基于Akt/Nrf2/GPX4信号通路探究养血柔筋方含药血清对IL-1β致大鼠退变软骨细胞铁死亡的影响  

Study on the Effect of Yangxue Roujin Formula Drug-containing Serum on IL-1β-Induced Iron Death of Rats Degenerated Chondrocytes Based on Akt/Nrf2/GPX4 Signaling Pathway

作　　者：沈锦涛 华茂奇[1] 张北 朱云麒 王上增 SHEN Jin-tao;HUA Mao-qi;ZHANG Bei;ZHU Yun-qi;WANG Shang-zeng(Henan Provincial Hospital of Traditional Chinese Medicine/The Second Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450011,China;Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南省中医院/河南中医药大学第二附属医院,河南郑州450011 [2]河南中医药大学,河南郑州450046

出　　处：《中药材》2024年第7期1793-1799,共7页Journal of Chinese Medicinal Materials

基　　金：河南省体育局2023年体育课题研究项目(202301);河南省卫生健康委国家中医临床研究基地科研专项(2021JDZX2039);河南中医药大学2021年度“科研苗圃工程”项目(MP2020-9)。

摘　　要：目的:探究养血柔筋方含药血清对Akt/Nrf2/GPX4信号通路的影响。方法:30只雄性Wistar大鼠分别连续灌胃给予低(9.18 g/kg)、中(18.36 g/kg)、高(36.72 g/kg)剂量养血柔筋方,2 w后收集血清。分离软骨细胞,①为研究铁死亡在IL-1β致软骨细胞死亡中的影响,在软骨细胞培养过程中分别加入50、100μmol/L的铁离子螯合剂;②为研究养血柔筋方含药血清在IL-1β诱导软骨细胞损伤中的作用,将所分离的细胞分为正常对照组、模型组及低、中、高剂量养血柔筋方含药血清组;③为研究Akt/Nrf2/GPX4信号在养血柔筋方含药血清改善软骨细胞铁死亡中的作用,培养过程中加入PI3K抑制剂(LY294002),分为正常对照组、模型组、养血柔筋方含药血清组、养血柔筋方含药血清+LY294002组。CCK-8法检测细胞存活率;试剂盒检测Fe^(2+)、MDA水平;流式细胞术检测ROS水平;Western Blot检测GPX4、p-Akt、Nrf2蛋白表达;免疫荧光法检测GPX4、MMP-13、CollagenⅡ蛋白表达。结果:与正常对照组比较,模型组细胞存活率、GPX4、p-Akt、Nrf2、CollagenⅡ蛋白表达显著降低,Fe^(2+)、MDA、ROS水平及MMP-13蛋白表达显著升高(P<0.05);与模型组比较,铁离子螯合剂组细胞存活率显著升高,Fe^(2+)、MDA水平显著降低(P<0.05)。与模型组比较,各剂量养血柔筋方含药血清组细胞存活率、GPX4、p-Akt、Nrf2、CollagenⅡ蛋白表达显著升高,Fe^(2+)、MDA、ROS水平及MMP-13蛋白表达显著降低(P<0.05)。与养血柔筋方含药血清组比较,养血柔筋方含药血清+LY294002组细胞存活率、GPX4、p-Akt、Nrf2、CollagenⅡ蛋白表达显著降低,MMP-13蛋白表达及MDA、Fe^(2+)水平显著升高(P<0.05)。结论:养血柔筋方含药血清可通过激活Akt/Nrf2/GPX4信号通路抑制膝骨关节炎软骨细胞铁死亡。Objective:To investigate the effect of Yangxue roujin formula drug-containing serum on Akt/Nrf2/GPX4 signaling pathway.Methods:30 male wistar rats were given low(9.18 g/kg),medium(18.36 g/kg)and high(36.72 g/kg)doses of Yangxue roujin formula,serum was collected after 2 weeks.Chondrocyte was isolated.In order to study the effect of iron death on chondrocyte death induced by IL-1β,50 and 100μmol/L iron chelating agents were added to chondrocyte culture.For investigating the effect of Yangxue roujin formula drug-containing serum on IL-1β-induced chondrocyte injury,the isolated cells were divided into normal control group,model group and low,medium,high doses of Yangxue roujin formula drug-containing serum groups.In order to study the effect of Akt/Nrf2/GPX4 signaling on the improvement of iron death of chondrocytes in Yangxue roujin formula drug-containing serum,PI3K in-hibitor(LY294002)was added during the culture process,the patients were divided into normal control group,model group,Yangxue roujin formula drug-containing serum group,Yangxue roujin formula drug-containing serum with LY294002 group.Cell survival rate was detected by CCK-8 method.Fe^(2+)and MDA levels were detected by the kit.Flow cytometry was used to detect ROS level.The protein ex-pressions of GPX4,p-Akt and Nrf2 were detected by Western Blot.The protein expressions of GPX4,MMP-13,CollagenⅡwere detec-ted by immunofluorescence.Results:Compared with normal control group,cell survival rate,protein expressions of GPX4,p-Akt,Nrf2 and CollagenⅡwere significantly decreased in model group,while Fe^(2+),MDA and ROS levels and protein expression of MMP-13 were significantly increased(P<0.05).Compared with the model group,the cell survival rate in the iron chelating agent group was signifi-cantly increased,and Fe^(2+)and MDA levels were significantly decreased(P<0.05).Compared with model group,the cell survival rate,protein expressions of GPX4,p-Akt,Nrf2 and CollagenⅡin each dose of Yangxue roujin formula drug-containing serum group were significa

关 键 词：养血柔筋方含药血清 膝骨关节炎 铁死亡 Akt/Nrf2/GPX4信号通路 

分 类 号：R285.5[医药卫生—中药学]