Salvianolic acid B attenuates cellular senescence and age-related decline in muscle function via dual mTOR/TP53INP2-autophagy regulation

Salvianolic acid B was purchased from Selleck (#S4735, HPLC: 99.73%).

Construction of the replicative senescent cell model involved continuous passaging of BJ cells until 45 PD (Population Doubling), at which point the cells lost their proliferative capacity and were used for subsequent processing; low-passage BJ cells (20–25 PD) were used as the control group. For the construction of the irradiation-induced senescent cell model, low-passage BJ cells (20–25 PD) were exposed to X-rays at a dose of 10 Gy and 25 mA using a Rad Source Technologies (United States) X-ray irradiator. Subsequently, the irradiated cells were cultured for 1-week post-irradiation to allow senescence induction before further processing.

The BJ and HAEC cells were purchased from Chinese Academy of Sciences of Type Culture Collection (CTCC). In an incubator with 5% CO2, the cells were cultivated in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 37 °C. Salvianolic acid B (Selleck, United States) were dissolved in DMSO to form a reserve solution. 100 μM and 200 μM Salvianolic acid B were utilized in BJ and HAEC cells for 24 h. A parallel DMSO vehicle control group was included, with the final DMSO concentration in all groups controlled at ≤ 0.2% (v/v) to exclude solvent-related interference.

8-week-old male C57BL/6J mice were purchased from GemPharmatech Co.,Ltd. Under SPF conditions, five mice per cage were allowed a 12-h light-dark cycle. All animal experiments were conducted following the guidelines and protocols approved by the Animal Protection and Ethics Committee of Jinan University (approval number 20240428-10). To establish a premature aging mouse model, X-rays (Rad Source Technologies, United States) were used at a dose of 4.5 Gy, 25 mA, followed by normal feeding for 2 months post-irradiation. After 2 months, SAB (Selleck, United States) was administered via intraperitoneal injection every 2 days for a total duration of 28 days.

Cell viability experiments were conducted using the CCK-8 assay kit (Beyotime, China). Cells were seeded at a density of 4,000 cells per well in a 96-well plate and incubated at 37 °C in a cell culture incubator for 24 h. After 24 h, the corresponding solvent or SAB (Selleck, United States) was added to each well and incubated at 37 °C in the cell culture incubator for another 24 h. Following this, the cell culture medium was aspirated from the 96-well plate, and 100 μL of 10% CCK-8 reagent was added to each well, followed by incubation of the cells at approximately 1 h at 37 °C in the cell culture incubator. Subsequently, the absorbance values were measured at a wavelength of 450 nm using a microplate reader (Biotek, United States) to assess cell viability. Cell viability (%) was calculated using the formula: (Experimental group absorbance value - Background absorbance value)/(Control group absorbance value - Background absorbance value) × 100%.

Briefly, cells were seeded at a density of 5 × 10⁴ cells per well and allowed to adhere for 24 h. They were then treated with the corresponding solvent or SAB under identical conditions to the CCK-8 assay. After 24 h of treatment, the medium was aspirated, and cells were washed twice with pre-warmed PBS. Subsequently, 0.5 mL of 0.25% trypsin-EDTA was added to each well and incubated at 37 °C for 3–5 min until complete detachment. Digestion was terminated by adding 1 mL of complete DMEM medium, and a single-cell suspension was prepared by gentle pipetting. A 10 μL aliquot of the cell suspension was analyzed using an automated cell counter to determine the viable cell concentration. Collect cells and wash with PBS, centrifuge and discard the supernatant. Incubate cells with DCFH-DA (Beyotime, China) in serum-free medium at 37 °C for 30 min. Subsequently, wash the cells three times with serum-free medium, and detect cellular ROS using flow cytometry.

To control for potential confounding effects of mitochondrial metabolic activity on the interpretation of CCK-8 results, a cell counting assay was performed. The experiment was under the same grouping and treatment conditions as the CCK-8 assay.

The intracellular ATP levels were determined utilizing an ATP assay kit (Beyotime, China). BJ cells were lysed as per the manufacturer’s guidelines. Specifically, cells in a 6-well plate were lysed in 200 μL of lysis buffer, followed by centrifugation at 4 °C and 12,000×g for 5 min. Subsequently, 20 μL of the resulting supernatant was utilized for ATP quantification. The ATP concentration was assessed using a microplate reader from (Biotek, United States), normalized to nmol/mg of protein, and the relative ATP levels were calculated.

Proteins were extracted using RIPA lysis buffer (Beyotime) containing 1% protease and phosphatase inhibitor cocktails (MCE). Protein concentrations were determined by BCA assay (Thermo Fisher), and 30 μg of each sample was separated by SDS-PAGE for proteins of interest. Electrophoresis was conducted at 80 V for 30 min followed by 120 V for 90 min. Proteins were transferred to PVDF membranes (Millipore) via wet transfer in Tris-glycine buffer with 20% methanol at 90 V (60 min) for low-molecular-weight proteins or 100 V (90 min) for larger proteins. After blocking with 5% BSA for 1 h, membranes were incubated with primary antibodies against LC3B (1:1,000, Proteintech), p62 (1:1,000, MCE), β-actin (1:200,000, Abclonal), p-mTOR(1:1,000,CST), p-S6K(1:1,000, CST), 4E-BP1 (1:1,000, CST) and GAPDH (1:200,000, Abclonal) at 4 °C overnight, followed by HRP-conjugated secondary antibody (1:2000, CST) incubation for 2 h at 37 °C. Signals were detected by ECL (Millipore) and quantified using ImageJ after imaging with a ChemiDoc XRS + system (Bio-Rad).

Total RNA was extracted from cultured BJ cells (proliferative, replicative senescent, or irradiation-induced senescent) using Trizol reagent (Takara) immediately after harvesting from culture dishes (24 h post-SAB or vehicle treatment). Subsequently, 1 μg of RNA was utilized for cDNA synthesis utilizing a First-Strand cDNA Synthesis kit following the manufacturer’s protocols (Vazyme, China). Real-time RT-PCR analysis was conducted using the Real-Time System (Thermo Fisher, Waltham, United States) and SYBR Green Mix. The relative expression levels of the target genes were determined using the relative delta-delta-Ct method.

Using the keyword “Salvianolic acid B,” relevant target points were searched in the Herb, GeneCards, and Swiss Target Prediction databases. Similarly, using the keyword “Senescence cell,” relevant target points were searched in the GeneCards database. The identified target points were then intersected to find common interacting genes. Subsequently, the common genes were inputted into the STRING database (version 12.0) to construct a protein-protein interaction (PPI) network with “Homo sapiens” as the reference species. The resulting network was visualized using Cytoscape 3.7.1 software. GO pathway enrichment analysis of the intersecting targets was performed using the clusterProfiler(4.6.2) R package.

The experiment was outsourced to Novogene Bioinformatics Technology Co. Ltd. Transcriptome sequencing data were analyzed for differential gene expression using the DESeq2 package (v 1.38.3). The volcano plot was utilized to display the numbers of upregulated and downregulated genes, while the heatmap was employed to illustrate the trends of gene upregulation and downregulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the clusterProfiler package (v 4.6.2) in conjunction with the org. Hs.e.g.,.db (3.16.0) and org. Mm.e.g.,.db (3.16.0) R packages.

The treadmill (Anhui Zhenghua Biological Instrument Equipment Co., Ltd., China) was positioned at a 15° angle relative to the ground. Prior to the formal treadmill testing, mice underwent continuous running adaptability training for 3 days, with the following running speed and duration: 5 rpm for 2 min, 7 rpm for 2 min, and 9 rpm for 1 min. During the actual experiment, the initial running speed for the mice was set at 5 rpm, with an increase of 2 rpm every 2 min until the mice reached exhaustion, and the distance run by the mice was recorded. Exhaustion was defined as the mice stopping running for 3 s or more even under electrical stimulation. The work done by the mice during running (kJ) was calculated as the product of the mouse’s body weight (kg), the distance run by the mouse (m), gravitational acceleration (g = 9.8 m/s^2), and sin(15°).

The mouse’s limbs were placed on a grip strength meter grid (Shanghai Xinruan, China), and the mouse’s tail was gently pulled backward until the mouse’s limbs detached from the grid. The grip strength meter recorded the instant grip force (gf) at the moment when the mouse’s limbs detached from the grid.

Fresh muscle tissues were fixed in 4% paraformaldehyde for 24 h, followed by dehydration in a graded alcohol series. The tissues were embedded in paraffin blocks for sectioning. Muscle tissues were sliced into 4 µm thick sections. After dewaxing paraffin sections, the nuclei were stained with hematoxylin and the cytoplasm with eosin. Following staining, the sections were dehydrated, sealed, and dried to obtain image information. Subsequently, the sections were scanned using panoramic scanning (3D HISTECH, Hungary) to acquire high-definition images for observation and analysis.

Cells were fixed for 15 min at room temperature using 4% paraformaldehyde solution, followed by washing the cells with PBS. The cells were then treated with a staining solution containing X-gal according to the instructions provided by SA-β-Gal assay kit (Beyotime, China). After staining, the cells were incubated at 37 °C for 12 h. Following incubation, the cells were washed with PBS to remove unreacted substrates and stains. Microscope was used to observe the cells, and the positivity rate was calculated from 100 cells per group.

The cells were exposed to a culture medium containing EdU for 4 h. After the incubation, the cells were gently washed with 3% BSA to remove unbound EdU. The cells were then permeabilized using PBS containing 0.3% Triton X-100. Subsequently, the cells were fixed for 15 min at room temperature using 4% paraformaldehyde solution. After washing, the cells were treated with the reaction mixture from an EdU staining kit (Beyotime, China) and incubated in the dark for 30 min. Following the washing of the cells, the cells were stained with Hoechst. T for 10 min. The cells were observed under a fluorescence microscope, and the positivity rate was calculated from 3 non-overlapping fields per group.

Data were analyzed using GraphPad Prism (Version 9.0.0, United States) and are shown as means ± standard deviations. An unpaired 2-tailed Student’s t-test was used to compare two experimental groups. Differences in three groups were assessed by a one-way ANOVA test. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To identify new compounds that can effectively modulate senescent cells, we screened 24 natural compounds. We utilized a normal human skin fibroblast cell line, BJ, and human aortic endothelial cells, HAEC, as a cellular model for this study. Cellular senescence was confirmed by the upregulation of p16 and p21 mRNA expression (Figures 1A–D) and increased SA-β-Gal staining (Figures 1E,F). During the screening phase, replicative senescent cells were treated with various natural compounds for 24 h. Based on the reduced expression of typical SASP factors, IL6 and IL1B, SAB was identified as a potential anti-aging compound (Figures 1G,H). To further validate the inhibitory effect of SAB on SASP in senescent cells, additional tests were performed on various SASP cytokines in replicative senescent cells (Figure 1I; Supplementary Figure S1A). Similar assessments were conducted on irradiation-induced senescent cells (Figure 1J). The results demonstrate that SAB suppresses SASP expression in both cell types.

To explore the potential effects of SAB on other senescence phenotypes, we first performed CCK8 assays to determine the safe concentration range of SAB for both proliferative and senescent cells. The results of the CCK8 assays indicated that SAB at concentrations below 200 μM did not cause significant cytotoxicity in either proliferative or senescent cells (Figures 2A,B). This finding was corroborated by a cell counting assay performed to rule out confounding effects from altered metabolic activity (Figures 2C,D). For the subsequent cell experiments, SAB concentrations of 100 μM and 200 μM were selected. To evaluate cellular senescence markers, we examined the expression of p16 and p21, the activity of β-galactosidase, and the status of cell proliferation. It is showed that SAB treatment did not significantly reduce p16 and p21 levels (Figures 2E,F) or alter β-galactosidase activity (Figures 2G,H). Furthermore, no significant changes in cell proliferation rates were observed compared to untreated controls (Figures 2I,J). These findings indicate that SAB cannot rescue cell cycle arrest in senescent cells or reverse cellular senescence. Although SAB did not significantly rescue cell cycle arrest in senescent cells, it was observed to inhibit excessive ROS production, thereby mitigating ROS-induced damage (Figures 2K,L). Additionally, SAB enhanced cellular metabolism by increasing ATP production, providing the energy required for cellular functions (Figure 2M). The above results indicate that although SAB is unable to rescue cell cycle arrest or reverse cellular senescence, it does have a certain degree of effect in slowing the cellular senescence phenotype, which aids in alleviating chronic inflammation, improving mitochondrial function, and enhancing cellular metabolism. SAB ameliorates senescent phenotypes through dual regulation of mTOR-dependent SASP suppression and TP53INP2-mediated autophagy activation, with concurrent reduction of intracellular ROS and enhancement of mitochondrial ATP production. These effects are not limited to antioxidant activity but reflect coordinated targeting of core senescence pathways—consistent with in vivo improvements in muscle function and reduction of age-related inflammation. Unlike pure antioxidants that only scavenge ROS without addressing upstream senescence drivers, SAB directly modulates mTOR and autophagy, validating its role as a senomorphic compound.

To identify the targets through which SAB downregulates SASP-related genes, we conducted systematic network analyses. We first retrieved SAB-related and senescence-associated targets from Herb, SwissTargetPrediction, and GeneCards databases, resulting in 50 SAB-related targets, 5,664 senescence-related targets, and 75 overlapping targets (Figure 3A). Subsequently, we conducted a GO analysis using these 75 targets, which indicated that SAB may influence the regulation of inflammatory responses (Figure 3B). To further elucidate the specific target on which SAB acts, we utilized STRING to analyze the associations between these 75 overlapping targets (Figure 3C). Red nodes indicate a more significant correlation between SAB and cellular senescence, whereas nodes in lighter shades represent a lesser or weaker correlation. To identify the most likely target of SAB action, we employed four distinct calculation methods to screen the top 15 targets (Figure 3D). Additionally, we selected the common genes among these four groups for molecular docking (Figure 3E). Binding energy analysis identified mTOR as the highest-affinity target for SAB (Figure 3F). Molecular modeling suggested SAB binding to mTOR at specific residues including GLY-169, LYS-170, VAL-171, ILE-172, GLU-2032, PHE-2039, THR-2098, TRP-2101, ASP-2102, and TYR-2105 (Figure 3G). To verify whether the inhibitory effect of SAB on SASP is dependent on mTOR, we used the mTOR inhibitor Rapamycin (Figure 3H). SASP suppression was achieved with either SAB alone or rapamycin alone. Combined treatment showed no additive inhibitory effect compared to individual agents. This non-additive effect suggests SAB-mediated SASP reduction operates through mTOR-dependent pathways. To further provide direct biochemical evidence for mTOR pathway inhibition, we performed Western blot analysis of key downstream effectors. The results demonstrated that SAB treatment (100 μM, 200 μM) dose-dependently reduced the phosphorylation levels of mTOR, S6K, and 4E-BP1 in senescent BJ cells, with an efficacy comparable to the rapamycin positive control (Figure 3I) Considering the high score of their combination, it is plausible that SAB can directly interact with mTOR to inhibit the mTOR’s effect in reducing the expression of SASP.

To investigate the mechanisms by which SAB ameliorates the senescence phenotype, we conducted RNA-seq analysis on senescent BJ cells treated with SAB. The results revealed that SAB treatment significantly upregulated 253 genes, downregulated 303 genes, and left 22,482 genes unchanged (Figure 4A). GO enrichment analysis of the significantly upregulated genes (Figure 4B) indicated that most changes were associated with autophagy-related pathways, including autophagy, processes utilizing autophagic mechanisms, macroautophagy, and regulation of autophagy. By intersecting the genes significantly altered in these four pathways, we identified 46 genes (Figure 4C). Among these 46 genes, TP53INP2, a gene involved in autophagy regulation, was identified (Figure 4D). Recent studies suggest that TP53INP2 is linked to muscle aging, and its upregulation in the muscles of aged mice promotes muscle cell autophagy, improving muscle function and exercise capacity. Therefore, we investigated whether SAB could enhance TP53INP2 expression and promote autophagy at the cellular level. Real-time quantitative PCR analysis confirmed that SAB treatment significantly increased TP53INP2 expression (Figure 4E). To further confirm the role of SAB in promoting cellular autophagy, we measured the protein expression levels of autophagy-related genes LC3B and p62. Western blot analysis revealed that SAB treatment significantly increased the LC3B-II/LC3B-I ratio and decreased p62 levels (Figures 4F–H). These results collectively indicate that SAB can effectively activate the cellular autophagy process. SAB targets senescent cells by enhancing TP53INP2-dependent autophagy (a key deficit in senescence), which in turn: (1) clears dysfunctional mitochondria to reduce ROS production and restore ATP metabolism; (2) suppresses mTOR-dependent SASP/inflammation; and (3) ultimately ameliorates age-related muscle dysfunction—without altering core senescence arrest markers (p16/p21, SA-β-Gal), consistent with its senomorphic identity. SAB ameliorates the muscle function and exercise capacity of aging mice.

To evaluate the efficacy of SAB in improving muscle function and exercise capacity in aging mice, we first established a premature aging mouse model through irradiation (Figure 5A) and administered SAB via intraperitoneal injection every 2 day. After irradiation, the average body weight of premature aging mice was approximately 5 g lower than that of young mice. Following 1 month of drug intervention, no significant weight gain was observed in the premature aging mice (Figure 5B). Subsequently, we evaluated the exercise capacity of the mice. In the treadmill test (Figure 5C), we compared the exercise performance and energy expenditure between the premature aging mice and young mice. The results revealed a significant decline in exercise capacity and energy expenditure in premature aging mice. Compared to solvent-injected mice, low-dose SAB treatment showed a trend toward improved exercise capacity and increased energy expenditure, while high-dose SAB treatment significantly enhanced exercise capacity and energy expenditure. In the grip strength test (Figure 5D), similar results were observed: the grip strength of the premature aging mice was lower than that of the young mice, and after SAB injection, the grip strength of the low-dose group increased slightly, while that of the high-dose group was significantly greater than the solvent group. Furthermore, analysis of HE-stained sections revealed that the gastrocnemius muscle cross-sectional area (CSA) was significantly smaller in premature aging mice than in young mice (Figures 5E,F). After SAB treatment, the low-dose group showed an upward trend in muscle CSA, while the high-dose group exhibited a significantly larger CSA than the solvent group. These results collectively indicate that SAB can effectively enhance muscle function and exercise capacity in aging mice.

To evaluate the efficacy of SAB in improving muscle function and exercise capacity in aging mice, we first established a premature aging mouse model through irradiation (Figure 5A) and administered SAB via intraperitoneal injection every 2 day. After intraperitoneal injection of SAB in mice, we conducted transcriptome sequencing analysis on the gastrocnemius muscle. The results revealed that 731 genes were significantly altered in the treated muscle tissue, including 33 downregulated and 698 upregulated genes (Figure 6A). KEGG enrichment analysis of the significantly upregulated genes identified the top 8 most significant pathways (Figure 6B), including the AMPK signaling pathway. Consistent with previous studies, activated AMPK is known to promote autophagy through multiple mechanisms. Additionally, AMPK enhances autophagy by inhibiting mTOR activity. Moreover, GO enrichment analysis of all significantly altered genes highlighted two pathways with significant changes: regulation of autophagy and positive regulation of autophagy (Figure 6D). Based on these findings, we propose that SAB can stimulate muscle autophagy. We also conducted KEGG enrichment analysis on the significantly downregulated genes and identified the top 8 most significant pathways (Figure 6C), including the TNF signaling pathway, suggesting that SAB may inhibit muscle inflammation. Furthermore, we measured the expression of Tp53inp2, a key autophagy regulator involved in muscle aging, and found that SAB treatment increased Tp53inp2 expression in mouse muscle (Figure 6E). We also analyzed SASP-related gene expression in muscle and observed that SAB treatment reduced the levels of Il6, Il1b, and Tgfb in mouse muscle (Figures 6F–H). These findings suggest that SAB ameliorates muscle aging by promoting autophagy through Tp53inp2 upregulation. Additionally, the anti-inflammatory and SASP-suppressing effects of SAB may contribute to the amelioration of muscle aging.

After intraperitoneal injection of SAB in mice, we conducted transcriptome sequencing analysis on the gastrocnemius muscle. The results revealed that 731 genes were significantly altered in the treated muscle tissue, including 33 downregulated and 698 upregulated genes (Figure 6A). KEGG enrichment analysis of the significantly upregulated genes identified the top 8 most significant pathways (Figure 6B), including the AMPK signaling pathway. Consistent with previous studies, activated AMPK is known to promote autophagy through multiple mechanisms. Additionally, AMPK enhances autophagy by inhibiting mTOR activity. Moreover, GO enrichment analysis of all significantly altered genes highlighted two pathways with significant changes: regulation of autophagy and positive regulation of autophagy (Figure 6D). Based on these findings, we propose that SAB can stimulate muscle autophagy. We also conducted KEGG enrichment analysis on the significantly downregulated genes and identified the top 8 most significant pathways (Figure 6C), including the TNF signaling pathway, suggesting that SAB may inhibit muscle inflammation. Furthermore, we measured the expression of Tp53inp2, a key autophagy regulator involved in muscle aging, and found that SAB treatment increased Tp53inp2 expression in mouse muscle (Figure 6E). We also analyzed SASP-related gene expression in muscle and observed that SAB treatment reduced the levels of Il6, Il1b, and Tgfb in mouse muscle (Figures 6F–H). These findings suggest that SAB ameliorates muscle aging by promoting autophagy through Tp53inp2 upregulation. Additionally, the anti-inflammatory and SASP-suppressing effects of SAB may contribute to the amelioration of muscle aging.

The data that support the findings of this study are openly available in NCBI database at https://www.ncbi.nlm.nih.gov/sra/PRJNA1241894↗.