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[1]王會(huì)含,王永堂,苗建華,等.川芎嗪對(duì)人骨關(guān)節(jié)炎軟骨細(xì)胞的影響及作用機(jī)制研究[J].中醫(yī)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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川芎嗪對(duì)人骨關(guān)節(jié)炎軟骨細(xì)胞的影響及作用機(jī)制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期數(shù):
2021年07期
頁碼:
4-10
欄目:
基礎(chǔ)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:
王會(huì)含; 王永堂; 苗建華; 李鳳新
(鄭州大學(xué)附屬鄭州中心醫(yī)院,河南 鄭州 450007)
Author(s):
WANG Huihan; WANG Yongtang; MIAO Jianhua; LI Fengxin
Zhengzhou Central Hospital affiliated to Zhengzhou University,Zhengzhou 450007,Henan,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  川芎嗪;  細(xì)胞凋亡;  軟骨細(xì)胞;  Trx-2/ASK-1/Caspase-3信號(hào)通路
Keywords:
osteoarthris;  tetramethylpyrazine;  apoptosis;  chondrocytes;  Trx-2/ASK-1/Caspase-3 signaling pathway
摘要:
目的:探討川芎嗪對(duì)人骨關(guān)節(jié)炎軟骨細(xì)胞的影響及其作用機(jī)制。方法:收集接受膝關(guān)節(jié)置換術(shù)患者的膝關(guān)節(jié)軟骨組織,分離并培養(yǎng)軟骨細(xì)胞,采用腫瘤壞死因子-α誘導(dǎo)建立骨關(guān)節(jié)炎軟骨細(xì)胞模型; 將骨關(guān)節(jié)炎軟骨細(xì)胞隨機(jī)分為對(duì)照組和川芎嗪低、中、高濃度組,對(duì)照組加入正常培養(yǎng)基進(jìn)行培養(yǎng),川芎嗪低、中、高濃度組分別加入含川芎嗪濃度為25 μg·mL-1、50 μg·mL-1、100 μg·mL-1 的培養(yǎng)基進(jìn)行培養(yǎng)。培養(yǎng)24 h后,分別采用異硫氰酸熒光素標(biāo)記的膜聯(lián)蛋白V/碘化丙啶雙染色法和MTT法測(cè)定軟骨細(xì)胞的凋亡率和活力,分別采用實(shí)時(shí)定量PCR和蛋白印跡法分析軟骨細(xì)胞凋亡相關(guān)基因硫氧還蛋白(thioredoxin,Trx)-2、凋亡信號(hào)調(diào)節(jié)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和蛋白表達(dá)。結(jié)果:①軟骨細(xì)胞培養(yǎng)結(jié)果。原代細(xì)胞培養(yǎng)至第15天,可見組織塊周圍有細(xì)胞成簇生長(zhǎng),細(xì)胞呈梭形、三角形或多角形; 第3代軟骨細(xì)胞多呈長(zhǎng)梭形。②軟骨細(xì)胞鑒定結(jié)果。甲苯胺藍(lán)染色顯示,細(xì)胞呈長(zhǎng)梭形,有1～3個(gè)細(xì)胞核,細(xì)胞質(zhì)呈藍(lán)色; 免疫組化染色顯示,細(xì)胞Ⅱ型膠原蛋白呈陽性,細(xì)胞質(zhì)內(nèi)可見黃色顆粒,細(xì)胞核無著色,表明為軟骨細(xì)胞。③軟骨細(xì)胞凋亡率測(cè)定結(jié)果。4組軟骨細(xì)胞凋亡率比較,差異有統(tǒng)計(jì)學(xué)意義[(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、高濃度組軟骨細(xì)胞凋亡率均低于對(duì)照組(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、高濃度組軟骨細(xì)胞凋亡率均低于川芎嗪低濃度組(LSD-t=13.826,P=0.000; LSD-t=21.349,P=0.000),川芎嗪高濃度組軟骨細(xì)胞凋亡率低于川芎嗪中濃度組(LSD-t=10.875,P=0.000)。④軟骨細(xì)胞活力測(cè)定結(jié)果。4組軟骨細(xì)胞活力比較,差異有統(tǒng)計(jì)學(xué)意義(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、高濃度組軟骨細(xì)胞活力均高于對(duì)照組(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、高濃度組軟骨細(xì)胞活力均高于川芎嗪低濃度組(LSD-t=10.277,P=0.000; LSD-t=19.000,P=0.000),川芎嗪高濃度組軟骨細(xì)胞活力高于川芎嗪中濃度組(LSD-t=6.000,P=0.000)。⑤軟骨細(xì)胞凋亡相關(guān)基因mRNA和蛋白表達(dá)分析結(jié)果。4組軟骨細(xì)胞Trx-2、ASK-1及Caspase-3的mRNA和蛋白相對(duì)表達(dá)量比較,組間差異均有統(tǒng)計(jì)學(xué)意義。川芎嗪低、中、高濃度組軟骨細(xì)胞Trx-2的mRNA和蛋白相對(duì)表達(dá)量均高于對(duì)照組(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、高濃度組軟骨細(xì)胞Trx-2的mRNA和蛋白相對(duì)表達(dá)量均高于川芎嗪低濃度組(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),川芎嗪高濃度組軟骨細(xì)胞Trx-2的mRNA和蛋白相對(duì)表達(dá)量均高于川芎嗪中濃度組(LSD-t=3.994,P=0.004; LSD-t=18.605,P=0.000); 川芎嗪低、中、高濃度組軟骨細(xì)胞ASK-1和Caspase-3的mRNA和蛋白相對(duì)表達(dá)量均低于對(duì)照組(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、高濃度組軟骨細(xì)胞ASK-1和Caspase-3的mRNA和蛋白相對(duì)表達(dá)量均低于川芎嗪低濃度組(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),川芎嗪高濃度組軟骨細(xì)胞ASK-1和Caspase-3的mRNA和蛋白相對(duì)表達(dá)量均低于川芎嗪中濃度組(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)。結(jié)論:川芎嗪能夠抑制人骨關(guān)節(jié)炎軟骨細(xì)胞的凋亡,提高軟骨細(xì)胞活力,且該作用在一定濃度范圍內(nèi)呈濃度依賴性; 其作用機(jī)制可能與調(diào)控Trx-2/ASK-1/Caspase-3信號(hào)通路有關(guān)。
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...

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