[1]王会含,王永堂,苗建华,等.川芎嗪对人骨关节炎软骨细胞的影响及作用机制研究[J].中医正骨,2021,33(07):4-10.
　WANG Huihan,WANG Yongtang,MIAO Jianhua,et al.A study of the effects of tetramethylpyrazine on human osteoarthritic chondrocytes and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2021,33(07):4-10.
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川芎嗪对人骨关节炎软骨细胞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33卷
期数:
2021年07期
页码:
4-10
栏目:
基础研究
出版日期:
2021-07-20

文章信息/Info

Title:
A study of the effects of tetramethylpyrazine on human osteoarthritic chondrocytes and its mechanism of action
作者:
王会含; 王永堂; 苗建华; 李凤新
(郑州大学附属郑州中心医院,河南 郑州 450007)
Author(s):
WANG Huihan; WANG Yongtang; MIAO Jianhua; LI Fengxin
Zhengzhou Central Hospital affiliated to Zhengzhou University,Zhengzhou 450007,Henan,China
关键词:
骨关节炎;  川芎嗪;  细胞凋亡;  软骨细胞;  Trx-2/ASK-1/Caspase-3信号通路
Keywords:
osteoarthris;  tetramethylpyrazine;  apoptosis;  chondrocytes;  Trx-2/ASK-1/Caspase-3 signaling pathway
摘要:
目的:探讨川芎嗪对人骨关节炎软骨细胞的影响及其作用机制。方法:收集接受膝关节置换术患者的膝关节软骨组织,分离并培养软骨细胞,采用肿瘤坏死因子-α诱导建立骨关节炎软骨细胞模型; 将骨关节炎软骨细胞随机分为对照组和川芎嗪低、中、高浓度组,对照组加入正常培养基进行培养,川芎嗪低、中、高浓度组分别加入含川芎嗪浓度为25 μg·mL-1、50 μg·mL-1、100 μg·mL-1 的培养基进行培养。培养24 h后,分别采用异硫氰酸荧光素标记的膜联蛋白V/碘化丙啶双染色法和MTT法测定软骨细胞的凋亡率和活力,分别采用实时定量PCR和蛋白印迹法分析软骨细胞凋亡相关基因硫氧还蛋白(thioredoxin,Trx)-2、凋亡信号调节激酶(apoptosis signal regulating kinase,ASK)-1、半胱氨酸天冬氨酸蛋白酶(cysteine aspartic acid specific protease,Caspase)-3的mRNA和蛋白表达。结果:①软骨细胞培养结果。原代细胞培养至第15天,可见组织块周围有细胞成簇生长,细胞呈梭形、三角形或多角形; 第3代软骨细胞多呈长梭形。②软骨细胞鉴定结果。甲苯胺蓝染色显示,细胞呈长梭形,有1～3个细胞核,细胞质呈蓝色; 免疫组化染色显示,细胞Ⅱ型胶原蛋白呈阳性,细胞质内可见黄色颗粒,细胞核无着色,表明为软骨细胞。③软骨细胞凋亡率测定结果。4组软骨细胞凋亡率比较,差异有统计学意义[(25.10±0.47)%,(22.08±0.25)%,(19.37±0.36)%,(16.05±0.58)%,F=13.776,P=0.000]。川芎嗪低、中、高浓度组软骨细胞凋亡率均低于对照组(LSD-t=12.685,P=0.000; LSD-t=21.642,P=0.000; LSD-t=27.107,P=0.000),川芎嗪中、高浓度组软骨细胞凋亡率均低于川芎嗪低浓度组(LSD-t=13.826,P=0.000; LSD-t=21.349,P=0.000),川芎嗪高浓度组软骨细胞凋亡率低于川芎嗪中浓度组(LSD-t=10.875,P=0.000)。④软骨细胞活力测定结果。4组软骨细胞活力比较,差异有统计学意义(吸光度值:0.25±0.04,0.41±0.02,0.54±0.02,0.60±0.01,F=131.875,P=0.000),川芎嗪低、中、高浓度组软骨细胞活力均高于对照组(LSD-t=8.000,P=0.000; LSD-t=14.500,P=0.000; LSD-t=18.981,P=0.000),川芎嗪中、高浓度组软骨细胞活力均高于川芎嗪低浓度组(LSD-t=10.277,P=0.000; LSD-t=19.000,P=0.000),川芎嗪高浓度组软骨细胞活力高于川芎嗪中浓度组(LSD-t=6.000,P=0.000)。⑤软骨细胞凋亡相关基因mRNA和蛋白表达分析结果。4组软骨细胞Trx-2、ASK-1及Caspase-3的mRNA和蛋白相对表达量比较,组间差异均有统计学意义。川芎嗪低、中、高浓度组软骨细胞Trx-2的mRNA和蛋白相对表达量均高于对照组(mRNA:LSD-t=6.925,P=0.000; LSD-t=15.581,P=0.000; LSD-t=16.046,P=0.000; 蛋白:LSD-t=2.479,P=0.000; LSD-t=23.000,P=0.000; LSD-t=33.988,P=0.000),川芎嗪中、高浓度组软骨细胞Trx-2的mRNA和蛋白相对表达量均高于川芎嗪低浓度组(mRNA:LSD-t=7.044,P=0.000; LSD-t=9.581,P=0.000; 蛋白:LSD-t=6.149,P=0.000; LSD-t=15.321,P=0.000),川芎嗪高浓度组软骨细胞Trx-2的mRNA和蛋白相对表达量均高于川芎嗪中浓度组(LSD-t=3.994,P=0.004; LSD-t=18.605,P=0.000); 川芎嗪低、中、高浓度组软骨细胞ASK-1和Caspase-3的mRNA和蛋白相对表达量均低于对照组(mRNA:LSD-t=8.808,P=0.000; LSD-t=10.398,P=0.000; LSD-t=19.350,P=0.000; LSD-t=3.796,P=0.000; LSD-t=5.096,P=0.000; LSD-t=10.028,P=0.000; 蛋白:LSD-t=5.041,P=0.001; LSD-t=13.466,P=0.000; LSD-t=21.719,P=0.000; LSD-t=2.481,P=0.038; LSD-t=7.286,P=0.001; LSD-t=16.865,P=0.000),川芎嗪中、高浓度组软骨细胞ASK-1和Caspase-3的mRNA和蛋白相对表达量均低于川芎嗪低浓度组(mRNA:LSD-t=3.385,P=0.000; LSD-t=20.466,P=0.000; LSD-t=3.400,P=0.000; LSD-t=9.701,P=0.000; 蛋白:LSD-t=8.296,P=0.000; LSD-t=17.303,P=0.000; LSD-t=6.228,P=0.000; LSD-t=17.365,P=0.000),川芎嗪高浓度组软骨细胞ASK-1和Caspase-3的mRNA和蛋白相对表达量均低于川芎嗪中浓度组(mRNA:LSD-t=9.550,P=0.005; LSD-t=3.619,P=0.007; 蛋白:LSD-t=14.017,P=0.005; LSD-t=4.985,P=0.001)。结论:川芎嗪能够抑制人骨关节炎软骨细胞的凋亡,提高软骨细胞活力,且该作用在一定浓度范围内呈浓度依赖性; 其作用机制可能与调控Trx-2/ASK-1/Caspase-3信号通路有关。
Abstract:
To explore the effects of tetramethylpyrazine(TMP)on human osteoarthritic(OA)chondrocytes and its mechanism of action.Methods:The chondrocytes were isolated from knee articular cartilage tissues collected from patients undergoing knee replacement and were cultured,and the third-generation ones were intervened by tumor necrosis factor(TNF)-α for inducing OA chondrocytes.The OA chondrocytes were randomly divided into control group,TMP low-concentration group,TMP middle-concentration group and TMP high-concentration group.The OA chondrocytes in control group were cultured in normal Dulbecco’s Modified Eagle’s Medium(DMEM),while the ones in TMP low-,middle- and high-concentration group were cultured in DMEM supplemented with TMP with concentration of 25,50 and 100 μg/mL respectively.After 24-hour culture,the chondrocyte apoptosis rate and viability were detected by using annexin V-fluorescein isothiocyanate(Annexin V-FIIC)/propidium iodide(PI)double-staining method and MTT method respectively; and the mRNA and protein expression levels of apoptosis-related genes,including thioredoxin(Trx)-2,apoptosis signal regulating kinase(ASK)-1 and cysteine aspartic acid specific protease(Caspase)-3,were analyzed by using real-time quantitative PCR(RT-qPCR)and Western-blot assays respectively.Results:After 15-day culture,the primary cells shaped as fusiform,triangle or polygon and clustered-growned around the tissue blocks,and the third-generation ones shaped mainly as long fusiform.The results of toluidine blue staining showed that the cells,with 1-3 nuclei and blue cytoplasm,shaped as long fusiform; and the immunohistochemical staining exhibited the results as positive typeⅡcollagen,yellow particles within cytoplasm and unstained nucleus...

参考文献/References:

[1] 王科艇,楼红侃,叶海.四妙汤口服联合腕踝针治疗轻中度膝骨关节炎湿热蕴结证[J].中医正骨,2019,31(5):41-43.
[2] 张琥,张旻,龚幼波,等.太极拳对早期膝骨关节炎患者步态影响的研究[J].中医正骨,2018,30(9):40-44.
[3] HE B,JIANG D M.HOTAIR-induced apoptosis is mediated by sponging miR-130a-3p to repress chondrocyte autophagy in knee osteoarthritis[J].Cell Biol Int,2020,44(2):524-535.
[4] CHEN R X,YE B Q,XIE H,et al.MiR-129-3p alleviates chondrocyte apoptosis in knee joint fracture-induced osteoarthritis through CPEB1[J].J Orthop Surg Res,2020,15(1):552.
[5] YU S Y,WU M F,ZHOU G M,et al.Potential utility of anti-TNF drugs in synovial chondromatosis associated with ankylosing spondylitis[J].Int J Rheum Dis,2019,22(11):2073-2079.
[6] ZHAO D Y,YU D D,REN L,et al.Ligustilide protects PC12 cells from oxygen-glucose deprivation/reoxygenation-induced apoptosis via the LKB1-AMPK-mTOR signaling pathway[J].Neural Regen Re,2020,15(3):473-481.
[7] 朱海泉,刘子敏,孟祥圣,等.川芎嗪抑制NF-κB p65磷酸化对LPS诱导的骨关节炎软骨细胞凋亡和炎症反应的调节作用[J].中国免疫学杂志,2019,35(2):181-185.
[8] 袁发浒,胡松,黄丽霞,等.基于基因芯片的TNF-α诱导骨关节炎细胞模型生物信息学分析[J].中国比较医学杂志,2019,29(7):53-60.
[9] 罗淦,汤样华,康仪,等.骨关节炎相关信号通路的研究进展[J].中医正骨,2019,31(5):31-37.
[10] 潘秀臣.自体脂肪干细胞结合PRP修复膝关节软骨[J].医药界,2019(6):180-181.
[11] 蒋翠云,任少琳,张纯萍,等.二氮嗪对氧化损伤状态下软骨细胞增殖和凋亡的影响机制研究[J].中国药房,2019,30(14):1890-1893.
[12] LIU W J,ZHA Z Q,WANG H T.Upregulation of microRNA-27a inhibits synovial angiogenesis and chondrocyte apoptosis in knee osteoarthritis rats through the inhibition of PLK2[J].J Cell Physiol,2019,234(12):22972-22984.
[13] ZHANG H,TANG W W,WANG S,et al.Tetramethylpyrazine inhibits platelet adhesion and inflammatory response in vascular endothelial cells by inhibiting P38 MAPK and NF-κB signaling pathways[J].Inflammation,2020,43(1):286-297.
[14] 段超,周细江,陈焱,等.人参皂苷Rgl对兔膝骨性关节炎关节软骨细胞凋亡的影响[J].中医学报,2018,33(12):2387-2392.
[15] FAN X S,WANG E S,HE J X,et al.Ligustrazine protects homocysteine-induced apoptosis in human umbilical vein endothelial cells by modulating mitochondrial dysfunction[J].J Cardiovasc Transl Res,2019,12(6):591-599.
[16] 曾利红,沈佳炎,方亮,等.川芎嗪联合塞来昔布对早期膝骨关节炎大鼠的影响[J].中国临床药理学杂志,2019,35(13):1363-1365.
[17] 车选义,赵清侠,李迪.硫氧还蛋白-2在氧化损伤的人晶状体上皮细胞中的表达及其意义[J].中南大学学报(医学版),2018,43(3):253-259.
[18] 余光书,林焱斌,熊国胜,等.补骨颗粒含药血清对大鼠软骨细胞凋亡及Trx2信号通路的影响[J].中国骨质疏松杂志,2019,25(6):27-31.
[19] BRANCO V,COPPO L,SOLá S,et al.Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury[J].Redox Biol,2017,13:278-287.

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通讯作者:李凤新 E-mail:jrzc2088@163.com
更新日期/Last Update: 1900-01-01