Meng Ying, Li Wei-Zhu, Shi You-Wei, Zhou Bing-Feng, Ma Rong, Li Wei-Ping
Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei, Anhui 230032, P.R. China.
Department of Cardiovascular Medicine, Binhu Hospital of Hefei City, Hefei, Anhui 230011, P.R. China.
Int J Mol Med. 2016 Jan;37(1):258-66. doi: 10.3892/ijmm.2015.2419. Epub 2015 Nov 25.
Ischemia-reperfusion (I/R) plays an important role in myocardial injury. In the present study, we aimed to examine the protective effects of Danshensu (DSS) against I/R injury and to elucidate the underlying mechanisms. For this purpose, H9c2 cells were cultured in hypoxic solution in a hypoxic incubator for 2 h, and then cultured in a high oxygen incubator for various periods of time and pre-treated with or without DSS, ammonium pyrrolidine dithiocarbamate (PDTC) or SP600125 [a c-Jun N-terminal kinase (JNK) inhibitor]. Cell apoptosis and cytosolic free Ca2+ ([Ca2+]i) levels were analyzed by flow cytometry. The protein expression levels of JNK, phosphorylated (p-)JNK, nuclear factor-κB (NF-κB) and transient receptor potential cation channel, subfamily C, member 6 (TRPC6) were measured by western blot analysis. The mRNA expression levels of JNK were measured by RT-qPCR. The results revealed that TRPC6 protein expression, the cell apoptotic rate and the [Ca2+]i levels increased in a time-dependent manner in the H9c2 cells following the induction of I/R injury. The apoptotic rate and TRPC6 protein expression decreased when the cells were treated with DSS prior to the induction of I/R injury. The knockdown of JNK expression by siRNA decreased the p-JNK and TRPC6 protein expression levels in the H9c2 cells subjected to I/R injury. The protein expression levels of p-JNK and NF-κB in the nucleus increased significantly when the H9c2 cells were subjected to I/R injury, whereas NF-κB expression in the cytoplasm decreased in a time‑dependent manner. However, p-JNK, NF-κB and TRPC6 protein expression, the [Ca2+]i level and cell apoptosis decreased when the H9c2 cells were pre-treated with DSS or SP600125. Therefore, our data suggest that DSS prevents myocardial I/R injury by inhibiting p-JNK activation and NF-κB translocation, which potentially upregulate TRPC6 expression, increase the [Ca2+]i level, and result in the apoptosis of H9c2 cells.
缺血再灌注（I/R）在心肌损伤中起重要作用。在本研究中，我们旨在研究丹参素（DSS）对I/R损伤的保护作用，并阐明其潜在机制。为此，将H9c2细胞在缺氧培养箱中的缺氧溶液中培养2小时，然后在高氧培养箱中培养不同时间，并分别用或不用DSS、吡咯烷二硫代氨基甲酸铵（PDTC）或SP600125 [一种c-Jun氨基末端激酶（JNK）抑制剂]进行预处理。通过流式细胞术分析细胞凋亡和胞质游离Ca2+（[Ca2+]i）水平。通过蛋白质印迹分析测量JNK、磷酸化（p-）JNK、核因子-κB（NF-κB）和瞬时受体电位阳离子通道亚家族C成员6（TRPC6）的蛋白表达水平。通过RT-qPCR测量JNK的mRNA表达水平。结果显示，在诱导I/R损伤后H9c2细胞中，TRPC6蛋白表达、细胞凋亡率和[Ca2+]i水平呈时间依赖性增加。在诱导I/R损伤前用DSS处理细胞时，凋亡率和TRPC6蛋白表达降低。用siRNA敲低JNK表达可降低遭受I/R损伤的H9c2细胞中的p-JNK和TRPC6蛋白表达水平。当H9c2细胞遭受I/R损伤时，细胞核中p-JNK和NF-κB的蛋白表达水平显著增加，而细胞质中NF-κB表达呈时间依赖性降低。然而，当H9c2细胞用DSS或SP600125预处理时，p-JNK、NF-κB和TRPC6蛋白表达、[Ca2+]i水平和细胞凋亡均降低。因此，我们的数据表明，DSS通过抑制p-JNK激活和NF-κB易位来预防心肌I/R损伤，这可能上调TRPC6表达、增加[Ca2+]i水平并导致H9c2细胞凋亡。
