Effect And Mechanism Of Astragalus Saponins On Primary Sarcopenia Induced By D-galactose

Objective:The effect of Astragaloside(AST)on d-galactose(D-gal)-induced skeletal muscle cell reduction and its mechanism were investigated by in vitro culture of mouse C2C12 myoblasts and in vivo experiment of C57BL/6 mice.Methods:Experiment 1: In vitro cell experiments to explore the effect of astragalus saponins on Dgalactose induced reduction of C2C12 cells and its mechanism.1.Cell counting kit-8:Mouse C2C12 myoblasts were cultured in vitro,and the 96-well cell culture plate was used for culture,proliferation and differentiation.Absorbance was detected by Cell counting kit-8(CCK-8)reagent to explore whether AST could promote proliferation and differentiation of C2C12 myoblasts treated with D-gal and the appropriate range of drug concentration for AST to interfere with C2C12 myoblasts.2.HE staining: ematoxylin-eosin staining(HE staining)was used to cultivate cells with24-well cell culture plates,and the effects of different concentrations of AST on D-gal induced skeletal muscle cell depletion model were observed from the aspects of cell morphology and cell muscular-tube fusion.3.Immunofluorescence technique: Cell sliders of C2C12 myoblasts were cultured in vitro and immunofluorescence labeling experiments were performed.The differentiation index was analyzed using Image J 1.48 software(National Institutes of Health,Bethesda,MD,USA).Myotube fusion was expressed by the percentage of myosin Heavy chain(MHC)positive cells.4.Western Blot: C2C12 myoblasts were treated with 20mg/ m L D-gal and with or without different concentrations of AST for intervention.Cells were cultured with 6-well cell culture plates to extract cell proteins.Western Blot(WB)was used to investigate the molecular mechanism of AST improving D-gal induced C2C12 myoblast reduction.5.Pathway inhibitor experiment: In D-gal + AST group,Akt inhibitor MK2206 and pAMPK inhibitor Compound C were added,and the correlation between the effect of AST on D-gal treated myoblasts and Akt activation and AMPK phosphorylation was verified by Western Blot analysis.6.Annexin V-FITC /PI staining: Annexin V-FITC /PI staining was used to investigate the effect of AST on myotube apoptosis of C2C12 myoblasts treated with D-gal.Experiment 2: In vivo animal experiment to explore the effect of astragalus saponins on D-gal induced primary sarcopenia in mice and its mechanismTwenty-four three-month-old male C57BL/6 mice with an average weight of about 30 g were selected for animal experiment.Mice were randomly divided into 4 groups(n=6),which were respectively set as normal saline control group,D-gal model group,D-gal + AST lowconcentration(10mg/kg)treatment group,and D-gal + AST high-concentration(20mg/kg)treatment group.D-gal model group,D-gal + AST low concentration(10mg/kg)treatment group,D-gal + AST high concentration(20mg/kg)treatment group were intraperitoneal injection of D-gal at 120mg/kg/day to induce the formation of primary sarcopenia model.Meanwhile,mice in control group were injected with the same volume of normal saline intraperitoneally for continuous modeling for 8 weeks.The modeling effect was based on the running table test and DXA scan data.Eight weeks later,the low-concentration D-gal + AST(10mg/kg)treatment group and the high-concentration D-gal + AST(20mg/kg)treatment group were treated with the corresponding concentration of AST,and the other animal groups were given the same volume of normal saline,intraperitoneal injection for 4 weeks.After a total of12 weeks of intervention,animal skeletal muscle was collected.Tibialis anterior(TA)muscle,quadriceps femoris and gastrocnemius muscles of each mouse were weighed,and the total weight and wet weight of each skeletal muscle tissue of each mouse were recorded.One side of the removed skeletal muscle tissue was immediately placed into GD tissue fixative for overnight fixation,paraffin embedding,tissue sections of 5μm were made,and HE staining was performed.Random visual field images of the upper,lower,left and right,and middle sections of each profile were taken.Image J software was used to calculate the percentage of skeletal muscle myotube positive area.Immunohistochemical staining was performed with p-Akt antibody and p-AMPK antibody diluted 1:100-1:250,and the corresponding protein expression level in TA muscle was analyzed by Image J software;Skeletal muscle tissue on the other side was immediately placed in liquid nitrogen and stored at-80℃.Later,tissue protein was extracted by tissue grinding and molecular mechanism analysis was performed by WB.Results:Experiment 1:1.Cell counting kit-8:C2C12 myoblasts were given 20mg/ml D-gal,and different concentrations of AST were detected.CCK-8 experiment showed that compared with the control group,the absorbance of cells treated with 20mg/ml D-gal was significantly reduced.The absorbance of C2C12 myoblasts treated with D-gal was significantly increased by AST intervention,and the absorbance of C2C12 myoblasts treated with D-gal was the highest when AST concentration was 0.08mg/ ml.Therefore,AST concentration in subsequent cell experiments was set to 0.08mg/ ml.2.HE staining: Microscopic observation of HE staining showed that compared with D-gal group,AST intervention in D-gal treated C2C12 myoblasts increased the myotube positive area,and the differentiation degree of myoblasts was significantly improved.3.Immunofluorescence staining: Immunofluorescence staining was used to label MHC factors,and the results showed that AST significantly improved D-gal induced myotube positive area compared with D-gal group.4.Western blot detection: WB results showed that the expression levels of muscle-specific proteins such as MHC,Myogenin and Myo D were significantly decreased and the expression of Myostatin was significantly increased compared with the control group,while AST significantly reversed the expression levels of the above protein factors,suggesting that AST promoted myoduct synthesis of C2C12 myoblasts.Compared with D-gal group,AST significantly increased the phosphorylation level of FOX3 a and decreased the protein expression of atrogin-1 and MURF1,suggesting that AST can reduce protein degradation of C2C12 myoblasts.Meanwhile,D-gal treated myoblasts reduced the levels of p-Akt/Akt,pMTOR/m TOR,p-P70S6K/P70S6 K and p-Foxo3a/Fox O3 a in the myoblasts,while AST improved the expression of these proteins,suggesting that AST promoted protein synthesis of C2C12 myoblasts.Moreover,AST can promote the expression levels of AMPK,SIRT-1 and PGC-1α,suggesting that AST can promote mitochondrial energy homeostasis of C2C12 myoblasts.5.Pathway inhibitor experiment: The results showed that compared with D-gal + AST group,the differentiation index of C2C12 myoduct was decreased after the addition of Akt inhibitor MK2206.In addition,the expression of p-m TOR /m TOR,p-P70S6 K /P70S6 K and p-Fox O3 A /Fox O3 a in myoducts was decreased to a certain extent after AST treatment.After the addition of p-AMPK inhibitor Compound C,the phosphorylation level of AMPK and the expression of SIRT1 and PGC-1α were significantly decreased compared with the D-gal + AST group.6.Annexin V-FITC /PI staining: Annexin V-FITC /PI staining showed that the apoptosis rate of D-gal group was significantly increased compared with the control group,while AST reduced the percentage of apoptosis induced by D-gal.Meanwhile,the expression of BAX,Caspase 3,and Bcl-2 genes related to myoblast apoptosis were detected by WB,and the results showed that AST inhibited BAX,and cleaved caspase-3 expression in D-gal induced C2C12 myotube,and upregulated bcl-2 expression.These results indicated that AST could improve the C2C12 myotube apoptosis induced by D-gal.Experiment 2:DXA scan results showed that D-gal significantly reduced the lean tissue proportion(expressed as the percentage of lean tissue in total body weight)of mice,and AST treatment significantly increased the lean tissue proportion of mice.The results of treadmill test showed that the optimal running time of mice in D-gal group was significantly reduced,and the optimal running time of mice was improved after AST treatment,suggesting that AST can improve the skeletal muscle endurance loss caused by D-gal.HE staining results of mouse TA showed that muscle fiber diameter in D-gal model group was smaller than that in control group,while AST could increase muscle fiber diameter in D-gal model group.Western blot results showed that AST increases the expression levels of MHC,Myogenin,and Myo D that are reduced by D-gal,and increases the phosphorylation levels of Akt,m TOR,S6 K,and Fox O3 a in mouse skeletal muscle tissue,and reduces D-gal-induced elevation of Bax and cleaved caspase-3.Conclusion:1.AST saponins can inhibit the reduction of C2C12 myoblasts induced by D-gal in vitro and promote the proliferation and differentiation of C2C12 myoblasts.2.AST saponins regulate protein synthesis and degradation through PI3K/AKT signaling pathway to improve D-gal induced primary sarcopenia.3.AST saponins promote mitochondrial energy homeostasis through AMPK/ SIRT-1/PGC-1α signaling pathway and improve D-gal induced primary sarcoma.4.AST saponins can improve muscle function in D-gal induced C57BL/6 sarcopenia mouse model.