Role And Mechanism Of TLR9-mediated Skeletal Muscle Fibrosis In Sarcopenia

Objective:1.To investigate the effect of TLR9 on skeletal muscle mass and strength in sarcopenic mice.2.To clarify the role of TLR9 activation in skeletal muscle fibrosis in sarcopenic mice.3.To investigate the protective effect and molecular mechanism of Sirt1 in collagen deposition of skeletal muscle in sarcopenic mice.Methods:1.Twenty male C57 mice were divided into 2 groups,the young control group(YC group,2 months old)and the old control group(OC group,18 months old),10 in each group.Thirty male TLR9 KO mice were divided into 3 groups,young TLR9 KO group(YT group,2 months old),old TLR9 KO group(OT group,18 months old),old TLR9KO+Ex527group(OTE group,18 months old).2.The OTE group received Ex527(5 mg/kg)intraperitoneal injection,and the other groups received the same amount of normal saline for 6weeks.3.The maximum grip strength of the mice was measured by electronic grip strength meter.The body composition of the mice was analyzed by DXA,and the weight of gastrocnemius muscle and body weight of the mice were measured by analytical balance.4.ECM related proteins in the gastrocnemius of mice,and the fibrosis of gastrocnemius muscle was measured by Western blot,Sirius red staining and immunohistochemistry staining.5.After treatment with the Sirt1 inhibitor Ex527 in the OTE group,the expression of protein and mRNA in the TLR9-Sirt1 mediated fibrosis related pathway was detected by Western blot and qPCR.Results:1.The muscle mass and grip strength in the sarcopenic mice constructed by natural aging were significantly lower than those in the young control group.Sirius red staining and Western blot results showed that the fibrosis was manifested in the gastrocnemius muscle tissue.It suggests that skeletal muscle fibrosis may be the cause of the decrease in skeletal muscle mass and strength in sarcopenic mice.2.Immunohistochemistry and Western blot showed that TLR9 was highly expressed in a sarcopenic mice.Pearson correlation analysis showed that the expression of TLR9 was highly linearly related to fibrosis-related proteins.It suggests that aging may cause abnormal activation of TLR9 and further lead to skeletal muscle fibrosis.3.Analysis of body composition,grip strength and gastrocnemius weight in mice of YC,YT,OC and OT groups showed that knocking out TLR9 could significantly improve the gastrocnemius muscle mass,the gastrocnemius muscle mass index,and the lean mass/fat mass ratio,as well as absolute and relative grip caused by aging.This change is not apparent in young mice.It suggests that TLR9 may be the cause of the onset of sarcopenia in aged mice.4.Western blot,Sirius red and immunohistochemical staining of OC group and OT group showed that after TLR9 knockout,the levels of ECM related protein and skeletal muscle fibrosis in aged mice were significantly decreased,suggesting that TLR9 could be one of the cause of sarcopenia.An important cause of muscle fibrosis,and inhibition of TLR9 may be an effective means to reduce ECM deposition in mice with sarcopenia.5.Western blot and Sirius red staining of OC group,OT group and OTE group showed that inhibition of Sirt1 reversed the improvement of skeletal muscle fibrosis in mice by TLR9 knockout,suggesting that Sirt1 was a key regulatory proteinin in the fibrosis process.6.Western blot and qPCR in OC group,OT group and OTE group showed that TLR9 knockout could significantly increase the expression and activity of Sirt1,while Sirt1 could improve tissue fibrosis by inhibiting TGF-?/Smad pathway.Inhibition of Sirt1 reversed the protective effect of knockout of TLR9 and increased the expression of TGF-?/Smad pathwayrelated proteins.Conclusion:Aging causes abnormal activation of TLR9 and further lead to skeletal muscle fibrosis Skeletal muscle fibrosis may be responsible for the decrease in skeletal muscle mass and strength in sarcopenic mice.TLR9 could accelerate the progression of skeletal muscle fibrosis in sarcopenic mice by inhibiting Sirt1 up-regulating the expression of TGF-?/Smad pathway related proteins.Inhibition of TLR9 may be an effective means to reduce ECM deposition and improve the symptoms in sarcopenic mice.