基于ERK/mTOR信号通路探讨六味地黄丸对氧化应激状态下成骨细胞自噬的影响  

Liuwei Dihuang pills induce osteoblast autophagy under oxidative stress via ERK/mTOR signaling pathway

作　　者：谢丽华 郭澜 陈赛楠 李生强 陈娟 葛继荣 XIE Lihua;GUO Lan;CHEN Sainan;LI Shengqiang;CHEN Juan;GE Jirong(The Key Research Laboratory of Osteoporosis Syndrome Genomics,Fujian Academy of Chinese Medical sciences,Fuzhou 350003;Fujian Key Laboratory of Integrated Traditional Chinese and Western Medicine for Osteoporosis(Fujian Academy of Chinese Medical Sciences,Rehabilitation Hospital Affiliated to Fujian University of TCM),Fuzhou 350003;Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]福建省中医药科学院骨质疏松证候基因组学研究室,福建福州350003 [2]福建省中西医结合防治骨质疏松重点实验室(福建省中医药科学院、福建中医药大学附属康复医院),福建福州350003 [3]福建中医药大学,福建福州350122

出　　处：《中国骨质疏松杂志》2023年第1期1-5,共5页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金项目(81873323);福建省自然科学基金项目(2020J01725,2022J01863)。

摘　　要：目的从ERK/mTOR信号通路观察六味地黄丸对氧化应激状态下MC3T3-E1细胞自噬的影响,探讨其治疗绝经后骨质疏松症的作用机制。方法用过氧化氢(H 2O 2)模拟氧化应激状态,六味地黄丸(LWDH)含药血清干预MC3T3-E1细胞,将细胞分为Model组(H 2O 2干预)、Blank组(空白血清)、LWDH组(LWDH含药血清)、Rap组(mTOR通路抑制剂)、U0126组(ERK通路抑制剂)、Rap+LWDH组(mTOR通路抑制剂、LWDH含药血清)、U0126+LWDH组(ERK通路抑制剂、LWDH含药血清);以不做干预的Control组作为对照。通过细胞活性氧(reactive oxygen species,ROS)检测细胞ROS水平;检测自噬蛋白LC3B及ERK/mTOR信号通路相关蛋白mTOR、p-mTOR、ERK1/2和p-ERK1/2的表达。结果ROS结果显示,与Model组相比,LWDH组、Rap组、U0126等各组细胞的ROS水平皆降低(P<0.05)。Western blot结果显示,与Model组相比,LWDH组、Rap组、LWDH+Rap组蛋白LC3Ⅱ/Ⅰ增高(P<0.05);与Model组相比,LWDH组、Rap组与Rap+LWDH组的p-mTOR蛋白表达下调(P<0.05),LWDH组、U0126组的p-ERK1/2蛋白表达下调(P<0.05)。结论六味地黄丸治疗绝经后骨质疏松症的机制可能与抑制ERK/mTOR信号通路诱导氧化应激状态下成骨细胞自噬相关。Objective To investigate the effect of Liuwei Dihuang pill-containing serum on the autophagy in MC3T3-E1 cells under oxidative stress via ERK/mTOR signaling pathway,and to explore the mechanism of its treating of osteoporosis.Methods Hydrogen peroxide(H 2O 2)was used to simulate oxidative stress.Cells were divided into Control group(no intervention),Model group(H 2O 2 intervention),Blank group(blank serum,H 2O 2 intervention),LWDH group(LWDH-containing serum,H 2O 2 intervention),Rap group(mTOR pathway inhibitor,H 2O 2 intervention),U0126 group(ERK pathway inhibitor,H 2O 2 intervention),Rap+LWDH group(mTOR pathway inhibitor,LWDH-containing serum,H 2O 2 intervention),and U0126+LWDH group(ERK pathway inhibitor,LWDH-containing serum,H 2O 2 intervention).Cellular ROS levels were detected with cellular reactive oxygen species(ROS).The expressions of autophagy protein LC3B and ERK/mTOR signaling pathway-related proteins mTOR,p-mTOR,ERK1/2,and p-ERK1/2 were detected with Western blotting.Results ROS result showed that the ROS level in LWDH group,Rap group,and U0126 group was lower than that in Model group(P<0.05).Western blotting result showed that the LC3II/Ⅰprotein increased in the LWDH,Rap,and LWDH+Rap groups compared to that in the Model group(P<0.05).The p-mTOR protein decreased in the LWDH,Rap,and Rap+LWDH groups compared to that in the Model group(P<0.05).The p-ERK1/2 protein expression was down-regulated in the LWDH group and U0126 group(P<0.05).Conclusion The mechanism of Liuwei Dihuang pills for the treatment of postmenopausal osteoporosis may be related to the autophagy in osteoblasts under oxidative stress by inhibiting ERK/mTOR signaling pathway.

关 键 词：六味地黄丸 成骨细胞 氧化应激 自噬 ERK/mTOR信号通路 

分 类 号：R681[医药卫生—骨科学] R285.5[医药卫生—外科学]