Zheng Daofeng, Li Zhongtang, Wei Xufu, Liu Rui, Shen Ai, He Diao, Tang Chengyong, Wu Zhongjun
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Department of Hepatobiliary surgery, Chongqing Cancer Institute, Chongqing, China.
Cell Physiol Biochem. 2018;49(5):2060-2072. doi: 10.1159/000493716. Epub 2018 Sep 21.
BACKGROUND/AIMS: Hepatic ischemia-reperfusion (I/R) injury, which is mainly induced by inflammation and unstable intracellular ions, is a major negative consequence of surgery that compromises hepatic function. However, the exact mechanisms of liver I/R injury have not been determined. Positive crosstalk with the Ca2+/CaMKII pathway is required for complete activation of the TLR4 pathway and inflammation. We previously found that miR-148a, which decreased in abundance with increasing reperfusion time, targeted and repressed the expression of CaMKIIα. In the present study, we examined the role of the miR-148a machinery in I/R-induced Ca2+/CaMKII and TLR4 signaling changes, inflammation, and liver dysfunction in vivo and in vitro.
Liver function was evaluated by serum aminotransferase levels and hematoxylin-eosin (HE) staining. Inflammatory factors were detected by enzyme-linked immunosorbent assay. Gene and protein expression were assessed by RT-PCR and western blot. Small interfering RNA was used to silence target gene expression. HE staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to measure hepatic tissue apoptosis. These assays were performed to identify factors upregulated in hepatic I/R injury and downregulated by miR-148a.
We manifested that expression of CaMKIIα and phosphorylation of TAK1 and IRF3 were elevated in hypoxia/reoxygenation (H/R)-treated primary Kupffer cells (KCs) and liver tissue of I/R-treated mice, but these effects were attenuated by treatment with miR-148a mimic and were accompanied by the alleviation of liver dysfunction and hepatocellular apoptosis. Luciferase reporter experiments showed that miR148a suppressed luciferase activity by almost 60%. Moreover, knockdown of CaMKIIα in H/R KCs led to significant deficiencies in p-TAK1, P-IRF3, IL-6, and TNF-α, which was consistent with the effects of miR-148a overexpression. Otherwise, the same trend of activation of TAK1 and IRF3 and inflammatory factors in vitro was observed in the siTAK1 + siIRF3 group compared with the siCaMKIIα group.
Taken together, we conclude that miR-148a may mitigate hepatic I/R injury by ameliorating TLR4-mediated inflammation via targeting CaMKIIα in vitro and in vivo.
背景/目的：肝缺血再灌注（I/R）损伤主要由炎症和不稳定的细胞内离子诱导，是影响肝功能的手术的主要负面后果。然而，肝I/R损伤的确切机制尚未确定。TLR4通路的完全激活和炎症需要与Ca2+/CaMKII通路进行正向串扰。我们之前发现，miR-148a的丰度随着再灌注时间的增加而降低，它靶向并抑制CaMKIIα的表达。在本研究中，我们在体内和体外研究了miR-148a机制在I/R诱导的Ca2+/CaMKII和TLR4信号变化、炎症及肝功能障碍中的作用。
通过血清转氨酶水平和苏木精-伊红（HE）染色评估肝功能。采用酶联免疫吸附测定法检测炎症因子。通过RT-PCR和蛋白质印迹法评估基因和蛋白质表达。使用小干扰RNA沉默靶基因表达。采用HE染色和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法检测肝组织凋亡。进行这些检测以鉴定肝I/R损伤中上调且被miR-148a下调的因子。
我们发现，在缺氧/复氧（H/R）处理的原代库普弗细胞（KC）和I/R处理小鼠的肝组织中，CaMKIIα的表达以及TAK1和IRF3的磷酸化水平升高，但用miR-148a模拟物处理可减弱这些作用，并伴随着肝功能障碍和肝细胞凋亡的减轻。荧光素酶报告实验表明，miR148a使荧光素酶活性降低了近60%。此外，在H/R KC中敲低CaMKIIα导致p-TAK1、P-IRF3、IL-6和TNF-α显著缺乏，这与miR-148a过表达的作用一致。此外，与siCaMKIIα组相比，在siTAK1 + siIRF3组中观察到体外TAK1和IRF3以及炎症因子的激活趋势相同。
综上所述，我们得出结论，miR-148a可能通过在体外和体内靶向CaMKIIα改善TLR4介导的炎症来减轻肝I/R损伤。
