川芎嗪对白细胞介素-1β诱导的软骨细胞凋亡和氧化应激的影响及作用机制研究  

Effects and mechanism of tetramethylpyrazine on chondrocyte apoptosis and oxidative stress induced by interleukin-1β:an experimental study

作　　者：李科[1] 曹玉净[1] 钱亚男 李光辉[1] 周松林[1] LI Ke;CAO Yujing;QIAN Yanan;LI Guanghui;ZHOU Songlin(Henan Province Hospital of TCM,Zhengzhou 450002,Henan,China;College of Orthopaedics and Traumatology of Henan University of Chinese Medicine,Zhengzhou 450002,Henan,China)

机构地区：[1]河南省中医院,河南郑州450002 [2]河南中医药大学骨伤学院,河南郑州450002

出　　处：《中医正骨》2025年第2期21-27,共7页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：河南省中医药科学研究专项课题(2024ZYZD06,2023ZY1008,2019ZYBJ15)。

摘　　要：目的:观察川芎嗪(tetramethylpyrazine,TMP)对白细胞介素(interleukin,IL)-1β诱导的软骨细胞凋亡和氧化应激的影响,并探讨其作用机制。方法:选用ATDC5小鼠软骨细胞进行实验,加入IL-1β模拟骨关节炎环境,通过测定不同浓度TMP干预后的细胞存活率确定本实验中TMP的浓度为5μg·mL^(-1)和10μg·mL^(-1)。将ATDC5小鼠软骨细胞分为4组。对照组常规培养,模型组、TMP低剂量组、TMP高剂量组均按照10μmol·L^(-1)加入IL-1β,TMP低剂量组、TMP高剂量组在此基础上分别按照5μg·mL^(-1)和10μg·mL^(-1)加入TMP。采用CCK-8法测定细胞增殖抑制率,采用Western Blot法检测B细胞淋巴瘤-2相关X(B-cell lymphoma-2 Associated X,Bax)蛋白含量、B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)蛋白含量确定细胞凋亡情况,采用ELISA测定技术检测炎症因子含量[IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)],分别采用硫代巴比妥酸法、黄嘌呤氧化酶法和比色法测定丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽(glutathione,GSH)等氧化应激指标含量,采用Western Blot法检测核转录因子红系2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)信号通路相关蛋白[Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)、Nrf2、血红素加氧酶-1(heme oxygenase-1,HO-1)、SOD2]含量。将ATDC5小鼠软骨细胞分为4组。空白组常规培养;诱导组按照10μmol·L^(-1)加入IL-1β;TMP组按照10μmol·L^(-1)加入IL-1β,并按照10μg·mL^(-1)加入TMP;Nrf2抑制剂组先按照10μmol·L^(-1)加入IL-1β、按照5μg·mL^(-1)加入Nrf2抑制剂ML385,最后按照10μg·mL^(-1)加入TMP,采用Western Blot法检测Nrf2下游蛋白(HO-1、SOD2)含量。结果:①软骨细胞增殖情况检测结果。模型组的细胞增殖抑制率高于对照组(P=0.043),TMP低剂量组和TMP高剂量组的细胞增殖抑制率均低于模型组(P=0.030,P=0.033),TMP低剂量组的细胞增殖�Objective:To observe the effects of tetramethylpyrazine(TMP)on interleukin(IL)-1β-induced chondrocyte apoptosis and oxidative stress,and to explore its underlying mechanism.Methods:The ATDC5 mouse chondrocytes(ATDC5 cells)were selected and cultured in the Dulbecco’s Modified Eagle’s Medium(DMEM)added with IL-1βaimed at simulating the environment of osteoarthritis.The concentrations of TMP for the following experiment were determined to be 5 and 10μg/mL by measuring the cell survival rate after intervention with different concentrations of TMP.The ATDC5 cells were divided into control group,model group,low-dose TMP(L-TMP)group,and high-dose TMP(H-TMP)group.The ATDC5 cells in the control group were cultured in the conventional DMEM;while the ones in mo-del group,L-TMP group,and H-TMP group in the DMEM adding with IL-1βwith concentration of 10 ng/mL;and the DMEM in L-TMP group and H-TMP group were further added with TMP with concentration of 5 and 10μg/mL,respectively.After 24-hour culture,the pro-liferation inhibition rate of the ATDC5 cells in each group was determined by using the cell counting kit-8(CCK-8)assay;the levels of B-cell lymphoma-2 Associated X(Bax)protein and B-cell lymphoma-2(Bcl-2)protein in the ATDC5 cells were detected by using Western blotting to determine the cellular apoptosis;the levels of inflammatory cytokines including IL-6 and tumor necrosis factor-α(TNF-α)in the ATDC5 cells were detected by using enzyme-linked immunosorbent assay(ELISA);the levels of oxidative stress markers including malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)in the ATDC5 cells were determined by using the thiobarbituric acid(TBA)method,xanthine oxidase(XO)method,and colorimetry,respectively;and the levels of nuclear factor-erythroid 2-related factor 2(Nrf2)signaling pathway-related proteins including Kelch-like ECH-associated protein 1(Keap1),Nrf2,heme oxygenase-1(HO-1),and SOD2 in the ATDC5 cells were detected by using Western blotting.Furthermore,another ATDC5 cells were divided int

关 键 词：川芎嗪 骨关节炎 软骨细胞 细胞凋亡 氧化性应激 白细胞介素-1β 核转录因子红系2相关因子2 体外试验 

分 类 号：R285.5[医药卫生—中药学]