二十五味鬼臼丸激活PI3K/Akt/mTOR介导的自噬缓解去势大鼠骨质疏松作用

Ershiwuwei Guijiu Pill Activates PI3K/Akt/mTOR-mediated Autophagy to Alleviate Osteoporosis in Ovariectomized Rats

目的:探讨二十五味鬼臼丸通过激活磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路抑制过度自噬防治绝经后骨质疏松(PMOP)症的作用机制。方法:将雌性SD大鼠通过手术去势,随机分为假手术(Sham)组、手术(OVX)组、二十五味鬼臼丸(GJ)组、盐酸雷洛昔芬(RLX)组,每组10只。用酶联免疫吸附测定法(ELISA)、比色法检测血清中雌激素、骨代谢标志物和胫骨组织中总超氧化物歧化酶(T-SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)水平,流式细胞术检测骨髓间充质干细胞内活性氧(ROS)水平,马松(Masson)染色观察胫骨近端病理变化,微计算机断层扫描技术(Micro-CT)观察胫骨微结构参数变化,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测胫骨组织中自噬效应蛋白(Beclin1)、微管相关蛋白1A/1B-轻链3(LC3)、微管相关蛋白5(Atg5)、PI3K、Akt、mTOR的表达情况。结果:与Sham组比较,OVX组大鼠血清雌二醇(E_(2))、钙离子(Ca^(2+))及骨组织T-SOD、GSH-Px、PI3K、Akt、mTOR mRNA明显下降(P<0.05,P<0.01);胫骨内骨密度(BMD)、骨表面积和骨体积比(BS/BV)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)、骨小梁连接性(Con)明显下降(P<0.05,P<0.01),骨骺生长板较薄,骨髓腔内充满脂肪空泡;磷(P)、MDA、ROS和Beclin1、LC3、Atg5 mRNA、蛋白水平及骨小梁分离度(Tb.Sp)明显升高(P<0.05,P<0.01)。与OVX组比较,GJ、RLX组大鼠血清E_(2)、Ca^(2+)及骨组织SOD、GSH-Px、PI3K、Akt、mTOR mRNA水平明显上升(P<0.05,P<0.01);胫骨内BMD、BS/BV、Tb.Th、Tb.N、Con明显升高,骨骺生长板相对增厚,骨髓腔内脂肪空泡明显减少(P<0.05,P<0.01);P、MDA、ROS、Beclin1、LC3、Atg5 mRNA、蛋白及Tb.Sp水平明显降低(P<0.05,P<0.01);p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR信号通路,在OVX组中显著降低(P<0.01),在GJ、RLX组中显著增高(P<0.01)。结论:二十五味鬼臼丸降低氧化应激抑制自噬,防治绝经后骨质疏松,其机制可能激活PI3K/Akt/mTOR信号通路从而抑制自噬有关。

Objective:To investigate the mechanism of Ershiwuwei Guijiu pill in preventing and treating postmenopausal osteoporosis(PMOP)by activating the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway and inhibiting excessive autophagy.Method:Female SD rats were ovariectomized and randomly divided into the sham operation group(Sham),the operation group(OVX),the Ershiwuwei Guijiu pill(GJ)group,and the raloxifene hydrochloride(RLX)group,with 10 rats in each group.Enzyme-linked immunosorbent assay(ELISA)and colorimetric methods were used to detect the levels of estrogen,bone metabolism markers in serum,and total superoxide dismutase(T-SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)in tibial tissue.Flow cytometry was used to detect reactive oxygen species(ROS)levels in bone marrow mesenchymal stem cells.Masson staining was used to observe pathological changes in the proximal tibia,and micro-computed tomography(Micro-CT)was used to observe changes in tibial microstructural parameters.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to detect the expression of autophagy-related proteins Beclin1,microtubule-associated protein 1A/1B-light chain 3(LC3),autophagyrelated 5(Atg5),as well as PI3K,Akt,and mTOR in tibial tissue.Result:Compared with the Sham group,the OVX group showed a significant decrease in serum levels of estradiol(E_(2))and calcium ion(Ca^(2+)),and T-SOD,GSH-Px,PI3K,Akt,and mTOR mRNA levels in bone tissue(P<0.05,P<0.01),significantly reduced bone mineral density(BMD),bone surface/bone volume(BS/BV),trabecular thickness(Tb.Th),trabecular number(Tb.N),and trabecular connectivity(Con)in the tibia(P<0.05,P<0.01),thinner epiphyseal growth plate,and the bone marrow cavity filled with fat vacuoles.Moreover,the levels of phosphorus(P),MDA,ROS,and mRNA and protein expression of Beclin1,LC3,and Atg5,as well as trabecular separation(Tb.Sp)were significantly elevated(P<0.05,P<0.01).Compared with the OVX group,the GJ and RLX groups showed significant increases in serum E_(2) and Ca^(2+),and bone tissue levels of SOD,GSH-Px,and the mRNA levels of PI3K,Akt,and mTOR(P<0.05,P<0.01),significantly increased BMD,BS/BV,Tb.Th,Tb.N,and Con in the tibia,thickened epiphyseal growth plate,and significantly reduced fat vacuoles in the bone marrow cavity(P<0.05,P<0.01).Additionally,the levels of P,MDA,ROS,Beclin1,LC3,Atg5 mRNA and proteins,and Tb.Sp were significantly decreased(P<0.05,P<0.01).The ratios of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR,which were significantly reduced in the OVX group(P<0.01),were significantly increased in the GJ and RLX groups(P<0.01).Conclusion:The Ershiwuwei Guijiu pill reduces oxidative stress and inhibits autophagy,thereby preventing and treating postmenopausal osteoporosis.Its mechanism may be related to the activation of the PI3K/Akt/mTOR signaling pathway,which inhibits autophagy.