PDF(Pubmed)
Abstract：
Activated macrophages serve a key role in various inflammatory diseases, such as atherosclerosis and septic shock. Tripartite motif-containing protein 65 (TRIM65) has been previously reported to participate in tumor progression and lung inflammation. However, the molecular mechanisms that controls its expression under inflammatory conditions and its consequences in activated macrophages remain poorly understood. The present study first collected the tissues of C57BL/6J mice, smooth muscle cells, macrophages and endothelial cells to determine the expression and distribution of TRIM65 by reverse transcription-quantitative (RT-q) PCR and western blotting. Mouse and human macrophages were treated with LPS and C57BL/6J mice were intraperitoneally injected with LPS followed by isolation of spleen, lung, aorta and bone marrow. Following treatment, TRIM65 mRNA and protein level was examined by RT-qPCR and western blotting. The results showed that TRIM65 was highly expressed in organs of the immune system, such as the spleen, lymph node and thymus, but lowly expressed in heart, liver, brain and kidneys. TRIM65 was also highly expressed in macrophages and endothelial cells. TRIM65 mRNA and protein expression levels were found to be decreased in LPS-treated macrophages in vitro and in tissues isolated from C57BL/6J mice intraperitoneally injected with LPS in vivo. In addition, to identify the signaling pathways by which LPS regulates TRIM65 expression, inhibitors of MAPK and Akt signaling pathways were used to treat macrophages followed by examination the expression of TRIM65 by western blotting. The results demonstrated that LPS-inhibited TRIM65 expression was blocked by treatment with the ERK1/2 inhibitor U0126. Moreover, the RT-qPCR results showed that TRIM65 knockout potentiated LPS-induced expression of inflammatory cytokines in macrophages. Taken together, data from the present study suggest that LPS decreased TRIM65 expression in macrophages and C57BL/6J mouse by activating the ERK1/2 signaling pathway, whilst TRIM65 knockout promoted macrophage activation. This information may facilitate the development of potential therapeutic strategies for the prevention and treatment of inflammatory diseases, such as atherosclerosis.
摘要：
活化的巨噬细胞在各种炎性疾病中起关键作用，如动脉粥样硬化和感染性休克。先前已经报道了含三方基序的蛋白65(TRIM65)参与肿瘤进展和肺部炎症。然而,在炎症条件下控制其表达的分子机制及其在活化巨噬细胞中的作用仍知之甚少.本研究首先收集了C57BL/6J小鼠的组织,平滑肌细胞，巨噬细胞和内皮细胞通过逆转录定量(RT-q)PCR和蛋白质印迹法测定TRIM65的表达和分布。小鼠和人巨噬细胞用LPS处理，C57BL/6J小鼠腹膜内注射LPS，然后分离脾脏，肺，主动脉和骨髓.治疗后,通过RT-qPCR和蛋白质印迹检测TRIM65mRNA和蛋白质水平。结果表明，TRIM65在免疫系统器官中高表达，比如脾脏，淋巴结和胸腺,但内心低落地表达，肝脏,大脑和肾脏.TRIM65在巨噬细胞和内皮细胞中也高表达。发现TRIM65mRNA和蛋白质表达水平在体外经LPS处理的巨噬细胞中以及在体内腹膜内注射LPS的C57BL/6J小鼠中分离的组织中降低。此外,为了确定LPS调节TRIM65表达的信号通路，使用MAPK和Akt信号通路抑制剂治疗巨噬细胞,然后通过蛋白质印迹法检测TRIM65的表达.结果表明,用ERK1/2抑制剂U0126处理阻断了LPS抑制的TRIM65表达。此外，RT-qPCR结果显示TRIM65基因敲除增强了LPS诱导的巨噬细胞炎性细胞因子的表达。一起来看,本研究的数据表明，LPS通过激活ERK1/2信号通路降低了巨噬细胞和C57BL/6J小鼠的TRIM65表达，而TRIM65敲除促进巨噬细胞活化。这些信息可能有助于开发预防和治疗炎症性疾病的潜在治疗策略。如动脉粥样硬化。
