于海杰,关启刚,郭亮,张心刚,孙国哲
YU Haijie,GUAN Qigang,GUO Liang,ZHANG Xingang,SUN Guozhe

• 1:中国医科大学附属第一医院

摘要(Abstract)：

目的:探讨雷公藤内酯醇(TPL)对H9c2细胞缺氧/复氧(H/R)损伤的保护作用机制。方法:将体外培养的H9c2大鼠心肌细胞分为对照组、H/R组及TPL+H/R组,TPL+H/R组用TPL预处理48 h后,再进行H/R处理;各组细胞处理结束后,CCK-8法检测H9c2细胞增殖情况,ELISA法检测H9c2细胞培养上清液中MDA、SOD及促炎因子TNF-α、IL-6、IL-1β的含量,Western blot检测H9c2细胞中Nrf2、HO-1、TLR4、MyD88及胞核中NF-κB p65的表达水平。结果:与对照组相比,H/R组H9c2细胞增殖受到明显抑制,细胞上清液中MDA和促炎因子含量增加,SOD活力下降,Nrf2、HO-1、TLR4、MyD88及胞核中NF-κB p65蛋白表达水平均显著上调;与H/R相比,TPL+H/R组细胞增殖抑制情况得到缓解,细胞上清液中MDA与促炎因子含量下降,SOD活力上升,Nrf2、HO-1蛋白水平进一步上调,而TLR4、MyD88及胞核中NF-κB p65表达水平显著下降。结论:TPL可能通过激活Nrf2/HO-1信号通路提高心肌细胞抗氧化应激的能力,同时可能通过抑制TLR4/MyD88/NF-κB p65信号通路缓解炎症反应,从而发挥心肌保护作用。
Objective: To investigate the protective mechanism of triptolide( TPL) on hypoxia/reoxygenation( H/R) injury in H9 c2 cells. Methods: The H9 c2 rat cardiomyocytes cultured in vitro were divided into control group,H/R group and TPL + H/R group( H/R treatment after the treatment with TPL for 48 h). CCK8 assay was used to determine cell proliferation. MDA,SOD and pro-inflammatory cytokines TNF-α,IL-6,IL-1β content in cell supernatants were detected using the corresponding ELISA kit. Western blot analysis was used to detect the protein expression levels of Nrf2,HO-1,TLR4,MyD88 in cells and NF-κB p65 in nuclei. Results: When compared to control group,H/R treatment caused shrinkage of H9 c2 cells,increased the number of round suspended cells,and significantly inhibited cell proliferation. The levels of MDA and pro-inflammatory cytokines in cellular supernatants were increased by H/R treatment. The activity of SOD was decreased. The expression levels of Nrf2,HO-1,TLR4,MyD88 in whole cell lysates and NF-κB p65 in nuclei were significantly upregulated. When Compared with H/R, TPL treatment significantly reversed H/R-mediated effects.Conclusion: TPL may increase the ability of anti-oxidative stress in cardiomyocytes by further activating the Nrf2/HO-1 signaling pathway,and at the same time,inhibit the inflammatory response by inhibiting the TLR4/MyD88/NF-κB p65 signaling pathway,thereby protecting the myocardium.

关键词(KeyWords)： 雷公藤内酯醇;心肌细胞;缺氧;复氧;氧化应激;炎症反应;信号传导
triptolide;cardiomyocytes;hypoxia;reoxygenation;oxidative stress;inflammatory response;signal transduction

Abstract:

Keywords:

基金项目(Foundation): 辽宁省自然科学基金资助项目(201602871)

作者(Author): 于海杰,关启刚,郭亮,张心刚,孙国哲
YU Haijie,GUAN Qigang,GUO Liang,ZHANG Xingang,SUN Guozhe

DOI: 10.19367/j.cnki.1000-2707.2019.02.013

参考文献(References)：

• [1] BHATNAGAR P,WICKRAMASINGHE K,WILLIAMSJ,et al. The epidemiology of cardiovascular disease inthe UK 2014[J]. Heart,2015,101(15):1182-1189.
• [2] THOMPSON S,JAMES M,WIEBE N,et al. Cause ofdeath in patients with reduced kidney function[J]. J AmSoc Nephrol,2015,26(10):2504-2511.
• [3]李卉,马敏,咸淑悦,等.加兰他敏对心肌缺血再灌注损伤大鼠血清CK-MB活性及TNF-α和IL-6水平的影响[J].贵州医科大学学报,2016,41(3):280-283.
• [4]徐彤彤.心肌缺血-再灌注损伤的瘦素机制研究新进展[J].中国急救医学,2010,30(6):548-551.
• [5]MAO X,TONG J,WANG Y,et al. Triptolide exhibitsantitumor effects by reversing hypermethylation of WIF1in lung cancer cells[J]. Mol Med Rep,2018,18(3):3041-3049.
• [6]HAO M,LI X,FENG J,et al. Triptolide protects againstischemic stroke in rats[J]. Inflammation,2015,38(4):1617-1623.
• [7] PENG W H,WANG J L,REN Y,et al. Inhibitoryeffects of PGA1 and TRI on the apoptosis of cardiac mi-crovascular endothelial cells of rats[J]. Exp Ther Med,2017,14(5):4288-4292.
• [8]GAO J M,MENG X W,ZHANG J,et al. Dexmedetomi-dine protects cardiomyocytes against hypoxia/reoxygen-ation injury by suppressing TLR4-MyD88-NF-kappaB sig-naling[J]. Biomed Res Int,2017,2017:1674613.
• [9]WANG D,CHEN T,LIU F. Betulinic acid alleviates my-ocardial hypoxia/reoxygenation injury via inducing Nrf2/HO-1 and inhibiting p38 and JNK pathways[J]. Eur JPharmacol,2018,838:53-59.
• [10]FRANTZ S,KOBZIK L,KIM Y D,et al. Toll4(TLR4)expression in cardiac myocytes in normal and failing myo-cardium[J]. J Clin Invest,1999,104(3):271-280.
• [11]蔺晓源,刘杰民. TLR4/MyD88/NF-κB信号通路与溃疡性结肠炎[J].胃肠病学,2013,18(4):244-246.
• [12]HUANG H C,NGUYEN T,PICKETT C B. Phosphoryla-tion of Nrf2 at Ser-40 by protein kinase C regulates an-tioxidant response element-mediated transcription[J]. JBiol Chem,2002,277(45):42769-42774.
• [13]LOBODA A,DAMULEWICZ M,PYZA E,et al. Role ofNrf2/HO-1 system in development,oxidative stress re-sponse and diseases:an evolutionarily conserved mecha-nism[J]. Cell Mol Life Sci,2016,73(17):3221-3247.
• [14]CHEN X,YAN L,GUO Z,et al. Adipose-derived mes-enchymal stem cells promote the survival of fat grafts viacrosstalk between the Nrf2 and TLR4 pathways[J]. CellDeath Dis,2016,7(9):e2369.
• [15]ZHANG B,CHEN Y,SHEN Q,et al. Myricitrin attenu-ates high glucose-induced apoptosis through activatingAkt-Nrf2 signaling in H9c2 cardiomyocytes[J]. Mole-cules,2016,21(7):901-906.
• [16]于佳佳.雌激素对Nrf2活化及HO-1,Cu/Zn-SOD表达的影响[D].长春:东北师范大学,2010.
• [17]ZHAO T T,YANG T L,GONG L,et al. Isorhamnetinprotects against hypoxia/reoxygenation-induced injure byattenuating apoptosis and oxidative stress in H9c2 cardio-myocytes[J]. Gene,2018,666:92-99.
• [18]乔丽杰,王延让,张明. Nrf2/HO-1通路在氧化损伤保护机制中研究进展[J].中国职业医学,2013,40(1):82-84.
• [19]FORESTI R,GOATLY H,GREEN C J,et al. Role ofheme oxygenase-1 in hypoxia-reoxygenation:requirementof substrate heme to promote cardioprotection[J]. Am JPhysiol Heart Circ Physiol,2001,281(5):H1976-H1984.
• [20]YU H,SHI L,ZHAO S,et al. Triptolide attenuates my-ocardial ischemia/reperfusion injuries in rats by inducingthe activation of Nrf2/HO-1 defense pathway[J]. Cardio-vasc Toxicol,2016,16(4):325-335.

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