HMGB1-TLR4促进缺氧复氧介导的大鼠肾小管上皮细胞凋亡

HMGB1-TLR4 promotes anoxia/reoxygenation injury-mediated apoptosis of rat proximal tubular epithelial cells

目的研究缺氧复氧后大鼠肾小管上皮细胞中高迁移率族蛋白1(HMGB1)和Toll样受体4(TLR4)的表达及其在肾小管上皮细胞凋亡中的调控机制。方法大鼠原代肾小管上皮细胞(PTECs)置于6孔培养板培养,随机分为对照组(control)、缺氧复氧组(A/R)、HMGB1抗体处理组(A/R+HMGB1)和TLR4抗体处理组(A/R+TLR4)(每组n=3)。用RT-PCR检测HMGB1和TLR4 mRNA的表达;Western blot法检测HMGB1、TLR4及凋亡相关分子Bcl-2、Bax、CHOP和caspase-8的蛋白表达;流式细胞术检测细胞凋亡。结果 A/R组HMGB1和TLR4的mRNA和蛋白的表达水平较对照组均显著上调(P<0.01),细胞凋亡水平较对照组显著增加(P<0.01),凋亡相关分子Bax、CHOP和caspase-8蛋白表达水平较对照组显著增加(P<0.01),Bcl-2蛋白表达较正常对照组下调(P<0.05)。经抗体处理后,HMGB1抗体处理组和TLR4抗体处理组PTECs细胞凋亡水平较缺氧复氧组显著下降(P<0.05),凋亡相关分子Bax、CHOP和caspase-8蛋白表达水平较缺氧复氧组下降(P<0.05),Bcl-2蛋白表达变化不明显。结论 HMGB1-TLR4轴可促进缺氧复氧介导的大鼠肾小管上皮细胞凋亡。

Objective To study the expression of high mobility group box-1 protein （HMGB1） and Toll-like receptor 4 （TLR4） in renal proximal epithelial cells following by anoxia/reoxygenation injury and the underlying mechanisms in cell apoptosis.Methods Rat primary proximal tubule epithelial cells （PTECs） were cultured and randomly divided into control,anoxia/reoxygenation group （A/R）,HMGB1 antibody treatment group （A/R ＋ HMGB1） and TLR4 antibody treatment group （A/R ＋TLR4）.RT-PCR was used to detect the mRNA level of HMGB1 and TLR4 ; Western blot was used to detect the protein expression of HMGB1,TLR4 and the apoptosis-related factors.Flow cytometry was used to detect apoptosis.Results The expression of mRNA and protein of TLR4 and HMGB1 significantly increased in A/R group as compared to control（P 〈 0.01）.The apoptosis cells in A/R group increased significantly compared as with control （P 〈 0.01）.The expression of Bax,CHOP and caspase-8 significantly increased as compared with control （P 〈 0.01）.While the expression of Bcl-2 decreased （P 〈 0.05）.After HMGB1 and TLR4 antibodies treatment,the apoptosis cells in A/R ＋ HMGB1 group and A/R ＋ TLR4 group significantly decreased as compared to A/R group （P 〈 0.05）.The expression of Bax,CHOP and caspase-8 also decreased significantly as compared with A/R group （P 〈 0.05）.The change of Bcl-2 protein expression was not obviously.Conclusions HMGB1-TLR4 axis promotes apoptosis of PTECs following A/R injury.