Department of Ultrasound, Weihai Municipal Hospital, Weihai, Shandong 264200, China.
Department of Cardiology, People's Hospital of Gaotang County, Liaocheng, Shandong 252800, China.
J Healthc Eng. 2022 Mar 26;2022:8124343. doi: 10.1155/2022/8124343. eCollection 2022.
The aim of this study was to evaluate whether myosin light chain kinase (MLCK) knockdown attenuated H9C2 cell hypoxia/reoxygenation (H/R) injury and downstream signaling pathway.
The MLCK expression in H/R injury model H9C2 cell was determined by western blot and qRT-PCR. H/R cells were transfected with si-MLCK in the presence of P38 inhibitor (SB203580) or ERK inhibitor (U0126). Then, cell apoptosis was verified by flow cytometry. Apoptosis-related proteins were detected by western blot. The contents of reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), interleukin-6 (IL-6), interleukin (IL)-1 (IL-1), and tumor necrosis factor- (TNF-) were measured using flow cytometry and colorimetric assays, respectively.
MLCK expression was higher in H/R cells. Knockdown of MLCK diminished the amounts of ROS, LDH, IL-6, IL-1 and TNF- and elevated the release of SOD in H/R model H9C2 cells. Additionally, H/R injury induced the cumulative expression and phosphorylation of ERK and the phosphorylation of P38, whereas MLCK siRNA-treated cells showed decreased ERK1/2 and P38 activation. Inversely, P38 inhibitor (SB203580) and ERK inhibitor (U0126) could reverse the cardioprotective effects induced by si-MLCK.
MLCK knockdown attenuated H/R injury in H9C2 cells via regulating the ERK/P38 signaling pathway. MLCK/ERK/p38 axis may provide novel insight into therapeutic targets to restrain I/R injury caused by revascularization therapy after acute myocardial infarction.
本研究旨在评估肌球蛋白轻链激酶（MLCK）敲低是否减轻 H9C2 细胞缺氧/复氧（H/R）损伤及其下游信号通路。
通过 Western blot 和 qRT-PCR 测定 H/R 损伤模型 H9C2 细胞中 MLCK 的表达。在存在 P38 抑制剂（SB203580）或 ERK 抑制剂（U0126）的情况下，用 si-MLCK 转染 H/R 细胞。然后，通过流式细胞术验证细胞凋亡。通过 Western blot 检测凋亡相关蛋白。使用流式细胞术和比色法分别测定活性氧（ROS）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、白细胞介素-6（IL-6）、白细胞介素-1（IL-1）和肿瘤坏死因子-（TNF-）的含量。
H/R 细胞中 MLCK 表达增加。MLCK 敲低减少了 H/R 模型 H9C2 细胞中 ROS、LDH、IL-6、IL-1 和 TNF-的含量，并提高了 SOD 的释放量。此外，H/R 损伤诱导 ERK 和 P38 的累积表达和磷酸化，而 MLCK siRNA 处理的细胞显示 ERK1/2 和 P38 激活减少。相反，P38 抑制剂（SB203580）和 ERK 抑制剂（U0126）可以逆转 si-MLCK 诱导的心脏保护作用。
MLCK 敲低通过调节 ERK/P38 信号通路减轻 H9C2 细胞的 H/R 损伤。MLCK/ERK/p38 轴可能为急性心肌梗死后再血管化治疗引起的 I/R 损伤提供新的治疗靶点。
