Division of Pediatric Cardiology, Department of Pediatrics, Changhua Christian Children's Hospital, Changhua 50050, Taiwan.
School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Molecules. 2019 Apr 8;24(7):1376. doi: 10.3390/molecules24071376.
To test whether KMUP-1 (7-[2-[4-(2-chlorophenyl) piperazinyl]ethyl]-1,3-dimethylxanthine) prevents myocardial ischemia-induced apoptosis, we examined KMUP-1-treated H9c2 cells culture. Recent attention has focused on the activation of nitric oxide (NO)-guanosine 3', 5'cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway triggered by mitogen-activated protein kinase (MAPK) family, including extracellular-signal regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 in the mechanism of cardiac protection during ischemia-induced cell-death. We propose that KMUP-1 inhibits ischemia-induced apoptosis in H9c2 cells culture through these pathways. Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and apoptotic evaluation was conducted using DNA ladder assay and Hoechst 33342 staining. The level of intracellular calcium was detected using - Fura2-acetoxymethyl (Fura2-AM) staining, and mitochondrial calcium with Rhod 2-acetoxymethyl (Rhod 2-AM) staining under fluorescence microscopic observation. The expression of endothelium NO synthase (eNOS), inducible NO synthase (iNOS), soluble guanylate cyclase α1 (sGCα1), PKG, Bcl-2/Bax ratio, ERK1/2, p38, and JNK proteins were measured by Western blotting assay. KMUP-1 pretreatment improved cell viability and inhibited ischemia-induced apoptosis of H9c2 cells. Calcium overload both in the intracellular and mitochondrial sites was attenuated by KMUP-1 pretreatment. Moreover, KMUP-1 reduced intracellular reactive oxygen species (ROS), increased plasma NOx (nitrite and nitrate) level, and the expression of eNOS. Otherwise, the iNOS expression was downregulated. KMUP-1 pretreatment upregulated the expression of sGCα1 and PKG protein. The ratio of Bcl-2/Bax expression was increased by the elevated level of Bcl2 and decreased level of Bax. In comparison with the ischemia group, KMUP-1 pretreatment groups reduced the expression of phosphorylated extracellular signal-regulated kinases ERK1/2, p-p38, and p-JNK as well. Therefore, KMUP-1 inhibits myocardial ischemia-induced apoptosis by restoration of cellular calcium influx through the mechanism of NO-cGMP-MAPK pathways.
为了测试 KMUP-1（7-[2-[4-(2-氯苯基)哌嗪基]乙基]-1,3-二甲基黄嘌呤）是否能预防心肌缺血诱导的细胞凋亡，我们检测了 KMUP-1 处理的 H9c2 细胞培养物。最近，人们的注意力集中在由丝裂原活化蛋白激酶（MAPK）家族激活的一氧化氮（NO）-鸟苷 3'，5'环单磷酸（cGMP）-蛋白激酶 G（PKG）信号通路，包括细胞外信号调节激酶 1/2（ERK1/2）、c-Jun N 末端激酶（JNK）和 p38 在缺血诱导的细胞死亡过程中心肌保护的机制中。我们提出，KMUP-1 通过这些途径抑制 H9c2 细胞培养物中缺血诱导的细胞凋亡。使用 MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐）测定法评估细胞活力，并通过 DNA 梯状测定法和 Hoechst 33342 染色进行凋亡评估。通过 - Fura2-乙酰氧基甲酯（Fura2-AM）染色检测细胞内钙离子水平，通过 Rhod 2-乙酰氧基甲酯（Rhod 2-AM）染色在荧光显微镜下观察线粒体钙离子。使用 Western blot 测定法测量内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）、可溶性鸟苷酸环化酶α1（sGCα1）、PKG、Bcl-2/Bax 比值、ERK1/2、p38 和 JNK 蛋白的表达。KMUP-1 预处理可改善 H9c2 细胞活力并抑制缺血诱导的细胞凋亡。细胞内和线粒体部位的钙超载均被 KMUP-1 预处理减弱。此外，KMUP-1 降低了细胞内活性氧（ROS）的水平，增加了血浆 NOx（亚硝酸盐和硝酸盐）水平，并增加了 eNOS 的表达。相反，iNOS 的表达被下调。KMUP-1 预处理上调了 sGCα1 和 PKG 蛋白的表达。Bcl-2/Bax 表达比值通过升高 Bcl2 水平和降低 Bax 水平来增加。与缺血组相比，KMUP-1 预处理组还降低了磷酸化细胞外信号调节激酶 ERK1/2、p-p38 和 p-JNK 的表达。因此，KMUP-1 通过 NO-cGMP-MAPK 通路的机制恢复细胞内钙内流，从而抑制心肌缺血诱导的细胞凋亡。
