左归丸下调自噬抑制破骨分化治疗老年性骨质疏松症

Zuogui Pills down-regulating autophagy to inhibit osteoclast differentiation in the treatment of senile osteoporosis

目的:探讨左归丸通过下调自噬抑制小鼠骨髓源性巨噬细胞(BMMs)破骨分化治疗老年性骨质疏松症(SOP)。方法:构建SOP小鼠模型,随机将20只小鼠分为2组,分别是衰老组(SOP组)和左归丸治疗组(左归丸组)。左归丸治疗2个月后,Micro-CT检测骨体积分数(BV/TV),TRAP染色检测骨小梁表面破骨细胞(OCs)数量,免疫组化(IHC)检测骨组织中P-BECLIN1的表达情况。提取小鼠BMMs,用不同浓度左归丸干预BMMs的破骨分化过程,TRAP染色观察OCs数量,RT-qPCR检测自噬标志基因Atg5、Atg12、LC3、P62 mRNA表达水平,Western Blot检测细胞中NFATC1、CTSK、LC3、P62和P-BECLIN1的蛋白表达水平。结果:体内实验中,与SOP组比较,左归丸组BV/TV明显增高(P<0.01);TRAP染色提示左归丸组骨小梁表面OCs数量明显少于SOP组(P<0.01);IHC检测发现左归丸组小鼠骨髓腔中P-BECLIN1表达量较SOP组显著下调(P<0.01)。体外实验中,与对照组比较,10、100、1000μg/mL左归丸均可抑制RANKL诱导的BMMs破骨分化(P<0.01),在抑制破骨分化过程中,自噬基因Atg5、Atg12、LC3 mRNA表达显著下降(P<0.05,P<0.01),P62 mRNA表达显著上升(P<0.01)。Western Blot显示左归丸可以下调NFATC1、CTSK、LC3、P-BECLIN1的蛋白表达,上调P62的蛋白表达。结论:左归丸下调自噬,抑制OCs形成,为临床治疗SOP提供新的思路。

Objective:To explore the effects of Zuogui Pills(ZGW)on senile osteoporosis(SOP)by down-regulating autophagy and inhibiting bone marrow-derived macrophage(BMMs)osteoclast differentiation in mice.Methods:The mouse model of senile osteoporosis was established and 20 mice were randomly divided into two groups:aging group(SOP group)and Zuogui Pills treatment group(ZGW group).After 2 months of treatment with Zuogui Pills,bone volume fraction(BV/TV)was measured by Micro-CT,the number of osteoclasts on the trabecular surface was detected by TRAP staining,and the expression of P-BECLIN1 in bone tissue was detected by immunohistochemistry(IHC).Mouse BMMs was extracted,and different concentrations of Zuogui Pills were used to interfere with the osteoclast(OCs)differentiation process of BMMs.The number of OCs was observed by TRAP staining,the mRNA expression of autophagy marker genes Atg5,Atg12,Lc3 and p62 was detected by RT-qPCR,and the protein expression levels of NFATC1,CTSK,LC3,P62 and P-BECLIN1 in cells were detected by Western Blot.Results:In vivo experiment,BV/TV in ZGW group was significantly higher than that in SOP group(P<0.01),the number of OCs on trabecular surface in ZGW group was significantly less than that in SOP group(P<0.01),and the expression of P-BECLIN1 in bone marrow cavity of ZGW group was significantly lower than that of SOP group(P<0.01).In vitro experiment,compared with the control group,10,100,1000μg/mL Zuogui Pills could inhibit the OCs differentiation of BMMs induced by RANKL(P<0.01).During the inhibition of OCs differentiation,the mRNA expression of autophagy genes Atg5,Atg12 and LC3 was decreased(P<0.05,P<0.01),while the mRNA expression of P62 was increased(P<0.01).Western Blot suggested that Zuogui Pills could inhibit the protein expression of NFATC1,CTSK,LC3 and P-BECLIN1 and up-regulate the protein expression of P62.Conclusion:Zuogui Pills can down-regulate autophagy and inhibit OCs formation,which can treat senile osteoporosis.Zuogui Pills is a potential drug for the treatment of SOP and provides a new idea for clinical treatment of SOP.