基于FOXO1的抗氧化作用探讨六味地黄方对去卵巢大鼠及H2O2诱导的氧化损伤MC3T3⁃E1细胞的干预作用
Intervention effect of Liuwei Dihuang Decoction on ovariectomized rats and H2O2⁃induced oxidative damaged MC3T3⁃E1 cells based on antioxidant effect of FOXO1
- 上海中医药杂志 2023年57卷第7期 页码：13-20
- 作者机构：
1.上海中医药大学附属曙光医院内分泌科（上海 201203）
- 作者简介：
陶乐维，男，博士，副主任医师，硕士研究生导师，主要从事糖尿病及其慢性并发症的临床与实验研究工作
- 基金信息：上海市自然科学基金项目(19ZR1458300);上海市科委科研计划项目(21S21900700);上海市中医糖尿病专科联盟建设项目（ZY〔2018-2020〕-FWTX-4011）;上海市临床重点专科项目(shslczdzk05401);上海市中医临床重点实验室项目(20DZ2272200);上海市浦东新区中医联合体建设项目(PDZY-2019-0601)
DOI：10.16305/j.1007-1334.2023.2203059
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陶乐维,韩煦,陈清光,等.基于FOXO1的抗氧化作用探讨六味地黄方对去卵巢大鼠及H2O2诱导的氧化损伤MC3T3⁃E1细胞的干预作用[J].上海中医药杂志,2023,57(7):13-20.
TAO Lewei,HAN Xu,CHEN Qingguang,et al.Intervention effect of Liuwei Dihuang Decoction on ovariectomized rats and H2O2⁃induced oxidative damaged MC3T3⁃E1 cells based on antioxidant effect of FOXO1[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(7):13-20.
陶乐维,韩煦,陈清光,等.基于FOXO1的抗氧化作用探讨六味地黄方对去卵巢大鼠及H2O2诱导的氧化损伤MC3T3⁃E1细胞的干预作用[J].上海中医药杂志,2023,57(7):13-20. DOI： 10.16305/j.1007-1334.2023.2203059.
TAO Lewei,HAN Xu,CHEN Qingguang,et al.Intervention effect of Liuwei Dihuang Decoction on ovariectomized rats and H2O2⁃induced oxidative damaged MC3T3⁃E1 cells based on antioxidant effect of FOXO1[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(7):13-20. DOI： 10.16305/j.1007-1334.2023.2203059.
摘要
目的,2,观察六味地黄方对去卵巢（OVX）大鼠氧化应激相关指标、骨转换标志物及股骨叉头框蛋白O1（FOXO1）表达的干预作用，并探讨其能否通过FOXO1来发挥抗氧化作用及其作用机制。,方法,2,（1）动物实验。将雌性Wistar大鼠分为正常组、假手术组、模型组、补佳乐组、N-乙酰半胱氨酸（NAC）组、六味地黄方大剂量及小剂量组，药物干预8周后检测骨髓活性氧（ROS）、血清抗氧化指标［超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-PX）、还原型谷胱甘肽（GSH）］、骨转换标志物［骨碱性磷酸酶（BALP）、骨钙素（OCN）、血清I型胶原羧基末端肽（CTX-I）］及股骨FOXO1蛋白表达。（2）细胞实验。制备对照组含药血清和低、中、高剂量含药血清。①将胚胎成骨细胞前体细胞（MC3T3-E1）分为正常组、模型组、对照含药血清组，以及低、中、高剂量含药血清组和NAC组，检测各组细胞FOXO1蛋白表达。②沉默MC3T3-E1细胞的,FOXO1,基因表达，以Western blot法验证后，将细胞分为正常组、模型组、NAC组、高剂量含药血清组、siNC组、siFOXO1组、siFOXO1+高剂量含药血清组，检测各组细胞的增殖及ROS水平。③将MC3T3-E1细胞分为正常组、模型组、NAC组、对照含药血清组，以及高剂量含药血清组，检测各组细胞p-FOXO1蛋白表达。,结果,2,（1）动物实验：与假手术组比较，模型组SOD、CAT、GSH-PX、GSH、BALP、OCN水平均明显降低（,P,<,0.05），CTX-I、ROS水平及FOXO1蛋白表达均明显升高（,P,<,0.05）；与模型组比较，六味地黄方大剂量组SOD、CAT、GSH-PX、GSH、BALP、OCN水平均明显升高（,P,<,0.05），CTX-I、ROS水平及FOXO1蛋白表达均明显降低（,P,<,0.05）。（2）细胞实验：①与正常组比较，经H,2,O,2,处理的MC3T3-E1细胞FOXO1蛋白表达明显增加（,P,<,0.05）；含药血清各剂量组FOXO1蛋白表达均低于模型组（,P,<,0.05）；②沉默MC3T3-E1细胞,FOXO1,基因后，siFOXO1组在48 h及72 h细胞增殖均明显低于模型组和siNC组（,P,<,0.05），ROS水平明显高于模型组和siNC组（,P,<,0.05）；siFOXO1+高剂量含药血清组在各时间点细胞增殖均明显高于模型组和siFOXO1组（,P,<,0.05），但都明显低于高剂量含药血清组（,P,<,0.05）；ROS水平明显低于模型组和siFOXO1组（,P,<,0.05），但明显高于高剂量含药血清组（,P,<,0.05）；③通过检测p-FOXO1发现，模型组p-FOXO1/GAPDH明显低于正常组（,P,<,0.05）；高剂量含药血清组p-FOXO1/GAPDH明显高于模型组（,P,<,0.05）。,结论,2,六味地黄方能抑制OVX大鼠骨髓ROS水平，提高血清抗氧化水平，促进OVX大鼠骨形成，抑制骨吸收，其机制可能部分与促进成骨细胞FOXO1磷酸化有关。
Abstract
Objective,2,To observe the intervention effect of Liuwei Dihuang Decoction on oxidative stress-related indices， bone turnover markers and the protein expression of FOXO1 in femoral in ovariectomized （OVX） rats. And to explore whether Liuwei Dihuang Decoction can exert antioxidant effect through FOXO1 and its mechanism.,Methods,2,（1） In animal experiment： Female Wistar rats were divided into normal group， sham-operated group， model group， Progynova group， N-acetylcysteine （NAC） group， Liuwei Dihuang Decoction high- and low-dose groups. Reactive oxygen species of bone marrow， serum antioxidant indices including SOD， CAT， GSH-PX， GSH and bone turnover markers including BALP， OCN， CTX-I， and the protein expression of FOXO1 in femoral were detected after 8 weeks of intervention. （2） In cell experiments， the decoction of Liuwei Dihuang was prepared and then the medicated serum of Liuwei Dihuang Decoction was collected， including medicated serum of control group， medicated serum groups with low-， medium-and high-dose. ①MC3T3-E1 cells were divided into normal group， model group， control medicated serum group， low-， medium- and high-dose medicated serum group of Liuwei Dihuang Decoction and NAC group. Protein expression of FOXO1 in each group was detected to verify the results of animal experiment. ②,FOXO1, gene expression of MC3T3-E1 cells was silenced. After verified by Western blot， the cells were divided into normal group， model group， NAC group， high-dose medicated serum group of Liuwei Dihuang Decoction， siNC group， siFOXO1 group， and siFOXO1+ high-dose group of Liuwei Dihuang Decoction medicated serum. The proliferation and ROS levels of cells in each group were detected. ③MC3T3-E1 cells were divided into normal group， model group， NAC group， control medicated serum group and high-dose medicated serum group of Liuwei Dihuang Decoction， and the protein expression of p-FOXO1 in each group was detected.,Results,2,（1） In animal experiment： Compared with the sham operation group， SOD， CAT， GSH-PX， GSH， BALP and OCN levels were significantly decreased （,P,<,0.05）， while CTX-I， ROS levels and FOXO1 protein expression were significantly increased in the model group （,P,<,0.05）. Compared with the model group， SOD， CAT， GSH-PX， GSH， BALP and OCN levels in high-dose group of Liuwei Dihuang Decoction were significantly increased （,P,<,0.05）， while CTX-I， ROS levels and FOXO1 protein expression were significantly decreased （,P,<,0.05）. （2） In cell experiment： ①Compared with the normal group， the protein expression of FOXO1 in MC3T3-E1 cells treated with H,2,O,2, was significantly increased （,P,<,0.05）. The protein expression of FOXO1 in each dose group of medicated serum of Liuwei Dihuang Decoction was lower than that in the model group （,P,<,0.05）. ②After silencing the ,FOXO1, gene in MC3T3-E1 cells， the cell proliferation in the siFOXO1 group at 48 h and 72 h was significantly lower than that in the model group and siNC group （,P,<,0.05）， and the level of ROS was significantly higher than that in the model group and siNC group （,P,<,0.05）. The cell proliferation in the siFOXO1+high-dose medicated serum of Liuwei Dihuang Decoction was significantly higher than that in the model group and siFOXO1 group at each time point （,P,<,0.05）， but significantly lower than that in the high-dose group of medicated serum of Liuwei Dihuang Decoction （,P,<,0.05）； the level of ROS was significantly lower than that in the model group and siFOXO1 group （,P,<,0.05）， but significantly higher than that in the high-dose group of medicated serum of Liuwei Dihuang Decoction （,P,<,0.05）. ③p-FOXO1/GAPDH in the model group was significantly lower than that in the normal group （,P,<,0.05）， and p-FOXO1/GAPDH in the high-dose group of medicated serum of Liuwei Dihuang Decoction was significantly higher than that in the model group （,P,<,0.05）.,Conclusion,2,Liuwei Dihuang Decoction can inhibit ROS levels in bone marrow of OVX rats， increase antioxidant levels in serum， promote bone formation and inhibit bone resorption in OVX rats. The mechanism may be partly related to promoting the phosphorylation of FOXO1 in osteoblasts.
关键词
骨质疏松症六味地黄方氧化应激大鼠模型经典名方中药研究
Keywords
osteoporosisLiuwei Dihuang Decoctionoxidative stressrat modelclassic formulatraditional Chinese herbal medicine research
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