中医正骨2025，Vol.37Issue(2)：21-27,7.
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 inter-leukin-1β:an experimental study
摘要
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 cul-tured 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 inter-vention 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-1 β 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 malondi-aldehyde(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,induc-tion 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 pro-liferation 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 com-pared 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 interven-tion 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 chondro-cytes.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Key words
tetramethylpyrazine/osteoarthritis/chondrocytes/apoptosis/oxidative stress/interleukin-1 beta/nuclear factor-erythroid 2-relat-ed factor 2/in vitro test引用本文复制引用
李科,曹玉净,钱亚男,李光辉,周松林..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(2):21-27,7.基金项目
河南省中医药科学研究专项课题(2024ZYZD06,2023ZY1008,2019ZYBJ15) （2024ZYZD06,2023ZY1008,2019ZYBJ15）
