肝脏线粒体DNA-TLR9-microRNA223环路负反馈调节对乙酰氨基酚诱导的急性肝损伤和炎症的机制研究

发布时间：2018-05-28 18:19

本文选题：APAP + miR-223　；参考：《安徽医科大学》2016年博士论文

【摘要】：对乙酰氨基酚(acetaminophen, APAP)过量使用是世界各地急性肝脏衰竭的主要诱因之一,其与肝脏内大量中性粒细胞的浸润密切相关。然而,有关肝脏内中性粒细胞浸润与炎症的具体机制仍不是很清楚。近年来研究发现微小RNA(microRNA, miRNA)可参与调控细胞增殖、分化、凋亡等多种重要生物学过程。髓系特异表达的miRNA-223被报道可以调节中性粒细胞的生成和功能,其是否参与调控APAP诱导的急性肝损伤及相关中性粒细胞的功能目前还未见文献报道。本研究构建miR-223敲除小鼠,以此深入探讨其对APAP诱导急性肝损伤及中性粒细胞功能的影响及其作用的分子机制。本实验分别对空腹过夜饥饿或正常饮食条件下野生型小鼠和miR-223敲除小鼠进行APAP腹腔注射以建立小鼠急性肝损伤模型。6小时或者24小时后,取小鼠血清进行肝脏转氨酶和miR-223检测,取肝组织进行病理组织学及相关免疫组化检测。采用磁珠分选方法分离肝脏,骨髓及外周血中中性粒细胞进行相关基因实时定量PCR或者western blot检测。结果发现,过量APAP注射能明显升高小鼠肝脏和血清中miR-223水平。更重要的是,相比于野生型小鼠,miR-223敲除小鼠血清中具有更高水平的ALT,AST,及更加严重的肝脏坏死,这提示miR-223敲除小鼠更易于APAP诱导的肝损伤。正如预期的一样,miR-223敲除小鼠肝脏中有更多的中性粒细胞和巨噬细胞的浸润。相对应的,miR-223敲除小鼠肝脏中炎症反应及氧化性损伤标记4-羟基壬烯酸(4-Hydroxynonenal,4-HNE)和N-硝基酪氨酸(N-nitrotyrosine)的表达明显高于野生型小鼠。有趣的是,腹腔注射坏死细胞可以引起小鼠腹腔中性粒细胞的大量浸润,而这种反应在miR-223敲除小鼠中更加明显。更有甚者,在体内或者体外,Toll样受体9(toll-like receptor 9, TLR9)配体或者坏死肝脏细胞释放的自由DNA通过TLR9/NF-κB通路诱导中性粒细胞miR-223的表达,而TLR9阻断剂可以抑制APAP诱导的中性粒细胞miR-223表达。此外,体外用TLR9配体刺激中性粒细胞后,缺乏miR-223的中性粒细胞产生更多的炎症细胞因子和趋化因子。机制上,miR-223被发现可以部分调控IKKa的表达负反馈控制TLR9/NF-κB介导的炎症反应。以上研究表明,miR-223的缺失增加肝脏中性粒细胞炎症反应和氧化应激反应,随后加重APAP诱导的肝毒性。这些发现提示,miR-223是控制急性中性粒细胞浸润和功能的重要调节分子,其可能作为治疗APAP诱导肝损伤的一个治疗靶点。
[Abstract]:Excessive use of acetaminophen (APAP) is one of the main causes of acute liver failure all over the world. It is closely related to the infiltration of a large number of neutrophils in the liver. However, the specific mechanisms of neutrophils infiltration and inflammation in the liver are still not clear. In recent years, small RNA (microRNA, miRNA) has been found. There are many important biological processes involved in regulating cell proliferation, differentiation, and apoptosis. Myeloid specific expression of miRNA-223 is reported to regulate the formation and function of neutrophils. Whether it participates in the regulation of APAP induced acute liver injury and related neutrophils has not yet been reported. This study constructs miR-223 knockout small In this study, the effect and the molecular mechanism of APAP induced acute liver injury and neutrophils function were investigated in this experiment. In this experiment, the mice were intraperitoneally injected with APAP in the wild and miR-223 knockout mice under empty stomach starvation or normal diet to establish a mouse model of acute liver injury for.6 hours or 24 hours. The liver transaminase and miR-223 were detected in the mice serum. The liver tissue was examined by histopathology and related immunohistochemical detection. The liver was separated by magnetic bead separation, and the neutrophils in the bone marrow and peripheral blood were detected in real-time quantitative PCR or Western blot. The results showed that excessive APAP injection could significantly increase the liver of mice. And miR-223 levels in the serum. More importantly, compared to the wild type mice, the miR-223 knockout mice had a higher level of ALT, AST, and more severe liver necrosis, suggesting that miR-223 knockout mice were more prone to APAP induced liver damage. As expected, there were more neutrophils and giant cells in the miR-223 knockout mice liver. The expression of inflammatory reaction and oxidative damage in the liver of miR-223 knockout mice, the expression of 4- hydroxyl NONYLIC acid (4-Hydroxynonenal, 4-HNE) and N- nitro tyrosine (N-nitrotyrosine) was significantly higher than that of wild type mice. The reaction is more obvious in miR-223 knockout mice. Furthermore, the free DNA released by Toll like receptor 9 (Toll-like receptor 9, TLR9) ligand or necrotic liver cells in vivo or in vitro can induce the expression of miR-223 in neutrophils through the TLR9/NF- kappa B pathway, and TLR9 blockers can inhibit APAP induced neutrophils. MiR-223 expression. In addition, after the stimulation of neutrophils with TLR9 ligands in vitro, more inflammatory cytokines and chemokines are produced by the neutrophils lacking miR-223. On the mechanism, miR-223 is found to partially regulate the negative feedback of IKKa to control the inflammatory response mediated by TLR9/NF- kappa B. The above study suggests that the absence of miR-223 increases the liver. The inflammatory and oxidative stress responses of neutrophils then aggravate the hepatotoxicity induced by APAP. These findings suggest that miR-223 is an important regulator to control the infiltration and function of acute neutrophils, and may be a therapeutic target for the treatment of APAP induced liver injury.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

【参考文献】