Rui Qianyun, Cao Shugang, Wang Xiaozhu, Duan Xiaoyu, Iao Xinyi, Dong Wanli, Fang Qi, Zhang Xueguang, Xue Qun
Department of Neurology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P.R. China.
Department of Neurology, The Second People's Hospital of Hefei, Hefei, Anhui 230011, P.R. China.
Exp Ther Med. 2021 Mar;21(3):219. doi: 10.3892/etm.2021.9621. Epub 2021 Jan 18.
Microglia activation plays vital roles in neuroinflammatory pathologys. Lemurs tyrosine kinase 2 (LMTK2) was reported to regulate NF-κB signals. In the present study, the roles of LMTK2 were investigated in lipopolysaccharide (LPS)-treated BV-2 cells. Reverse transcription-quantitative (RT-q)PCR and western blotting (WB) were utilized to analyze LMTK2 levels in LPS-treated BV2 cells. MTT assay determined cell viabilities. Nitric oxide (NO) and prostaglandin E2 (PGE2) levels were assessed through Griess and enzyme-linked immunosorbent assay (ELISA), respectively. The expression level of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected through RT-qPCR and WB. The release of inflammatory mediators under LPS stimulation, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-10, were analyzed through ELISA. WB was used to analyze the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1)/NAD(P)H dehydrogenase quinone 1 (NQO1) signal pathway. The results showed that the levels of the inflammatory mediators, iNOS, NO, COX-2 and PGE2, along with pro-inflammatory factors, TNF-α, IL-1β and IL-6, were significantly decreased following the induction of exogenous LMTK2 expression by LMTK2 overexpression plasmids in LPS-induced BV2 microglia. In contrast, anti-inflammatory factor IL-10 showed obvious decrease. Additionally, LMTK2 overexpression induced the elevation of Nrf2 in the cytoplasm and nucleus, along with the upregulation of HO-1 and NQO1 expression. In conclusion, LMTK2 is postulated to regulate neuroinflammation possibly through Nrf2 pathway. The present study is essential to reveal the underlying function of LMTK2 and to identify novel therapeutic targets for drug development in treating neuroinflammation.
小胶质细胞激活在神经炎症病理过程中发挥着至关重要的作用。据报道，狐猴酪氨酸激酶2（LMTK2）可调节核因子κB信号。在本研究中，研究了LMTK2在脂多糖（LPS）处理的BV-2细胞中的作用。采用逆转录定量（RT-q）PCR和蛋白质印迹法（WB）分析LPS处理的BV2细胞中LMTK2的水平。MTT法测定细胞活力。分别通过格里斯法和酶联免疫吸附测定（ELISA）评估一氧化氮（NO）和前列腺素E2（PGE2）水平。通过RT-qPCR和WB检测诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）的表达水平。通过ELISA分析LPS刺激下炎症介质肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、IL-6和IL-10的释放。采用WB分析核因子红细胞2相关因子2（Nrf2）/血红素加氧酶1（HO-1）/NAD（P）H脱氢酶醌1（NQO1）信号通路。结果显示，在LPS诱导的BV2小胶质细胞中，通过LMTK2过表达质粒诱导外源性LMTK2表达后，炎症介质、iNOS、NO、COX-2和PGE2的水平以及促炎因子TNF-α、IL-1β和IL-6均显著降低。相比之下，抗炎因子IL-10则明显升高。此外，LMTK2过表达诱导细胞质和细胞核中Nrf2升高，同时HO-1和NQO1表达上调。总之，推测LMTK2可能通过Nrf2途径调节神经炎症。本研究对于揭示LMTK2的潜在功能以及确定治疗神经炎症药物开发的新治疗靶点至关重要。
