Effect of Wenshen Tongluo Zhitong Recipe on the Intervention of Autophagy on Senile Osteoporosis Mice Model by AMPK/mTOR Signaling Pathway
Objective To study the intervention effects and mechanism of Wenshen Tongluo Zhitong Recipe(WTZR)on senile osteoporosis(SOP)model mice.Methods Sixty 6-month-old male C57BL/6J mice were randomly divided into six groups,i.e.,control group,model group,low-dose WTZR group,medium-dose WTZR group,high-dose WTZR group,and Alendronate Sodium group,10 mice in each group.The SOP mouse model was established by intraperitoneal injection of D-galactose(150 mg/kg per day for 8 weeks).After 8 weeks,mice in low,medium,and high dose WTZR groups were orally administered with 0.105,0.210,and 0.420 g/10 kg WTZR by gastrogavage every day.Mice in the control group and model group were administered with equal volume of physiological saline by gastrogavage.Mice in Alendronate Sodium group were administered with 1.530mg/kg Alendronate Sodium by gastrogavage every week.All administration lasted for a total of 12 weeks.Enzyme-linked immunosorbent assay(ELISA)was used to measure the serum levels of bone calmodulin-associated protein(OPG)and β-bone collagen cross-linked(β-CTX).Histopathological changes of the tibia were observed by HE staining.micro-CT was used to detect bone mineral density(BMD),bone volume fraction(BV/TV),trabecular(TB)number,TB thickness,and TB separation.Western Blot was used to detect the expression levels of p16,p53,Beclin-1,and microtubule-associated protein 1 in mouse femur bone marrow,as well as the levels of microtubule associated protein light chain 3-Ⅱ(LC3-Ⅱ),phosphorylated adenylate activated protein kinase(p-AMPK),AMP-activated protein kinase(AMPK),phosphorylated mammalian target protein of Rapa mold(p-mTOR),and mTOR protein.Results Compared with the control group,the serum level of OPG significantly decreased,while the level of β-CTX increased(P＜0.05,P＜0.01).The TB number decreased,TB thickness widened,the gap widened,narrowed,and broke more frequently.micro-CT scan results showed a significant reduction in the number of TB,TB thickness,BMD,BV/TV,andTB pattern factor in the femur tissue of the model group(P＜0.05,P＜0.01),while TB separation significantly increased(P＜0.01).The expression levels of age-related marker proteins p16,p53,and p-mTOR/mTOR ratio in the femur of the model group significantly increased(P＜0.05).The autophagy-associated proteins Beclin-1,LC3-Ⅱ,and p-AMPK/AMPK ratio significantly decreased(P＜0.01).Compared with the model group,the serum level of OPG in SOP model mice of medium/high dose WTZR groups and Alendronate Sodium group increased,while the level of β-CTX decreased(P＜0.05,P＜0.01).The expression levels of age-related marker proteins p16 and p53 in the femur significantly decreased(P＜0.05,P＜0.01).The expressions of Beclin-1 and LC3-Ⅱ proteins significantly increased(P＜0.05,P＜0.01).In the high-dose WTZR group and Alendronate Sodium group,the number of TB,TB thickness,BMD,BV/TV,and TB pattern factor significantly increased(P＜0.05),while TB separation significantly decreased(P＜0.05).Additionally,the p-AMPK/AMPK ratio significantly increased,while the p-mTOR/mTOR ratio significantly decreased(P＜0.01).Conclusions WTZR elevated BMD,improved bone microstructure,and stimulated bone formation in SOP mice.Its mechanism might be associated with regulating AMPK/mTOR signaling pathway,thus improving the autophagy level.
国家科技期刊平台
NSTL
万方数据
维普
