Huang Zhongyi, Zhao Dan, Wang Yongjian, Li Xiaolei, Li Jianqiu, Han Jie, Jiang Lisi, Ai Fen, Zhou Zhaoxiong
Department of Emergency, Shenzhen Hospital of Southern Medical University, Shenzhen, Guangdong 518000, P.R. China.
Department of Emergency, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430000, P.R. China.
Exp Ther Med. 2021 Oct;22(4):1139. doi: 10.3892/etm.2021.10573. Epub 2021 Aug 8.
C1q/TNF-related protein 9 (CTRP9) acts as an adipokine and has been reported to exert numerous biological functions, such as anti-inflammatory and anti-oxidative stress effects, in ischemic heart disease. In the present study, the role of CTRP9 in neonatal rat cardiomyocytes (NRCMs) following hypoxia/reoxygenation (H/R) and the underlying mechanism was investigated. Adenoviral vectors containing CTRP9 or green fluorescent protein were transfected into NRCMs. A H/R model was constructed 2 days after transfection by 2 h incubation under hypoxia followed by 4 h of reoxygenation. Lactate dehydrogenase (LDH), creatine kinase (CK) and CK-myocardial band (CK-MB) levels were detected by a biochemical analyzer using biochemical kits. In addition, cell viability was detected using trypan blue staining to determine the extent of cell injury. Inflammatory cytokines TNF-α, IL-6 and IL-10 were measured by ELISA. Western blotting and reverse transcription-quantitative PCR were used to evaluate the expression levels of CTRP9, toll-like receptor 4 (TLR4), myeloid differentiation primary response (MyD88) and NF-κB. The DNA binding activity of NF-κB was also detected using an electrophoretic mobility shift assay. The results indicated that transfection with adenoviral vectors containing CTRP9 could markedly enhance CTRP9 expression. CTRP9 overexpression increased cell viability and decreased the release of LDH, CK and CK-MB. In addition, CTRP9 overexpression reduced TNF-α and IL-6 levels whilst increasing IL-10 levels, but decreased the expression of TLR4, MyD88 and NF-κB. Furthermore, the DNA binding activity of NF-κB under H/R was also decreased by CTRP9 overexpression. In conclusion, the results of the present study suggested that CTRP9 could protect cardiomyocytes from H/R injury, which was at least partially due to the inhibition of the TLR4/MyD88/NF-κB signaling pathway to reduce the release of inflammatory cytokines.
C1q/TNF相关蛋白9（CTRP9）作为一种脂肪因子，据报道在缺血性心脏病中发挥多种生物学功能，如抗炎和抗氧化应激作用。在本研究中，研究了CTRP9在新生大鼠心肌细胞（NRCMs）缺氧/复氧（H/R）后的作用及其潜在机制。将含有CTRP9或绿色荧光蛋白的腺病毒载体转染到NRCMs中。转染2天后，通过缺氧孵育2小时，然后复氧4小时构建H/R模型。使用生化试剂盒通过生化分析仪检测乳酸脱氢酶（LDH）、肌酸激酶（CK）和肌酸激酶同工酶（CK-MB）水平。此外，使用台盼蓝染色检测细胞活力以确定细胞损伤程度。通过酶联免疫吸附测定法（ELISA）测量炎性细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-10（IL-10）。使用蛋白质免疫印迹法和逆转录定量聚合酶链反应（RT-qPCR）评估CTRP9、Toll样受体4（TLR4）、髓样分化初级反应蛋白（MyD88）和核因子κB（NF-κB）的表达水平。还使用电泳迁移率变动分析检测NF-κB的DNA结合活性。结果表明，用含有CTRP9的腺病毒载体转染可显著增强CTRP9表达。CTRP9过表达增加细胞活力并减少LDH、CK和CK-MB的释放。此外，CTRP9过表达降低TNF-α和IL-6水平，同时增加IL-10水平，但降低TLR4、MyD88和NF-κB的表达。此外，CTRP9过表达还降低了H/R条件下NF-κB的DNA结合活性。总之，本研究结果表明，CTRP9可保护心肌细胞免受H/R损伤，这至少部分归因于抑制TLR4/MyD88/NF-κB信号通路以减少炎性细胞因子的释放。
