六味地黄方含药血清对H_(2)O_(2)诱导的氧化损伤MC3T3-E1细胞Runx2、OPG/RANKL的干预作用及其机制探讨  

Intervention effects of medicated serum of Liuwei Dihuang Decoction on Runx2 and OPG/RANKL of oxidative damaged MC3T3-E1 cells induced by H_(2)O_(2) and investigating its mechanism

作　　者：陶乐维[1] 韩煦 陈清光[1] 徐隽斐[1] 陆灏[1] TAO Lewei;HAN Xu;CHEN Qingguang;XU Juanfei;LU Hao(Department of Endocrinology,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院内分泌科,上海201203

出　　处：《上海中医药杂志》2022年第8期102-106,共5页Shanghai Journal of Traditional Chinese Medicine

基　　金：上海市科委自然科学基金资助项目(19ZR1458300);上海市科委科研计划项目(21S21900700);上海市科委中医临床重点实验室项目(20DZ2272200);上海市卫健委进一步加快中医药事业发展三年行动计划项目(ZY[2018-2020]-FWTX-4011);上海市卫健委临床重点专科项目(shslczdzk05401);上海市浦东新区中医联合体建设项目(PDZY-2019-0601)

摘　　要：目的观察六味地黄方含药血清对过氧化氢(H_(2)O_(2))诱导的氧化损伤小鼠胚胎成骨细胞前体细胞(MC3T3-E1)Runt相关转录因子2(Runx2)及护骨素/核因子-κB受体活化因子配体(OPG/RANKL)的干预作用,并从抗氧化角度初步探讨其作用机制。方法制作六味地黄方汤剂,对成年Wistar大鼠进行灌胃,获取六味地黄方含药血清。除正常组外,其余各组先用1.0 mmol/L H_(2)O_(2)预处理MC3T3-E1细胞6 h,随后模型组更换正常培养基,N-乙酰半胱氨酸(NAC)组更换含有2.5 mmol/L NAC的培养基,六味地黄方含药血清组更换含有10%药物血清的培养基,各组均处理24 h。采用DCFH-DA荧光探针检测MC3T3-E1细胞内活性氧(ROS)水平。根据试剂盒说明书检测过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、还原型谷胱甘肽(GSH)。采用Western blot法检测Runx2、OPG、RANKL、肿瘤抑制蛋白P53及磷酸化状态的P53蛋白(p-P53)表达。结果与正常组比较,模型组ROS明显升高(P<0.05),同时CAT、SOD、GSH-PX、GSH均明显降低(P<0.05);在蛋白表达方面,与正常组相比,模型组Runx2、OPG蛋白表达均明显降低(P<0.05),RANKL蛋白表达明显升高(P<0.05),同时p-P53/P53也有明显升高(P<0.05)。与模型组相比,六味地黄方含药血清组GSH明显升高(P<0.05),ROS水平明显下降(P<0.05)。在蛋白表达方面,与模型组比较,六味地黄方含药血清组Runx2、OPG蛋白表达均明显升高(P<0.05),RANKL蛋白表达明显降低(P<0.05),同时p-P53/P53明显降低(P<0.05)。结论六味地黄方含药血清能促进H_(2)O_(2)致氧化损伤MC3T3-E1细胞内Runx2蛋白表达,升高OPG蛋白表达,降低RANKL蛋白表达,这可能与其升高GSH的含量、抑制P53蛋白的磷酸化,进而降低细胞内ROS水平有关。Objective To observe the intervention effects of medicated serum of Liuwei Dihuang Decoction on Runt-related transcription factor 2(Runx2)and osteoprotegerin/nuclear factor-κB receptor activator ligand(OPG/RANKL)in preparietal osteoblasts(MC3T3-E1)of mice with oxidative injury induced by hydrogen peroxide(H_(2)O_(2)),and to explore its mechanism from the point of view of antioxidation.Methods The Liuwei Dihuang Decoction was prepared and given to the adult Wistar rats by intragastric administration,and then the medicated serum of Liuwei Dihuang Decoction was collected.Except for the normal group,the other groups of MC3T3-E1 cells were first pretreated with 1.0 mmol/L H_(2)O_(2)for 6 h,and then the culture medium of the model group was replaced with normal culture medium,the N-acetylcysteine(NAC)group replaced with 2.5 mmol/L NAC and the medicated serum groups with 10%medicated serum.Each group was treated for 24 hours.The level of reactive oxygen species(ROS)in MC3T3-E1 cells was detected by DCFH-DA fluorescence probe.Catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GSH-PX)and reduced glutathione(GSH)were detected according to the kit instructions.The expressions of Runx2,OPG,RANKL,tumor suppressor protein p53 and phosphorylated p53 protein(p-P53)were detected by Western blot.Results Compared with the normal group,ROS in the model group was significantly increased(P<0.05),while CAT,SOD,GSHPX,and GSH were significantly decreased(all P<0.05).In terms of protein expression,compared with the normal group,the protein expressions of Runx2 and OPG in the model group were significantly decreased(all P<0.05),while the RANKL was significantly increased(P<0.05),and p-P53/P53 was also significantly increased(P<0.05).Compared with the model group,the GSH in the medicated serum group of Liuwei Dihuang Decoction was significantly increased(P<0.05)and the level of ROS was significantly decreased(P<0.05).In terms of protein expression,compared with the model group,the protein expressions of Runx2 and OPG in the med

关 键 词：骨质疏松 六味地黄方 Runt相关转录因子2 护骨素/核因子-κB受体活化因子配体 成骨细胞 中药研究 

分 类 号：R285.5[医药卫生—中药学]