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[1]李科,曹玉凈,錢亞男,等.川芎嗪對白細胞介素-1β誘導的軟骨細胞凋亡和氧化應激的影響及作用機制研究[J].中醫(yī)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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川芎嗪對白細胞介素-1β誘導的軟骨細胞凋亡和氧化應激的影響及作用機制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期數(shù):
2025年02期
頁碼:
21-27
欄目:
基礎研究
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; 曹玉凈1; 錢亞男2; 李光輝1; 周松林1
(1.河南省中醫(yī)院,河南 鄭州 450002; 2.河南中醫(yī)藥大學骨傷學院,河南 鄭州 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
關鍵詞:
川芎嗪;  骨關節(jié)炎;  軟骨細胞;  細胞凋亡;  氧化性應激;  白細胞介素-1β;  核轉錄因子紅系2相關因子2;  體外試驗
Keywords:
tetramethylpyrazine;  osteoarthritis;  chondrocytes;  apoptosis;  oxidative stress;  interleukin-1 beta;  nuclear factor-erythroid 2-related factor 2;  in vitro test
摘要:
目的:觀察川芎嗪(tetramethylpyrazine,TMP)對白細胞介素(interleukin,IL)-1β誘導的軟骨細胞凋亡和氧化應激的影響,并探討其作用機制。方法:選用ATDC5小鼠軟骨細胞進行實驗,加入IL-1β模擬骨關節(jié)炎環(huán)境,通過測定不同濃度TMP干預后的細胞存活率確定本實驗中TMP的濃度為5 μg·mL-1和10 μg·mL-1。將ATDC5小鼠軟骨細胞分為4組。對照組常規(guī)培養(yǎng),模型組、TMP低劑量組、TMP高劑量組均按照10 μmol·L-1加入IL-1β,TMP低劑量組、TMP高劑量組在此基礎上分別按照5 μg·mL-1和10 μg·mL-1加入TMP。采用CCK-8法測定細胞增殖抑制率,采用Western Blot法檢測B細胞淋巴瘤-2相關X(B-cell lymphoma-2 Associated X,Bax)蛋白含量、B細胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)蛋白含量確定細胞凋亡情況,采用ELISA測定技術檢測炎癥因子含量[IL-6、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)],分別采用硫代巴比妥酸法、黃嘌呤氧化酶法和比色法測定丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽(glutathione,GSH)等氧化應激指標含量,采用Western Blot法檢測核轉錄因子紅系2相關因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)信號通路相關蛋白[Kelch樣ECH相關蛋白1(Kelch-like ECH-associated protein 1,Keap1)、Nrf2、血紅素加氧酶-1(heme oxygenase-1,HO-1)、SOD2]含量。將ATDC5小鼠軟骨細胞分為4組。空白組常規(guī)培養(yǎng); 誘導組按照10 μmol·L-1加入IL-1β; TMP組按照10 μmol·L-1加入 IL-1β,并按照10 μg·mL-1加入TMP; Nrf2抑制劑組先按照10 μmol·L-1加入IL-1β、按照5 μg·mL-1加入Nrf2抑制劑ML385,最后按照 10 μg·mL-1 加入TMP,采用Western Blot法檢測Nrf2下游蛋白(HO-1、SOD2)含量。結果:①軟骨細胞增殖情況檢測結果。模型組的細胞增殖抑制率高于對照組(P=0.043),TMP低劑量組和TMP高劑量組的細胞增殖抑制率均低于模型組(P=0.030,P=0.033),TMP低劑量組的細胞增殖抑制率高于TMP高劑量組(P=0.049)。②軟骨細胞凋亡情況檢測結果。模型組的Bax/Bcl-2蛋白含量比值高于對照組、TMP低劑量組及TMP高劑量組(P=0.000,P=0.005,P=0.000),TMP高劑量組的Bax/Bcl-2蛋白含量比值低于TMP低劑量組(P=0.003)。③軟骨細胞中炎癥因子含量檢測結果。模型組的IL-6和TNF-α含量均高于對照組、TMP低劑量組及TMP高劑量組(IL-6:P=0.035,P=0.024,P=0.049; TNF-α:P=0.017,P=0.039,P=0.032); TMP低劑量組的IL-6和TNF-α含量均高于TMP高劑量組(P=0.019,P=0.028)。④軟骨細胞中氧化應激指標含量檢測結果。模型組的MDA含量高于對照組(P=0.027),SOD和GSH含量均低于對照組(P=0.013,P=0.028); TMP低劑量組和TMP高劑量組的MDA含量均低于模型組(P=0.020,P=0.040),SOD和GSH含量均高于模型組(SOD:P=0.048,P=0.039; GSH:P=0.031,P=0.022); TMP高劑量組的MDA含量低于TMP低劑量組(P=0.040),SOD和GSH含量均高于TMP低劑量組(P=0.026,P=0.038)。⑤軟骨細胞中Nrf2信號通路相關蛋白含量檢測結果。模型組的Keap1蛋白含量高于對照組(P=0.000),Nrf2、HO-1及SOD2蛋白含量均低于對照組(P=0.003,P=0.004,P=0.003); TMP低劑量組和TMP高劑量組的Keap1蛋白含量均低于模型組(P=0.002,P=0.000),Nrf2、HO-1及SOD2蛋白含量均高于模型組(Nrf2:P=0.002,P=0.008; HO-1:P=0.000,P=0.001; SOD2:P=0.002,P=0.000); TMP高劑量組的Keap1蛋白含量低于TMP低劑量組(P=0.034),Nrf2、HO-1及SOD2蛋白含量均高于TMP低劑量組(P=0.000,P=0.039,P=0.029)。⑥Nrf2抑制劑干預后軟骨細胞中Nrf2下游蛋白含量測定結果。誘導組的HO-1、SOD2蛋白含量均低于空白組(P=0.000,P=0.001),TMP組的HO-1、SOD2蛋白含量均高于誘導組(P=0.023,P=0.030),Nrf2抑制劑組的HO-1、SOD2蛋白含量均低于TMP組(P=0.040,P=0.000)。結論:TMP對IL-1β誘導的軟骨細胞凋亡和氧化應激具有保護作用,其作用機制可能與激活Nrf2信號通路,增強軟骨細胞抗氧化能力有關。
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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備注/Memo

備注/Memo:
基金項目:河南省中醫(yī)藥科學研究專項課題(2024ZYZD06,2023ZY1008,2019ZYBJ15)
通訊作者:曹玉凈 E-mail:[email protected]
更新日期/Last Update: 1900-01-01