基于PI3K/AKT/GSK‐3β信号通路的杜仲健骨方治疗骨质疏松症作用机制研究

Study on the mechanism of Duzhong Jiangu decoction in the treatment of osteoporosis based on the PI3K/Akt/GSK⁃3βsignaling pathway

目的基于PI3K/AKT/GSK‐3β信号通路探讨杜仲健骨方治疗骨质疏松症的作用机制。方法将60只SD大鼠按随机数字表法分成6组,每组10只,分别为:空白组、杜仲健骨方高(4.86 g/kg)、中(2.43 g/kg)、低剂量组(1.215 g/kg)、阿仑膦酸钠组(阳性药,1 mg/kg)、模型组(生理盐水),摘除双侧卵巢造模,制备骨质疏松症模型。空白组仅翻动两侧卵巢,不做任何处理。给药8周后,主动脉采血,ELISA法测定各组血清抗酒石酸酸性磷酸酶(Str ACP)、肿瘤坏死因子‐α(TNF‐α)、白介素‐1β(IL‐1β)、丙二醛(MDA)含量,以及超氧化物歧化酶(SOD)活性;骨密度扫描仪测定大鼠股骨骨密度值;实时荧光定量聚合酶链式反应(RT‐PCR)和蛋白免疫印迹法(Western blot)检测磷酸肌醇3‐激酶(PI3K)、蛋白激酶B(Akt)、糖原合成酶激酶‐3(GSK‐3β)mRNA和蛋白表达。结果模型组大鼠骨密度值、血清中Str ACP、TNF‑α、IL‐1β、MDA、SOD的水平,PI3K、AKT、GSK‐3βmRNA和蛋白表达,与空白组比较差异均有统计学意义(P<0.01);与模型组比较,杜仲健骨方剂量组能够提高骨密度值,血清SOD活性显著升高(P<0.05,P<0.01),血清TNF‐α、IL‐1β、Str ACP、MDA含量显著降低(P<0.05,P<0.01);与模型组比较,杜仲健骨方各剂量组中PI3K、AKT、GSK‐3βmRNA和蛋白表达差异均有统计学意义(P<0.05,P<0.01)。结论杜仲健骨方对骨质疏松症的治疗作用可能是通过调控信号通路PI3K/AKT/GSK‐3β来实现的。

Objective To investigate the mechanism of Duzhong Jiangu decoction in the treatment of osteoporosis based on the PI3K/Akt/GSK‐3βsignal pathway.Methods 60 SD rats were divided into six groups according to the random number table method,including the blank group,high(4.86 g/kg),medium(2.43 g/kg)and low doses(1.215 g/kg)of Duzhong Jiangu decoction groups,alendronate sodium group(positive drug,1 mg/kg)and the model group(normal saline).Rat bilateral ovaries were removed for modeling osteoporosis.In the blank group,only the ovaries on both sides were flipped without any treatment.After 8 weeks of administration,aortic blood was collected to measure serum anti‐tartaric acid phosphatase(Str ACP),TNF‐α,Il‐1β,malondialdehyde(MDA)content and superoxide dismutase(SOD)activity by ELISA.Bone mineral density value of rat femur was determined by bone mineral density scanner.mRNA and protein expressions of phosphatidylinositol 3‐kinase(PI3K),protein kinase B(Akt),glycogen synthasekinase 3β(GSK‐3β)were detected by real‐time quantitative PCR(RT‐PCR)and western blot.Results Bone mineral density,serum levels of Str ACP,TNF‐α,Il‐1β,MDA,SOD,and mRNA and protein expression of PI3K,AKT,GSK‐3βin the model group were significantly different with that in the blank group(P<0.01).Compared with the model group,the bone mineral density and the serum SOD activity were significantly increased in Duzhong Jianguo decoction groups,but the serum contents of TNF‐α,Il‐1β,Str ACP and MDA were significantly reduced in Duzhong Jianguo decoction groups(P<0.05,P<0.01).The mRNA and protein expressions of PI3K,Akt and GSK‐3βwere significantly different between Duzhong Jiangu decoction groups and the model group(P<0.05,P<0.01).Conclusion The therapeutic effect of Duzhong Jiangu decoction on osteoporosis may be related to regulation of the PI3K/AKT/GSK‐3βsignaling pathway.