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Received:February 18, 2020 Published Online:October 20, 2020
Received:February 18, 2020 Published Online:October 20, 2020
中文摘要: 目的 研究热休克蛋白22(HSP22)对氧化型低密度脂蛋白(ox-LDL)诱导的HBMEC人脑微血管内皮细胞(HBMEC)损伤的影响及其机制。
方法 使用含不同浓度(0、20、40、80、160 μg/ml)ox-LDL的培养基培养HBMEC细胞,Real-time PCR和Western blot检测HSP22和内皮型一氧化氮合酶(eNOS)mRNA和蛋白表达;ELISA法检测肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-1β含量;Griess法检测一氧化氮(NO)含量;在80 μg/ml ox-LDL培养的HBMEC细胞中,采用ELISA、Griess、Real-time PCR和Western blot法检测干扰或过表达HSP22后TNF-α、IL-6、IL-1β、NO含量和eNOS mRNA、蛋白表达的变化。
结果 ox-LDL能促进HBMEC细胞中HSP22表达(P<0.05),能诱导炎症因子TNF-α、IL-6、IL-1β释放,并抑制NO合成和eNOS mRNA表达,均呈浓度依赖性(P均<0.05)。在80 μg/ml ox-LDL培养的HBMEC细胞中,干扰HSP22,炎症因子TNF-α、IL-6、IL-1β分泌增加,NO合成减少,eNOS mRNA表达下调(P均<0.05);过表达HSP22则结果相反。
结论 ox-LDL能够诱导HBMEC细胞功能损伤,HSP22通过抑制TNF-α、IL-6、IL-1β分泌和NO合成并促进eNOS 表达,对HBMEC细胞发挥保护作用。
Abstract:Objective To investigate the effect of heat shock protein 22 (HSP22) on oxidized low density lipoprotein (ox-LDL)-induced human brain microvascular endothelial cells (HBMEC) and its molecular mechanism.
Methods HBEMC cells were cultured in medium containing different concentrations (0,20,40,80 or 160 μg/ml) of ox-LDL.The protein and mRNA expressions of HSP22 and endothelial nitric oxide synthase (eNOS) in HBEMC cells were detected by Western blot and Real-time PCR,respectively;the levels of TNF-α,IL-6 and IL-1β were respectively detected by ELISA,and the levels of NO was detected by Griess method.After knockdown or over-expression of HSP22 in HBMEC cells cultured in medium containing 80 μg/ml ox-LDL,the contents of TNF-α,IL-6,IL-1β,NO and expression of eNOS mRNA and protein were detected by ELISA,Griess Real-time PCR and Western blot,respectively.
Results Ox-LDL promoted the expression of HSP22 in HBMEC cells (P<0.05),induced the release of TNF-α,IL-6 and IL-1β and inhibited the synthesis of NO and expression of eNOS mRNA with concentration-dependent manner(all P<0.05).In HBMEC cells cultured in medium containing 80 μg/ml ox-LDL,the levels of TNF-α,IL-6 and IL-1β significantly increased and NO synthesis decreased and eNOS mRNA expression was down-regulated(all P<0.05) after knockdown of HSP22,however,the opposite results appeared after overexpression of HSP22 in HBMEC cells.
Conclusion Ox-LDL could induce functional damage of HBMEC cells,and HSP22 could protect HBMEC cells by inhibiting TNF-α,IL-6,IL-1β secretion and NO synthesis and promoting the expression of eNOS.
keywords: Human brain microvascular endothelial cells Oxidized low-density lipoprotein Heat shock protein 22 Inflammation factor Endothelial nitric oxide synthase
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