1 INTRODUCTION

As the human lifespan has significantly increased over the last century, so has the prevalence of age-associated diseases including malignant cancers.1 Aging is defined as a time-dependent decline in physiological functional associated with age-related conditions such as cardiovascular diseases, neurodegenerative diseases, and cancer. The capability to resist stress, damage, and disease degrades with age and most cells appeared as high levels of reactive oxygen species (ROS) and/or reactive nitrosative species.2 A growing body of evidence indicates that chronic inflammation and senescence are critical contributors to many age-related diseases. Chronic age-dependent inflammation, also known as “inflammaging,” is suspected as one of the main causes of biological aging.2 Cellular senescence is thought to play an important role in the aging process and senescent cells are characterized by several features including irreversible cell cycle arrest, elevated senescence-associated β-galactosidase (SA-β-gal) activity and the senescence-associated secretory phenotype (SASP).2, 3

Aging is the great risk factor for many liver disorders including nonalcoholic steatohepatitis and alcoholic, autoimmune, and cholestatic liver diseases.1, 3 Furthermore, older subjects are more vulnerable to viral infection that can accelerate the process of liver fibrosis.3 Senescent hepatocytes have been implicated in reduced liver regeneration and the etiologies of chronic liver diseases including cirrhosis and hepatocellular carcinoma (HCC).4

Macroautophagy (autophagy) is a lysosome-dependent catabolic mechanism that degrades cellular components to maintain cellular homeostasis under physiological and pathological conditions. This process supplies substrates for energy generation, plays a critical role in cell death and survival,5 and is associated with health and longevity.6, 7 Autophagy declines with age in many tissues including the liver.8 AMP-activated protein kinase (AMPK) is recognized as a critical regulator of energy metabolism, stress resistance, cellular proteostasis, and autophagy.9 One study demonstrated that the activation capacity of AMPK signaling declined with aging, which impaired cellular homeostasis and facilitates the aging process.10

Hepatocyte damage is a central mechanism involved in inflammation and disease progression in a variety of acute and chronic liver disorders.3 Inflammasomes are cytosolic protein complexes that play critical roles in host defense and cellular damage.11 Pyroptosis is a caspase-1-dependent form programmed cell death (also known as inflammatory necrosis) that depends on NOD-, LRR-, and pyrin domain-containing protein-3 (NLRP3) inflammasome formation.12 NLRP3 cleaves pro-caspase-1, then cleaved-caspase-1 transforms pro-interleukin (IL)-1β and pro-IL-18 into their mature forms (IL-1β and IL-18).13 Accumulating evidences indicate that NLRP3 inflammasome formation is stimulated by diverse triggers including adenosine triphosphate (ATP), cholesterol crystals, lipopolysaccharide, potassium efflux, mitochondrial DNA, lysosomal damage, and ROS.13

Astragalus polysaccharide (APS) is one of the extracts derived from Astragalus membranaceus9; this important component of herbal prescriptions has anti-inflammation,14 antioxidation, anti-hypertensive, and anti-tumor immune response effects.9

In the present study, we aimed to investigate the effects of APS on hydrogen peroxide (H2O2)-induced hepatocyte senescence and explore the molecular mechanism in vitro and in vivo. Particular emphasis was given to the novel mechanisms of cell senescence by detecting ROS levels, mitochondria function, pyroptosis, and apoptosis. Our results support a novel protective role of APS in ameliorating H2O2-induced hepatocyte senescence by activating autophagy signaling via the AMPK/mammalian target of rapamycin (mTOR) pathway, which could be exploited to promote healthy lifespan.

2 MATERIALS AND METHODS 2.1 Cell culture and treatment

In this study, Huh7 and LM3 cells were grown in Dulbecco's minimum essential medium (DMEM), while L02 cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines were kept in an incubator at 5% CO2, 37°C. Cells were primed with 100 μM APS for 48 h (Sigma, St. Louis, MO) prior to H2O2 exposure. A subset of cells was pretreated with the autophagy inhibitor chloroquine (10 μM for 2 h, Santa Cruz Biotechnology, Dallas, TX).

2.2 Animal treatment

Fifteen-month-old female C57BL/6 mice were purchased from the Beijing Zhishan Co. Ltd (Beijing, China). After adaptive feeding, the mice were randomly divided into three groups (n = 5 each): Control, APS, and APS + Chloroquine. In APS groups, mice were injected intraperitoneally with APS (200 mg/kg) for 7 consecutive days. Mice in the APS + Chloroquine groups were given intraperitoneal APS at the same dose (200 mg/kg) as well as chloroquine (80 mg/kg). Control mice were injected intraperitoneally with PBS. All animals were sacrificed at the indicated time points. Liver tissue samples were rapidly removed and stored in a −80°C freezer. All experimental procedures were approved by the laboratory animal ethical commissions of Shanghai Jiao Tong University (DWSY2021-011).

2.3 SA-β-galactosidase staining

After treating cells cultured in 24-well plates, SA–β-gal staining was performed with a kit (Solarbio, Beijing, China) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS and incubated in SA-β-gal solution (pH 6.0) for staining overnight at 37°C without CO2. Senescent cells were identified as blue-stained cells under light microscopy, a total of 1000 cells were counted in six random field to determine the percentage of SA-β gal-positive cells. For liver tissue staining, the tissue was embedded in optimal cutting temperature compound, sectioned at 7 mm and stained according to the manufacturer's instructions. The image was observed by light microscopy and the average number of positive cells was calculated every five high magnification field of view.

2.4 Cell viability assay

The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability according to the manufacturer's instructions (Dojindo, Kumamoto, Japan). L02, Huh7, and LM3 (5000/well) cells were seeded in 96-well plates overnight. To detect the effects of APS on viability, cells were first co-cultured with different concentrations (0, 10, 50, 100, 300 μM) of APS (Aladdin, Shanghai, China) for 48 h and then exposed to H2O2 for 24 h. At the prespecified time points, 10 μl of CCK-8 solution was added to the cells. After incubation for another 4 h, the optical density values were determined at 450 nm with a microplate reader (BioTek, Winooski, VT). Cell viability was calculated as (experimental group absorbance value/control group absorbance value) × 100%.

2.5 Intracellular ROS measurement

Intracellular ROS levels were measured with 2, 7-dichlorofluorescein diacetate (DCFH-DA) (Beyotime, Shanghai, China). Cells were seeded in 6-well plates overnight and subjected to different treatments. At the prespecified time points, cells were washed with sterile PBS, then incubated with 100 μl of 5 μM fresh DCFH-DA in PBS for 20 min at 37°C in the dark then washed twice with PBS. ROS concentrations were determined by a flow cytometer (Beckman Coulter, Brea, CA) and fluorescence microscopy (Leica, Wetzlar, Germany).

2.6 Apoptosis assay

Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kits (Invitrogen, Carlsbad, CA) were used to detect apoptosis ratios. Briefly, after treatment with H2O2 and APS, cells were washed with PBS and incubated with Annexin V-FITC and PI for 15 min at room temperature in the dark then detected by flow cytometer.

2.7 Transmission electron microscopy

After indicated treatment, L02 cells were harvested by centrifugation (1000 rpm for 5 min), sectioned, fixed with 4% glutaraldehyde overnight, dehydrated in a graded concentration of ethanol, and embedded in epoxy resin. Ultra-thin 70-nm sections were prepared and observed under transmission electron microscopy (TEM; EM 109, Carl Zeiss, Oberkochen, Germany).

2.8 Mito-Tracker green labeling

After treatment and washing in with PBS, cells were incubated with 50 nM Mito-Tracker Green for 30 min at 37°C to monitor mitochondrial content. Immediately after incubation, cells were photographed in fresh media under fluorescence microscopy.

2.9 Acridine orange staining

Acridine orange (AO; Invitrogen) was used to measure the number of acidic vesicular organelles (AVOs) in cells, which includes autophagolysosomes. After the indicated treatments, cells were incubated with AO (1 μg/ml) for 20 min at 37°C in the dark. After washing with PBS, a fluorescence microscope was used to observe the samples. Fluorescence intensity was obtained using the Image-Pro Plus6.0 software (Media Cybernetics, Rockville, MD).

2.10 Mitochondrial membrane potential measurement

After treatment, mitochondrial membrane potential (MMP) was assessed in cells using the JC-1 probe (Beyotime). Cells were incubated with JC-1 staining solution for 20 min at 37°C. After washing with PBS, the cells were subjected to detected by flow cytometry. Red and green fluorescence represent JC-1 aggregates and the monomeric form of JC-1, respectively. MMP was reflected by the ratio of red to green fluorescence intensity.

2.11 Enzyme-linked immunosorbent assay

Cells were seeded in 6-well plates and exposed to various treatments. After incubation, culture supernatants were collected, and concentrations of the inflammatory cytokines IL-6, IL-8, IL-1β, and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA) kits (Fushen, Shanghai, China) according to the manufacturer's instructions.

2.12 Histological analysis

Liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and cut transversely into 5-μm sections. Deparaffinized and rehydrated liver sections were stained with Masson's trichrome for pathological evaluation.

2.13 Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using RNA extraction kits (SparkJade, Shandong, China) according to manufacturer's instructions, and cDNA was synthesized using reverse transcriptase (ABClone, Wuhan, China). For analysis of indicated genes, a SYBR Green PCR kit (ABClone) was used to quantify relative mRNA levels, and the relative expression levels were calculated using the 2−ΔΔCT method. Real-time PCR primer sequences are shown in Table S1.

2.14 Western blotting

Protein was extracted using radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China) and quantified using Bicinchoninic Acid Protein Assay Kits (Pierce, Rockford, IL). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA). The membranes were incubated with the indicated primary antibodies overnight at 4°C: anti-p16 (ab25758, 1:1000), anti-p21 (1:1000), anti-p53 (1:1000), anti-p-AMPK (1:800), anti-AMPK (1:1000), anti-p-mTOR (1:1000), anti-mTOR (1:1000), anti-ASC (1:1000), anti-caspase-1 (1:1000), anti-cleaved-caspase-1 (1:1000), anti-NLRP3 (1:1000) and anti-gasdermin D (GSDMD, 1:1000) all purchased from Abcam (Cambridge, UK). β-actin (1:1000; Cell Signaling Technology, Danvers, MA) was used as the loading control. Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Absin, Shanghai, China), and exposed to enhanced chemiluminescent (ECL) reagent (NCM Biotech, Suzhou, China) for band detection. Immunoreactive bands were visualized using ECL detection reagent (Beyotime). Band intensity was quantified with the ImageJ software (National Institutes of Health, Bethesda, MD).

2.15 Statistical analysis

All experiments were repeated at least three times. Data are presented as mean ± standard deviation (SD) and were statistically analyzed by one-way analysis of variance (ANOVA) using the GraphPad 8.0 statistical software (GraphPad Inc., San Diego, CA). Differences were considered significantly different at p <0.05.

3 RESULTS 3.1 H2O2 induced cell senescence and the optimal protective concentration of APS

Cells were treated with H2O2 to mimic damage caused by oxidative stress. The human normal hepatocyte line L02 and hepatoma cell lines Huh7, LM3 were treated with different concentrations of H2O2 for 24 h. The concentration of H2O2 to induce cell senescence was relatively low, and too high of a dose could easily induce cell death. The results showed that 220 nM for L02 cells, 880 nM for LM3 cells, and 440–660 nM (we chose 660 nM) for Huh7 cells were the suitable concentrations, so these were used in subsequent experiments. Huh7 cells were incubated with 660 nM H2O2 for 6, 12, and 24 h. Senescent cells were observed at 12 and 24 h (Figure S1A). Next, three types of cells were treated with different concentrations of APS (0, 10, 50, 100, and 300 μM) for 48 h followed by induction with H2O2 for 24 h. The CCK-8 assay results showed that 100 μM APS exerted the strongest effect to resist cell cycle arrest (Figure S1B). The cell viabilities of all groups treated with APS were higher than those without APS, indicating that APS had protective effects against H2O2-induced damage.

3.2 APS ameliorated H2O2-induced cell senescence

Three cell lines were treated with 100 μM of APS for 48 h followed by 24 h of incubation with H2O2. mRNA expression levels of specific senescence markers (p16, p53, p21, IL-6, IL-8) were dramatically upregulated in the H2O2-treated group compared with Control group, but the effect was markedly decreased in the H2O2 + APS group (Figure 1A). To further confirm our results, the relative protein expressions were detected by Western blotting (WB) and ELISA. The results showed the cytokines IL-6 and IL-8 (Figure 1B) and protein levels of p16, p53, and p21 (Figure 1C,D) were increased in the H2O2-treated group compared with Control group, but the promoting effect was markedly decreased by APS in L02 and Huh7 cells. The percentage of SA-β-gal positive cells decreased in the presence of APS compared with H2O2 group (Figure 1E,F). Taken together, these data indicated that APS alleviated H2O2-induced liver cell impairment.

Astragalus polysaccharide alleviated cell senescence. (A) Relative mRNA expression levels of p16, p53, p21, IL6, and IL8 were measured by qRT-PCR. (B) Supernatant levels of IL6 and IL8 were measured by ELISA. (C,D) Representative western blotting and quantification of protein expression levels of p16, p53, and p21 were measured by western blotting. (E,F) Representative images showing SA-β-gal-positive cells. (*p <0.05, **p <0.01)

3.3 APS ameliorated cell death

Apoptosis plays an important role during liver aging and is related to many forms of liver dysfunction and disease.15, 16 The chronic, low-grade, sterile form of inflammation has been proposed as a mediator of human health span and aging.17 Pyroptosis is another form of programmed cell death distinct from apoptosis that involves the release of proinflammatory intracellular contents.5 We observed a significant increase in the percentage of apoptotic cells in the H2O2 group compared with the control group, but this was significantly attenuated in the presence of APS (Figure 2A,B). Furthermore, we clarified the effect of APS on pyroptosis by detecting the expression of NLRP3, ASC, IL-18, IL1β, caspase-1, and GSDMD using ELISA, qRT-PCR, and WB. The expression levels of all of these markers were significantly increased in the H2O2 group compared with control group, but APS reversed these effects (Figure 2C–E).

Astragalus polysaccharide ameliorated cell death. (A,B) Apoptosis rate of each group was evaluated by flow cytometer. (C,D) Representative western blotting and quantification of pyroptosis-related proteins: pro-GSDMD, cleaved-GSDMD, pro-caspase-1, cleaved-caspase-1, ASC, and NLRP3. (E) Quantification of inflammatory cytokines IL-18 and IL-1β in supernatant by ELISA. (*p <0.05, **p <0.01)

3.4 APS reduced ROS and protected mitochondria

Considering the remarkable in vitro effects of APS, we next investigated the detailed mechanism. High level of ROS is potentially critical for the induction and maintenance of cell senescence.2 Intracellular ROS levels were highest in the H2O2 group detected by flow cytometry (Figure 3A,C) and fluorescence microscopy (Figure S2). However, ROS levels were reduced in APS group. Mitochondria produces the majority of cellular energy and is also the primary source of ROS.18 As appropriate MMP is necessary for mitochondrial oxidative phosphorylation and ATP production,19 we measured MMP to investigate the effects of APS on mitochondrial homeostasis using the JC-1 probe. As shown in Figure 3B,D, H2O2 reduced the ratio of red to green fluorescence, indicating MMP polarization, while APS increased the ratio of red to green fluorescence and abolished the H2O2-induced damage of mitochondrial. Mitochondrial morphology was assessed with the MitoTracker Green probe. Mitochondria in the Control group exhibited tubular networks with smooth morphology; however, cells exposed to H2O2 had highly fragmented mitochondria in a discontinuous network (Figure 3F). Ultrastructural TEM analyses showed significant accumulation of damaged mitochondria that were longer and larger with swollen and disrupted cristae, and the number of mitochondria was reduced in the H2O2 group (Figure 3E). Notably, APS normalized mitochondria morphology, suggesting that it was able to alleviate damage.

Astragalus polysaccharide reduced ROS and protected mitochondria. (A,C) The ROS content in each group was detected by flow cytometry. (B,D) MMP in each group was detected by flow cytometry. (E) TEM showing the ultrastructural features of intracellular mitochondria. (Arrows: mitochondria; scale bar = 1 μm, 500 nm). (F) Mitochondria morphology was observed with Mito-Tracker Green. (scale bar: 100 μm. *p <0.05, **p <0.01)

3.5 APS alleviated cell injury by activating AMPK/mTOR to enhance autophagy

Mitophagy (a form of autophagy) was recently reported to play beneficial roles in eliminating damaged and unhealthy mitochondria and maintaining organelle quality and quantity.20 Damaged mitochondria can generate secondary ROS and perpetuate oxidative insults, which negatively affects healthy mitochondrial.21, 22 Therefore, timely and efficient removal of damaged mitochondria by mitophagy is crucial for mitochondrial quality control and maintenance of normal cell functions. We detected relative autophagy proteins by WB. LC3 conversion and p62 protein levels are considered the most reliable hallmarks of autophagy.7 APS significantly increased conversion of LC3-I to LC3-II and downregulated p62 expression compared with the H2O2 group (Figure 4A,B). AO staining was used to measure the number of AVOs in cells (bright red fluorescence) and evaluate cell death patterns. Cells in the H2O2 group exhibited bright congregated green fluorescence, a phenomenon of early apoptosis, because of congregated chromatin and nucleolus pyknosis (Figure 4C,D). The bright red fluorescence was most evident in the APS group, indicating that APS increased the number of autophagolysosomes, while the uniform green fluorescence showed that APS alleviated apoptosis.

Astragalus polysaccharide alleviated cell injury by activating AMPK/mTOR to enhance autophagy. (A,B) Representative western blotting and quantification of protein expression of LC3b, p62, Beclin-1, and ATG5 expressions. (C) Autophagolysosome formation was measured by AO staining. (Scale bar = 1 μm, 500 nm). (D) Quantification of autophagolysosome formation. (E) Ultrastructural features of autophagosomes on TEM. (Scale bar = 1 μm, 500 nm). (F,G) representative western blotting and quantification of protein expression of p-AMPK, AMPK, p-mTOR, and mTOR levels. (*p <0.05, **p <0.01)

To further confirm that APS induced autophagy, TEM was performed to observe autophagosome formation in L02 cell line. As shown in Figure 4E, we observed fewer autophagosomes in H2O2-treated cells and abundant autophagosomes in the cytoplasm of APS-treated cells. These results suggested that APS increased autophagosome formation. We also detected the expression of several autophagy-related proteins to confirm the effect of APS in three cell lines. The results showed that LC3 II, Atg5, and Beclin-1 expression increased with APS treatment. These data indicated that APS induced autophagy activation in liver cells. To further confirm the effect of autophagy on hepatocyte senescence, we used chloroquine to inhibit autophagy. The results showed chloroquine weakened the protective function of APS: expression levels of p16, p21, p53, IL-6, and IL-8 and SA-β-gal staining were increased compared with the APS group (Figure 1), and ROS levels and MMP were upregulated (Figure 3).

3.6 APS alleviated liver aging in vivo

To further confirm the protective effect, we intraperitoneally administrated APS to aged mice for 1 week prior to liver tissue collection. APS-treated mice showed significant reductions in expression levels of p16, p53, p21, IL-6, and IL-8 compared with PBS-treated mice (Figure 5A,E,F) as detected by qRT-PCR and WB. The number of SA-β-gal-positive cells also reduced with APS treatment (Figure 5H,I). Senescence in hepatocytes is well documented in cirrhosis,4 so we also measured the degree of fibrosis. Massive cytoplasmic vacuolation and fibrosis were observed between the portal tracts and around the central vein, and both changes were alleviated by APS (Figure 5G). The relative levels of pyroptosis-related proteins NLRP3, ASC, cleaved-caspase-1, cleaved-GSDMD, and cytokines IL-18, IL1β were lower in the APS group (Figure 5B–D), while LC3II expression increased. Chloroquine cotreatment increased the expression of p16, p53, p21, IL-6, and IL-8, which attenuated the protective effect of APS (Figure 5E,F). APS also increased p-AMPK levels and reduced expression of p-mTOR, suggesting that APS activated AMPK and inhibited mTOR to induce autophagy (Figure 5E–G).

Astragalus polysaccharide alleviated cell senescence in mouse liver in vivo. (A) p16, p53, p21, IL6, and IL8 gene expressions were measured by qRT-PCR. Average target gene/GAPDH ratios were plotted (n = 5 mice/group). (B,C) Representative western blotting and quantification of pyroptosis-related proteins including pro-GSDMD, cleaved-GSDMD, pro-caspase-1, cleaved-caspase-1, ASC, and NLRP3. (D) IL1β and IL18 gene expression levels were measured by qRT-PCR. (E,F) Representative western blotting and quantification of autophagy-related proteins including LC3b, P62, Beclin-1 and ATG5, p-AMPK, AMPK, p-mTOR, and mTOR. (G) Masson's trichrome-stained liver tissue (scale bar = 100 μm). (H,I) SA-β-gal-positive cells in liver sections. Yellow arrows: SA-β-gal positive cells (scale bar = 100 μm). (*p <0.05, **p <0.01)

4 DISCUSSION

Advances in science, medicine, and public policy have dramatically increased the number of people surviving into old age, which has led to considerable societal challenges. Indeed, aging is the single greatest risk factor for most complex chronic diseases, which carry profound social and economic costs.23, 24 Therefore, the pace of population aging is an important healthcare and social issue. Identifying new approaches to regulate the biology of aging could significantly control disease and improve the health of the elderly.1, 23

Senescence is not static; it is a progressive process that begins with a presenescent status and shifts to full senescence. p16 and/or p53–p21 pathways are classical avenues to reach stable cell-cycle arrest accompanied by production of SASP-associated proteins (IL-6, IL-8).23 We detected these markers as well as SA-β-galactosidase expression to confirm that H2O2 successfully induced senescence.25

APS is a natural product as a traditional Chinese medicine to treat cardiovascular diseases, immune system disease and cancer.14, 26 The effect of APS may be dose-dependent, with low concentrations (e.g., 0.1 μg/ml) providing cell protection,27 and high concentrations (e.g., 1000 μg/ml) inducing a direct killing effect and improving immune function.26

In our study, we employed three types of hepatocytes: L02 (immortalized liver cells), Huh7 (immortal cell line composed of epithelial-like, tumorigenic cells), and LM3 (metastatic HCC cells). Although our experiments were not performed in primary human hepatocytes, these cell lines recapitulate the condition of the aging liver. Concentration of genotoxic agents that trigger senescence to different types of cells are not equal.28 LM3 had the strongest ability to resist H2O2-induced injury. It is controversial whether senescent cells arrest or promote tumor cell growth. SASP-associated proteins released by senescent cells should be considered effective weapons against pretumorigenesis events rather than an anti-cancer mechanism acting on malignant cells28 given our finding that the L02 and Huh7 cell lines are more sensitive than LM3. Besides, LM3 produced minimal ROS in response to H2O2. Our results showed that APS alleviated cell senescence in all three types of cells. Nevertheless, p53 expression was not upregulated in the LM3 line treated with H2O2, possibly due to the high self-renewal capacity of LM3 and large functional p53 network.12, 29 In addition, certain senescence-like phenotypes may not reflect a terminal state of growth arrest, and cells with self-renewal capacity may ultimately contribute to disease recurrence.30

Several recent studies indicated that autophagy is substantially lower in the aged liver compared to the young liver.31 Our results demonstrated an essential function of APS in autophagy. It facilitated the removal of activated mitochondria and helped control oxidative metabolism, thereby delaying cell senescence. ROS, mitochondria, autophagy, and cell death (pyrolysis, apoptosis) are inextricably linked (Figure 6). Loss of mitochondrial homeostasis leads to high ROS levels.3 Excessive ROS production causes oxidative modifications in adjacent mitochondria.16 Pyroptosis and apoptosis are mediated by mitochondrial dysfunction and subsequent ROS production. Autophagic failure can result in accumulation of dysfunctional mitochondria that contribute to bioenergetic deficits and exacerbate oxidative stress.22 Autophagy influences mitochondrial recycling and can thus modulate hepatic apoptosis via the mitochondrial pathway. Inflammasome-dependent caspase-1 activation promotes pyroptotic cell death and proinflammatory cytokine secretion.32 Excessive inflammasome activation induces sterile inflammation that can eventually lead to acute or chronic hepatitis and liver fibrosis.13 A recent review described findings from several studies that have shown that reduced regenerative capacity of the aged remnant liver can be restored by promoting autophagy.1 To date, research into cell senescence mechanism has mainly concentrated on pro-inflammatory effects, oxidative stress, telomere shortening, DNA damage accumulation, abnormal oncogene activities, metabolic alterations, and excessive ROS generation. Given the important role of mitophagy in senescence, we speculated that APS may increase mitophagy to reduce cell senescence by eliminating ROS, reducing pyrolysis and apoptosis, and maintaining normal cellular metabolism. The APS groups showed reduced ROS (Figure 3A,C), less mitochondrial damage (Figure 3B,D,E,F), decreased pyroptosis and apoptosis (Figure 2), and increased autophagy (Figure 4). However, these effects were weakened by the autophagy inhibitor chloroquine, further highlighting that APS could induce autophagy and perhaps delay aging.

Schematic showing how Astragalus polysaccharide alleviates hepatocyte senescence

AMPK mediates metabolic changes to resist ROS accumulation.3 Previous studies reported that AMPK activation was a key contributor to APS-induced insulin sensitivity in adipocytes,9 APS activated the PI3K/AKT/mTOR pathway to increase cellular autophagy to slow the development of Parkinson disease,27 and APS alleviated pyroptosis of Leydig cells by promoting autophagy via the ROS–AMPK–mTOR axis.5 AMPK plays an important role in the autophagy process by remodeling transcriptional networks to control mitochondrial biogenesis and turnover via mitophagy.33, 34 We observed activation of AMPK with inhibition of mTOR, suggesting that APS may enrich autophagy via AMPK/mTOR pathway.

In conclusion, our results demonstrated that APS increased autophagy by upregulating AMPK/mTOR signaling to remove activated mitochondria, control oxidative metabolism, reduce cell death, and delay cell senescence.

CONFLICT OF INTEREST

All authors declare no conflict of interest.