| The growth and development of skeletal muscle is regulated by a variety of factors and signal pathways.It has a strong ability to regenerate,the regeneration of skeletal muscle depends on the ability of skeletal muscle satellite cells to exit the resting state,asymmetric division,proliferation and differentiation.As muscle-derived stem cells,skeletal muscle satellite cells have a repairing effect on damaged muscle tissue.Metformin is a specific drug for the treatment of type 2 diabetes,which can maintain the body’s blood sugar steady state in a variety of ways.In addition,metformin also plays a role in the treatment of obesity,various cancers and anti-aging.The aging process is usually accompanied by some skeletal muscle diseases,such as skeletal muscle reduction.,which indicates that metformin may also have a certain regulatory effect on the growth and development of skeletal muscle.Therefore,this study used bovine skeletal muscle satellite cells(BSMSCs)to induce myogenic differentiation in vitro to simulate the growth and development of skeletal muscle in vivo,and screened the optimal concentration of metformin by treating BSMSCs with different concentrations of metformin,and explored its effects on the proliferation and differentiation of BSMSCs and related mechanism;Due to the difficulty of handling on cattle,mice are further used to carry out in vivo verification experiments,aiming to provide some references for the muscle development and differentiation of cattle and the repair of muscle damage.1.Screening the optimal concentration of metformin to explore its effect on the proliferation and differentiation of BSMSc.First,the CCK-8 experiment was used to screen the optimal concentration of metformin on BSMSc.Then,real-time fluorescent quantitative PCR(qRT-PCR),Western blot,EdU cell proliferation experiment,cell growth and differentiation state were used to further explore its effects on the proliferation and differentiation of BSMSc.The results show that the optimal concentration of metformin on BSMSc is 2 mmol/L,which can inhibit the proliferation and differentiation of BSMSc.2.Explore the relevant mechanisms of metformin regulating the proliferation and differentiation of BSMSCs.Muscle satellite cells are muscle-derived stem cells,and the process of regulating the growth and development of skeletal muscle requires the participation of energy metabolism.Early experiments found that metformin regulates the proliferation and differentiation of bovine skeletal muscle satellite cells mainly due to changes in protein levels,indicating that protein metabolism is involved in the process of metformin regulating the proliferation and differentiation of bovine skeletal muscle satellite cells.Western blot was used to detect the changes in the expression of AMPK/mTOR/RPS6 signaling pathways in protein metabolism pathways at different stages after metformin treatment of BSMSCs,and explore the possible mechanism of metformin regulating bovine skeletal muscle satellite cells.The results showed that the levels of p-AMPK in the proliferation and differentiation phases of BSMSCs were significantly increased after metformin treatment,and the levels of p-RPS6 were significantly decreased,indicates that metformin may regulate the proliferation and differentiation of bovine skeletal muscle satellite cells by activating the AMPK/mTOR/RPS6 signaling pathway.In order to further study the mechanism of metformin regulating the proliferation and differentiation of bovine skeletal muscle satellite cells,the siRNA of RPS6 was designed and synthesized to detect the effect of interference with RPS6 on the proliferation and differentiation of bovine skeletal muscle satellite cells.The results found that interference with RPS6 also inhibited the proliferation and differentiation of BSMSCs.The above results indicate that metformin can activate AMPK to phosphorylate the active GTP-bound mTOR activator(Rheb)into an inactive GDP-bound form,thereby negative regulation of rapamycin target protein 1(mTORCl),which inhibits the mRNA translation process by inhibiting the phosphorylation of its downstream ribosomal S6 protein(RPS6),thereby inhibiting protein synthesis,play a role in regulating the proliferation and differentiation of bovine skeletal muscle satellite cells.3.In vivo experiments in mice verify the effect of metformin on skeletal muscle development.Ten 6-week-old SPF Kunming female mice weighing(25±0.4)g were selected,after one week of adaptive breeding,they were injected with 30 mg/kg metformin diluent into the muscle of the right thigh,and the same amount of physiological saline was injected on the left side,weighing and injection were performed every 3 days,after one month,the mice were sacrificed and the gastrocnemius muscle was taken to weigh,then the gastrocnemius muscle is prepared for tissue sectioning and RNA and protein extraction operations.The results showed that the gastrocnemius muscle wet weight and muscle fiber diameter of the metformin group were significantly lower than those of the control group;Western blot experiments also found that the skeletal muscle proliferation and differentiation marker factors of the metformin group were significantly lower than those of the control group,and the p-AMPK level was significantly increased,p-RPS6 levels are significantly reduced,the experimental results tend to be consistent with in vitro experiments,indicating that metformin can also regulate the growth and development of mouse skeletal muscle by activating the AMPK/mTOR/RPS6 signaling pathway in vivo.In summary,this study found that metformin can regulate the proliferation and differentiation of BSMSCs by activating the AMPK/mTOR/RPS6 signaling pathway.In vivo experiments show that it can also regulate the growth and development of mouse skeletal muscle by activating the AMPK/mTOR/RPS6 signaling pathway. |
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