目的:观察α2-肾上腺素能受体激动剂B-HT933对脂多糖(LPS)刺激的心肌细胞产生肿瘤坏死因子α(TNF-α)的影响,并初步分析其作用机制。方法:分离培养SD大鼠乳鼠心肌细胞。利用免疫荧光染色观察心肌α2A-肾上腺素能受体的分布;B-HT933和/或LPS处理心肌细胞一定时间后,用ELISA方法检测细胞培养液中TNF-α的含量、实时荧光定量PCR测定心肌细胞TLR4和TNF-αmRNA的表达、Western blot分析心肌细胞中相关信号分子的磷酸化水平。结果:免疫荧光染色证实乳鼠心肌细胞中存在α2A-肾上腺素能受体。LPS以剂量和时间依赖的方式刺激心肌细胞产生TNF-α,0.1μmol/L的B-HT933处理能显著抑制LPS诱导的TNF-αmRNA的表达和TNF-α蛋白的产生。而且,BHT能抑制LPS诱导的心肌细胞IκBα的磷酸化。结论:乳鼠心肌细胞上存在α2A-肾上腺素能受体,其激动剂B-HT933可能通过抑制心肌细胞IκBα的磷酸化来减少LPS诱导的TNF-α产生。
AIM:To observe the effect of B-HT933, a selective α2-adrenoceptor agonist, on lipopolysaccha-ride ( LPS )-induced TNF-αproduction in neonatal rat cardiomyocytes and to explore the underlying mechanisms . METHODS:The neonatal rat cardiomyocytes were cultured .The localization of α2A-adrenoceptor in the cardiomyocytes was examined by immunofluorescence staining .The cardiomyocytes were exposed to LPS or/and B-HT933 for different time.The level of TNF-αin the supernatants and the mRNA expression of TNF-αwere detected by ELISA and real-time PCR, respectively.In addition, LPS-associated signal molecules in the cardiomyocytes were also examined by Western blotting.RESULTS: Immunofluorescence staining showed that α2A-adrenoceptors were localized in the cardiomyocytes . LPS stimulated TNF-αproduction in the cardiomyocytes in a dose and time-dependent manner .B-HT933 pretreatment sig-nificantly inhibited the expression of TNF-αat mRNA and protein levels in LPS-treated cardiomyocytes .Furthermore, LPS exposure induced IκBαand p38 phosphorylation in cardiomyocytes and only IκBαphosphorylation was prevented by B-HT933 treatment.CONCLUSION:α2A-adrenoceptors are present in neonatal rat cardiomyocytes and its agonist B -HT933 inhibits LPS-induced TNF-αproduction in cardiomyocytes via suppressing IκBαphosphorylation .