Yu Jiangkun, Lu Yanyu, Li Yapeng, Xiao Lili, Xing Yu, Li Yanshen, Wu Leiming
Department of Cardiology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.
J Pharm Pharmacol. 2015 Sep;67(9):1240-50. doi: 10.1111/jphp.12415. Epub 2015 Apr 16.
S100A1 plays a crucial role in hypoxia-induced inflammatory response in cardiomyocytes. However, the role of S100A1 in hypoxia-induced inflammatory response in cardiomyocytes is still unknown.
enzyme-linked immunosorbent assay (ELISA) was performed for the determination of inflammatory cytokines. Immunocytochemistry and immunofluorescence, Western blot analysis and Real-time polymerase chain reaction (RT-PCR) were conducted to assess protein or mRNA expressions. Fluorogenic probe dihydroethidium (DHE) was used to evaluate the generation of reactive oxygen species (ROS) while Hoechst 33342 staining for apoptosis. Small interfering RNA (siRNA) for S100A1 was used to evaluate the role of S100A1.
The levels of ROS and inflammatory cytokine including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8 in H9c2 cells were increased remarkably by hypoxia. However, IL-37 protein or mRNA levels were decreased significantly. Both Toll-like receptor 4 (TLR4) inhibitor Ethyl (6R)-6-[N-(2-Chloro-4fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) treatment or siRNA S100A1 downregulated TLR4 expression and inflammatory cytokine level and mRNA in H9c2 cells, as well as weakening ROS and phospho-p65 Nuclear factor (NF)-κB levels. Further, S100A1 treatment significantly reduced TNF-α protein or mRNA level whereas enhanced IL-37 protein or mRNA level, and could attenuate ROS and phospho-p65 NF-κB levels.
Our results demonstrate that S100A1 can regulate the inflammatory response and oxidative stress in H9C2 cells via TLR4/ROS/NF-κB pathway. These findings provide an interesting strategy for protecting cardiomyocytes from hypoxia-induced inflammatory response.
S100A1在心肌细胞缺氧诱导的炎症反应中起关键作用。然而，S100A1在心肌细胞缺氧诱导的炎症反应中的作用仍不清楚。
采用酶联免疫吸附测定（ELISA）法测定炎性细胞因子。进行免疫细胞化学和免疫荧光、蛋白质印迹分析及实时聚合酶链反应（RT-PCR）以评估蛋白质或mRNA表达。用荧光探针二氢乙锭（DHE）评估活性氧（ROS）的生成，用Hoechst 33342染色检测细胞凋亡。使用针对S100A1的小干扰RNA（siRNA）评估S100A1的作用。
缺氧显著增加H9c2细胞中ROS及炎性细胞因子（包括肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6和IL-8）的水平。然而，IL-37的蛋白质或mRNA水平显著降低。Toll样受体4（TLR4）抑制剂乙基（6R）-6-[N-（2-氯-4-氟苯基）氨磺酰基]环己-1-烯-1-羧酸酯（TAK-242）处理或siRNA S100A1均可下调H9c2细胞中TLR4表达、炎性细胞因子水平及mRNA，同时减弱ROS和磷酸化p65核因子（NF）-κB水平。此外，S100A1处理显著降低TNF-α蛋白质或mRNA水平，而提高IL-37蛋白质或mRNA水平，并可减弱ROS和磷酸化p65 NF-κB水平。
我们的结果表明，S100A1可通过TLR4/ROS/NF-κB途径调节H9C2细胞中的炎症反应和氧化应激。这些发现为保护心肌细胞免受缺氧诱导的炎症反应提供了一种有趣的策略。
