[1]李科,曹玉净,钱亚男,等.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[J].中医正骨,2025,37(02):21-27.
　LI Ke,CAO Yujing,QIAN Yanan,et al.Effects and mechanism of tetramethylpyrazine on chondrocyte apoptosis and oxidative stress induced by interleukin-1β:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(02):21-27.
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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()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年02期
页码:
21-27
栏目:
基础研究
出版日期:
2025-02-20

文章信息/Info

Title:
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2; 李光辉1; 周松林1
(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)
Author(s):
LI Ke1; CAO Yujing1; QIAN Yanan2; LI Guanghui1; ZHOU Songlin1
1.Henan Province Hospital of TCM,Zhengzhou 450002,Henan,China 2.College of Orthopaedics and Traumatology of Henan University of Chinese Medicine,Zhengzhou 450002,Henan,China
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
Keywords:
tetramethylpyrazine;  osteoarthritis;  chondrocytes;  apoptosis;  oxidative stress;  interleukin-1 beta;  nuclear factor-erythroid 2-related factor 2;  in vitro test
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5 μg·mL-1和10 μg·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10 μmol·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5 μg·mL-1和10 μg·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10 μmol·L-1加入IL-1β; TMP组按照10 μmol·L-1加入 IL-1β,并按照10 μg·mL-1加入TMP; Nrf2抑制剂组先按照10 μmol·L-1加入IL-1β、按照5 μg·mL-1加入Nrf2抑制剂ML385,最后按照 10 μg·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抑制率高于TMP高剂量组(P=0.049)。②软骨细胞凋亡情况检测结果。模型组的Bax/Bcl-2蛋白含量比值高于对照组、TMP低剂量组及TMP高剂量组(P=0.000,P=0.005,P=0.000),TMP高剂量组的Bax/Bcl-2蛋白含量比值低于TMP低剂量组(P=0.003)。③软骨细胞中炎症因子含量检测结果。模型组的IL-6和TNF-α含量均高于对照组、TMP低剂量组及TMP高剂量组(IL-6:P=0.035,P=0.024,P=0.049; TNF-α:P=0.017,P=0.039,P=0.032); TMP低剂量组的IL-6和TNF-α含量均高于TMP高剂量组(P=0.019,P=0.028)。④软骨细胞中氧化应激指标含量检测结果。模型组的MDA含量高于对照组(P=0.027),SOD和GSH含量均低于对照组(P=0.013,P=0.028); TMP低剂量组和TMP高剂量组的MDA含量均低于模型组(P=0.020,P=0.040),SOD和GSH含量均高于模型组(SOD:P=0.048,P=0.039; GSH:P=0.031,P=0.022); TMP高剂量组的MDA含量低于TMP低剂量组(P=0.040),SOD和GSH含量均高于TMP低剂量组(P=0.026,P=0.038)。⑤软骨细胞中Nrf2信号通路相关蛋白含量检测结果。模型组的Keap1蛋白含量高于对照组(P=0.000),Nrf2、HO-1及SOD2蛋白含量均低于对照组(P=0.003,P=0.004,P=0.003); TMP低剂量组和TMP高剂量组的Keap1蛋白含量均低于模型组(P=0.002,P=0.000),Nrf2、HO-1及SOD2蛋白含量均高于模型组(Nrf2:P=0.002,P=0.008; HO-1:P=0.000,P=0.001; SOD2:P=0.002,P=0.000); TMP高剂量组的Keap1蛋白含量低于TMP低剂量组(P=0.034),Nrf2、HO-1及SOD2蛋白含量均高于TMP低剂量组(P=0.000,P=0.039,P=0.029)。⑥Nrf2抑制剂干预后软骨细胞中Nrf2下游蛋白含量测定结果。诱导组的HO-1、SOD2蛋白含量均低于空白组(P=0.000,P=0.001),TMP组的HO-1、SOD2蛋白含量均高于诱导组(P=0.023,P=0.030),Nrf2抑制剂组的HO-1、SOD2蛋白含量均低于TMP组(P=0.040,P=0.000)。结论:TMP对IL-1β诱导的软骨细胞凋亡和氧化应激具有保护作用,其作用机制可能与激活Nrf2信号通路,增强软骨细胞抗氧化能力有关。
Abstract:
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-1β aimed at simulating the environment of osteoarthritis.The concentrations of TMP for the following experiment were determined to be 5 and 10 μg/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-1β with 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 10 μg/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 into blank group,induction group,TMP group,and Nrf2 inhibitor group.The ATDC5 cells in the blank group were cultured in the conventional DMEM; while the ones in induction group in the DMEM adding with IL-1β with concentration of 10 μmol/L; the ones in TMP group in the DMEM adding with IL-1β with concentration of 10 μmol/L and TMP with concentration of 10 μg/mL; and the ones in Nrf2 inhibitor group in the DMEM adding with IL-1β with concentration of 10 μmol/L,ML385 with concentration of 5 μg/mL and TMP with concentration of 10 μg/mL.The levels of Nrf2 downstream proteins including HO-1 and SOD2 in the ATDC5 cells were detected by employing Western blotting.Results:①The proliferation of the ATDC5 cells.The proliferation inhibition rate of the ATDC5 cells was higher in model group compared to control group(P=0.043),and was lower in L-TMP group and H-TMP group compared to model group(P=0.030,P=0.033),and was higher in L-TMP group compared to H-TMP group(P=0.049).②The apoptosis of the ATDC5 cells.The ratio of Bax protein level to Bcl-2 protein level was greater in model group compared to control group,L-TMP group and H-TMP group(P=0.000,P=0.005,P=0.000),and was smaller in H-TMP group compared to L-TMP group(P=0.003).③The levels of inflammatory cytokines in the ATDC5 cells.The levels of IL-6 and TNF-α in the ATDC5 cells were higher in model group compared to control group,L-TMP group and H-TMP group(IL-6:P=0.035,P=0.024,P=0.049; TNF-α:P=0.017,P=0.039,P=0.032),and were higher in L-TMP group compared to H-TMP group(P=0.019,P=0.028).④The levels of oxidative stress markers in the ATDC5 cells.The level of MDA was higher,while the levels of SOD and GSH were lower in model group compared to control group(P=0.027,P=0.013,P=0.028).The level of MDA was lower,while the levels of SOD and GSH were higher in L-TMP group and H-TMP group compared to model group(MDA:P=0.020,P=0.040; SOD:P=0.048,P=0.039; GSH:P=0.031,P=0.022).The level of MDA was lower,while the levels of SOD and GSH were higher in H-TMP group compared to L-TMP group(P=0.040,P=0.026,P=0.038).⑤The levels of Nrf2 signaling pathway-related proteins in the ATDC5 cells.The level of Keap1 was higher,while the levels of Nrf2,HO-1 and SOD2 were lower in model group compared to control group(P=0.000,P=0.003,P=0.004,P=0.003).The level of Keap1 was lower,while the levels of Nrf2,HO-1 and SOD2 were higher in L-TMP group and H-TMP group compared to model group(Keap1:P=0.002,P=0.000; Nrf2:P=0.002,P=0.008; HO-1:P=0.000,P=0.001; SOD2:P=0.002,P=0.000).The level of Keap1 was lower,while the levels of Nrf2,HO-1 and SOD2 were higher in H-TMP group compared to L-TMP group(P=0.034,P=0.000,P=0.039,P=0.029).⑥The levels of Nrf2 downstream proteins in the ATDC5 cells after intervention with inhibitor.The levels of HO-1 and SOD2 in the ATDC5 cells were lower in induction group compared to blank group(P=0.000,P=0.001),and were higher in TMP group compared to induction group(P=0.023,P=0.030),and were lower in Nrf2 inhibitor group compared to TMP group(P=0.040,P=0.000).Conclusion:TMP has a protective effect against chondrocyte apoptosis and oxidative stress induced by IL-1β.It may exert the effects by activating Nrf2 signaling pathway and enhancing the antioxidant capacity of chondrocytes.

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备注/Memo

备注/Memo:
基金项目:河南省中医药科学研究专项课题(2024ZYZD06,2023ZY1008,2019ZYBJ15)
通讯作者:曹玉净 E-mail:bravecao@163.com
更新日期/Last Update: 1900-01-01