels

   Serum levels of IL-2, IL-4, and IL-10 were significantly lower, whereas
   IL-6 and TNF-α levels were significantly higher in the AM group
   compared to the AC group (p < 0.05, p < 0.01) ([168]Figures 8C–H).

   After drug administration, serum IL-2 levels significantly increased in
   both BZU-M and BZU-H groups (p < 0.01). The serum levels of IL-6 and
   IFN-γ in the BZU-H group were significantly reduced (p < 0.05). All BZU
   administration groups significantly increased IL-4 and decreased serum
   TNF-α levels (p < 0.05, p < 0.01).

3.10 Effect of BZU on spleen tissue morphology

   [169]Figure 8I’s HE staining pathological results revealed that the
   spleens of AC group rats exhibited a clear structure, closely arranged
   lymphocytes, and well-defined red and white pulp with distinct edges.
   The spleens in the AM group exhibited disrupted structures with
   indistinct margins between the white and red pulps. The white pulp
   appeared blurred and reduced in size, with a normal but sparse
   lymphocyte population. Compared with the AM group, the white pulp of
   the spleen of rats in the BZU-M and BZU-H groups was significantly
   enlarged, the red and white pulp structures were clearly demarcated,
   and the histopathological changes of the spleen were all significantly
   improved.

3.11 Localization and expression levels of PDK1, CD3, and IKKα/β proteins in
rat spleen tissue

   As shown in [170]Figure 8J, CD3^+T cells were predominantly distributed
   in the white pulp. In the AM group, the number of periarterial
   lymphatic sheath cells composed of T lymphocytes was reduced. The
   proteins IKKα/β and PDK1 exhibit predominant expression within the red
   pulp and cortical structures of splenic tissue. Compared with the AC
   group, the expression level of IKKα/β protein was significantly
   increased, and CD3 expression was significantly decreased in the AM
   group (p < 0.01). Post drug administration, CD3 protein expression
   significantly increased in the BZU-H group, while IKKα/β protein
   expression significantly decreased in the BZU-L and BZU-H groups (p <
   0.05).

3.12 Levels of PI3K, AKT, p-AKT, NF-κB, and CD3 as determined by western
blotting

   [171]Figures 9A–E show that compared with the AC group, the expression
   levels of PI3K and NF-κB proteins in the spleen tissue of the AM group
   were significantly increased, while the expression level of CD3 protein
   was significantly decreased (p < 0.05). The expression of p-AKT protein
   showed an upward trend, but there was no significant difference (p >
   0.05). After administration, the protein expression of PI3K, p-AKT, and
   NF-κB in the spleen tissue of rats in the BZU-H group was significantly
   reduced. The expression of CD3 proteins was significantly increased in
   all administration groups (p < 0.05). The immunoblot band diagram is
   shown in [172]Figure 9F.

FIGURE 9.

   [173]FIGURE 9
   [174]Open in a new tab

   BZU regulates the expression of PI3K/Akt/NF-κB pathway in the spleen.
   The protein expression of (A) PI3K, (B)AKT, (C) p-AKT, (D) CD3 and (E)
   NF-κB. (F) WB images of PI3K, AKT, p-AKT, CD3 and NF-κB protein
   expression. The mRNA expression of (G) Pik3cg, (H) Akt1, (I) Pdk1 and
   (J) Nfκb1. Statistical significance of differences between groups is
   depicted on the graphs. Data were shown as mean ± SD (Each group n =
   3). AC, Young control group; AM, Old model group; BZU-L, low-dose BZU
   group; BZU-M, medium-dose BZU group; BZU-H, high-dose BZU group. ^△ p <
   0.05, ^△△ p < 0.01. vs. control; *p < 0.05, **p < 0.01. vs. model.

3.13 mRNA expression levels of Pik3cg, Akt1, Pdk1 and Nfκb1 in tissues as
determined by qRT-PCR

   The AM group exhibited significantly elevated mRNA expression levels of
   Pik3cg, Akt1, Pdk1 and Nfκb1 compared to the AC group (p < 0.01). The
   BZU-H group exhibited significantly reduced mRNA expression levels of
   Pik3cg compared to the AM group (p < 0.01). Additionally, the BZU-M and
   BZU-H groups reduced Pdk1 mRNA expression, while the BZU-L and BZU-M
   groups reduced Akt1 mRNA expression (p < 0.05, p < 0.01). All BZU
   administration groups administration groups significantly reduced Nfκb1
   mRNA expression. ([175]Figures 9G–J).

4 Discussion

   According to relevant statistics, China’s elderly demographic, defined
   as individuals aged 65 and older, constituted 11.4% of the nation’s
   total population by the conclusion of 2017, amounting to 158 million
   individuals ([176]Qidi et al., 2021). Furthermore, projections indicate
   that between 2000 and 2055, the rate of population aging in China is
   expected to surge from 10% to 34%, surpassing the global average growth
   rate. A 2019 study in The Lancet identified stroke, ischemic heart
   disease, lung cancer, and Alzheimer’s disease as leading causes of
   mortality in China, highlighting the serious social issue of
   accelerated population aging ([177]Maigeng et al., 2017). Therefore,
   slowing down the aging process has become an important scientific
   problem that needs to be solved.

   Aging results in a gradual decline in physiological and organ
   functions, weakening physical and mental states. This decline may
   manifest in symptoms such as neurasthenia, cold intolerance, decreased
   physical strength, memory impairment, microcirculation issues, and back
   and leg pain ([178]Elsa et al., 2017). Therefore, this test evaluated
   the effect of BZU by observing these apparent indicators of aging. We
   evaluated autonomous activity times, body temperature, grip strength,
   swimming endurance, and microcirculatory blood flow in aging rats to
   examine the impact of BZU on mental mood, metabolic function, physical
   performance, and microcirculation. It was found that BZU could increase
   the times of autonomous activity, raise body temperature, enhance grip
   strength and fatigue resistance, and increase the microcirculatory
   blood flow of naturally aging rats. This suggests that BZU can modulate
   various functional impairments and improve biological indicators of
   aging in naturally aging rats.

   In addition, as humans age, the bone marrow undergoes many changes.
   Clinical studies have identified a differentiation shift in the
   hematopoietic system among the elderly, characterized by an increased
   proportion of myeloid progenitor cells and a decreased proportion of
   lymphoid progenitor cells ([179]Bowen et al., 2019). Red blood cells,
   hemoglobin and platelets tend to be lower in healthy elderly men than
   in young men, and anemia occurs in the elderly. The experiment
   indicated a tendency for increased RBC, HGB, and PLT levels in the
   peripheral blood of naturally aging rats, differing somewhat from those
   observed in healthy elderly humans. This suggests that there may be
   some differences in platelet number and function compensation between
   aged rats and the elderly. However, there are other studies that show
   that aging is also manifested by bone marrow and platelet skewing,
   inflammation, and expansion of clonal hematopoiesis. The inflammatory
   state encourages a constant differentiation of hematopoietic stem cells
   towards megakaryocytes, which ultimately leads to an elevated platelet
   count and a rise in the platelet ratio ([180]Siddhartha, 2020). This
   suggests that aged rats may be in a chronic inflammatory state and may
   also imply that aged rats may suffer from senile polycythemia vera
   (PV), which could potentially serve as an animal model for PV. The
   results showed that BZU significantly reduced whole blood viscosity and
   lowered HGB and PLT levels. This suggests that it may have an
   ameliorative effect on thrombosis and hematopoiesis and may have
   therapeutic effects on youth-related blood disorders.

   In our investigation utilizing UPLC-EIS-MS, we identified 69 active
   compounds within BZU. Through network pharmacological analysis of BZ’s
   anti-aging properties, 37 nodes were identified, including key targets
   such as AKT1, TNF, PTEN, FOS, and PARP1. GO and KEGG analyses indicate
   that the PI3K/AKT is an upstream signaling pathway capable of
   regulating NF-κB, both of which are crucial in combating
   immunosenescence. As one of the crucial kinases involved in T-cell
   priming signal transduction, PI3K plays a role in anti-apoptosis and
   promoting cell proliferation and differentiation. It is associated with
   cell survival, tumorigenesis, and apoptosis, and holds significant
   importance in the phosphorylation of Akt. Studies have indicated that
   during the aging process, the PI3K pathway becomes overactivated. In
   particular, the dominant activating mutation of PI3K leads to T-cell
   dysfunction and immunosenescence ([181]Carrie et al., 2013). When PI3K
   is activated, it generates phosphatidylinositol-3,4,5-trisphosphate
   (PIP3). PIP3 binds to the intracellular signaling protein PDK1, thereby
   activating AKT. The kinase PDK1 mediates the regulation of the PI3K/AKT
   signaling pathway, which is involved in the regulation of immune cell
   development as well as T cell subpopulation differentiation ([182]Zhen
   et al., 2021). Subsequently, activated PI3K and AKT participate in the
   phosphorylation and nuclear translocation of NF-κ B, promoting the
   production of inflammatory mediators, leading to an imbalance in the
   inflammatory regulatory network of the immune system, and ultimately
   leading to inflammatory aging of the body ([183]Claudio et al., 2018).

   We initially investigated BZU’s impact on the differentiation of
   peripheral blood immune cells, including leukocytes, B, NK, and T cells
   subsets. Recent research indicates a strong correlation between immune
   function decline and aging ([184]Anna et al., 2019; [185]Liying and
   Tao, 2020; [186]Zhong et al., 2020). In 1969, Professor Walfod proposed
   the doctrine of immunosenescence, which considers that the senescence
   of immune cells and the aging of immune organs are the essential causes
   of organismal aging ([187]Walford, 1969). It is well known that the
   level of WBC is an indicator of immune function. Moreover, research has
   indicated that the modulation of white blood cell levels can prevent
   chronic diseases and alleviate the burden of aging ([188]Gurum et al.,
   2020). Studies have also found that three dose groups of BZ water
   extract can significantly reduce the aging performance score and the
   number of spontaneous activities of naturally aging mice to a certain
   extent. Additionally, it can increase the number of whole blood WBC,
   RBC, HGB, and lymphocytes, regulate T-cell ratios, and improve
   age-related functional declines such as immunosuppression, insufficient
   hematopoiesis, and memory attenuation in naturally aging mice,
   demonstrating significant therapeutic advantages ([189]Qidi et al.,
   2021). The study demonstrated that BZU significantly increased WBC
   levels in naturally aging rats, indicating its potential to enhance
   immune function and delay aging by boosting leukocyte levels.

   In addition, as the body ages, the immune system exhibits a gradual
   decline in response to antigens and becomes less reactive to foreign
   antigens. T lymphocytes are key effector cells in immune regulation and
   cellular immunity, crucial for organismal defense. Aging prevents the
   development of naïve T cells and increases memory T cells as antigen
   stimulation increases due to decreased development and maturation
   ([190]Tae-Kyung et al., 2018). The decline of CD8^+T cells during
   senescence was more pronounced than that of CD4^+T cells
   ([191]Czesnikiewicz Guzik et al., 2008). Age-related changes in
   regulatory T cells were observed, marked by an increase in Treg cell
   numbers. The results of this experiment showed that the proportions of
   lymphocytes and T, Th, naïve Tc, naïve Th and effector Tc cells
   decreased significantly in naturally aging rats showed a decreasing
   trend, while the proportions of Treg, Tc, memory Tc and memory Th cells
   increased significantly, which is consistent with the dysregulation of
   cellular subsets in the elderly. The administration at BZU showed an
   increase in lymphocytes and various T cell subpopulations (Th, naïve
   Tc, naïve Th, effector Tc) and a significant decrease in Tc, memory Tc
   and memory Th cells. It appears that BZU increases the number of T
   lymphocytes and improves the dysregulation of T lymphocyte
   subpopulations in naturally aging rats, and regulates lymphocyte
   function.

   Numerous studies indicate that aging alters Th1 and Th2 cell counts,
   disrupting the Th1/Th2 homeostatic balance ([192]Huang et al., 2013).
   Transcriptome sequencing revealed significant upregulation of
   inflammation-related genes and signaling pathways in aging tissues
   ([193]Ahadi et al., 2020). Simultaneously, the elderly exhibit elevated
   blood levels of pro-inflammatory factors, including CRP, IL-6, and
   TNF-α. Inhibition of the PI3K/Akt signaling pathway reduced
   TNF-α-induced NP cell senescence ([194]Li et al., 2017). This study
   found that aging rats exhibited significantly reduced serum levels of
   IL-2, IL-4, and IL-10, alongside significantly increased serum levels
   of IL-6, IFN-γ, and TNF-α. After administration of BZU, serum levels of
   IL-2 and IL-4 significantly increased, whereas IL-6, IFN-γ, and TNF-α
   levels significantly decreased. This indicates that BZU can alter the
   levels of immune molecules in the aging body, inhibit inflammatory
   responses, and improve Th1 and Th2 cell imbalance.

   These studies suggest that BZU regulates the development and
   differentiation of peripheral blood immune cells. After that, we
   examined the impact of BZU on spleen pathology and immune cell
   proliferation. BZU significantly ameliorated aging rats’ spleen
   histopathology in HE studies. T and B lymphocytes, the primary cells of
   the immune system, can be activated by Con A and LPS, respectively.
   Their proliferative activity influences the level of immune response
   and is a common indicator of immune competence. It has been shown that
   the proliferative capacity of lymphocytes is significantly reduced in
   7-month-old mice compared to 2-month-old young C57BL/6J mice, and the
   proliferative capacity decreases with age ([195]Jing et al., 2017).
   Studies have indicated that BZ granules, BZ polysaccharides,
   Atractylenolide I, and Atractylenolide III can effectively promote the
   proliferation of splenic lymphocytes in normal mice within a
   concentration range of 0.2–125 μg/mL ([196]Cairan, 2016). Other
   research has found that water extracts of BZ can enhance the immune
   response of mice to foot-and-mouth disease vaccination, thereby
   increasing the proliferation rate of splenic lymphocytes ([197]Ruili et
   al., 2009). The experiment demonstrated that naturally aging rats
   exhibited a significant reduction in the proliferative capacity of T
   and B lymphocytes, whereas BZU administration notably enhanced this
   capacity.

   The above results indicate that BZU has a modulating effect on the
   differentiation and proliferation of immune cells and T-cell
   subpopulations in the peripheral blood and spleen of naturally aging
   rats and can balance the levels of immune molecules. Network
   pharmacology enrichment analysis identified the PI3K/AKT/NF-κB
   signaling pathway as crucial. According to reports, pterostilbene
   alleviates chondrocyte senescence induced by IL-1β through inhibiting
   the PI3K/AKT/NF-κB signaling pathway ([198]Linjie et al., 2021). Other
   studies have found that ginsenoside Rk1 can inhibit the PI3K/AKT/NF-κB
   signaling pathway both in vitro and in vivo, regulate the secretion of
   various proinflammatory cytokines, alleviate ultraviolet-induced
   inflammatory responses, and improve skin aging caused by ultraviolet
   radiation ([199]Yannan et al., 2022). Consequently, we employed
   quantitative RT-PCR, Western blotting, and immunohistochemistry to
   examine protein and mRNA expression within this pathway. The results of
   this experiment were consistent with the fact that, after aging, the
   expression level of CD3 protein decreased but activated the PI3K/AKT
   pathway. Significant increases in PI3K and NF-κB protein levels were
   observed, while IKK/α/β protein expression levels were seen to increase
   as well. The AM group showed significantly elevated mRNA expression
   levels of Pik3cg, Akt1, Pdk1, and Nfκb1 compared to the AC group. AKT
   activates the NF-κB signaling pathway by reducing IκB-mediated
   inhibition, leading to increased expression of NF-κB proteins and mRNAs
   in the nucleus. After BZU administration, CD3 protein expression was
   significantly upregulated, and PI3K, p-AKT, IKKα/β, and NF-κB protein
   levels were significantly downregulated. Furthermore, mRNA expression
   levels of Pik3cg, Akt1, Pdk1, and Nfκb1 significantly decreased.

   In summary, our study suggests that BZU may boost lymphocyte
   proliferation by inhibiting the PI3K/Akt/NF-κB signaling pathway,
   correcting immune cell imbalances, reducing inflammatory responses, and
   ultimately enhancing immune function and potentially delaying aging.
   This discovery provides a comprehensive theoretical basis for future
   research on BZ’s improvement of immunosenescence. However, it's worth
   noting that this study did not explore the main pharmacologically
   active components in BZU, which represents a limitation of this
   research and an area worthy of further exploration in future studies.
   Meanwhile, in terms of animal model selection, it is necessary to
   include animals from multiple age groups to determine whether the drug
   has a specific age-independent immunomodulatory effect on elderly mice,
   which also requires additional research. This study demonstrates that
   BZ exhibits significant efficacy in enhancing immunity and delaying
   aging, providing a certain basis for the development, application, and
   clinical use of BZ-related anti-aging products.

Funding Statement

   The author(s) declare that financial support was received for the
   research and/or publication of this article. This study was supported
   by the National Natural Science Foundation of China (82274139,
   82003977), the Huadong MedicineJoint Funds of the Zhejiang Provincial
   Natural Science Foundation of China (LHDMZ22H280002), the Key Research
   and Development Program of Zhejiang Province (2020C04020) and the Key
   Laboratory of Zhejiang Province (2012E10002).

Data availability statement

   The original contributions presented in the study are included in the
   article/[200]supplementary material, further inquiries can be directed
   to the corresponding authors.

Ethics statement

   No human studies are presented in the manuscript. The animal study was
   approved by The research was approved by a university ethics committee
   (Zhejiang Chinese Medical University Reference ZSLL-2016-118). The
   study was conducted in accordance with the local legislation and
   institutional requirements. No potentially identifiable images or data
   are presented in this study.

Author contributions

   WY: Data curation, Writing – original draft, Writing – review and
   editing. SJ: Methodology, Project administration, Writing – original
   draft. GS: Data curation, Software, Writing – original draft. ZN:
   Investigation, Supervision, Writing – review and editing. YZ: Data
   curation, Resources, Writing – original draft. Suhong Chen: Resources,
   Writing – original draft. LG: Methodology, Writing – original draft,
   Writing – review and editing.

Conflict of interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Generative AI statement

   The authors declare that no Generative AI was used in the creation of
   this manuscript.

Publisher’s note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations, or those of the publisher, the editors and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

Supplementary material

   The Supplementary Material for this article can be found online at:
   [201]https://www.frontiersin.org/articles/10.3389/fphar.2025.1550357/fu
   ll#supplementary-material
   [202]Table3.xlsx^ (197.8KB, xlsx)
   [203]Table2.docx^ (61.5KB, docx)
   [204]Table4.xlsx^ (20.6KB, xlsx)
   [205]Table1.xlsx^ (65KB, xlsx)

Abbreviations

   AKT, protein kinase B; BP, biological process; BZ, Atractylodis
   macrocephalae rhizoma; BZU, BZ ultrafine powder; CC, Cellular
   component; GO, Gene ontology; HE, Hematoxylin-eosin; HGB, hemoglobin;
   HPLC, High-performance liquid chromatography; IFN-γ, Interferon-γ;
   IL-2, Interleukin 2; IL-4, Interleukin 4; IL-6, Interleukin 6; IL-10,
   Interleukin 10; KEGG, Kyoto encyclopedia of genomes; MF, molecular
   function; PDK1, phosphatidylkinase-dependent kinase 1; PI3K,
   phosphatidylinositol 3-kinase; PLT, blood platelet; PPI,
   Protein-protein interaction; RBC, Red blood cell; SEM, Scanning
   electron microscope; Tc, CD8^+, Cytotoxic T cells; Th, CD4^+, T helper
   cells; Th1, Helper T cells 1; Th2, Helper T cells 2; TNF-α, Tumor
   necrosis factor-α; Treg, CD4^+CD25^+Foxp3^+; Regulatory T cells; WBC,
   white blood cell.

References