Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage Polarization.
Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage Polarization.
复制标题
巨噬细胞 Regnase-1 缺失通过调节巨噬细胞极化加剧肝脏缺血/再灌注损伤
DOI：
10.3389/fphys.2020.582347
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发表时间：
2020
影响因子：
4
通讯作者：
Lianbao K
中科院分区：
文献类型：
--
作者：
Xiaoming A;Wenbo J;Jinyi W;Bin W;Chunyang H;Qi C;Lianbao K
Regnase-1（MCPIP）已被鉴定为抗炎剂，但其对肝脏缺血/再灌注（I/R）损伤的影响知之甚少。巨噬细胞可以演变成双相反应并分化成显著的极化，在肝I/R损伤期间极大地促进了不受控制的炎症级联反应。因此，本研究的目的是探讨regnase-1是否通过操纵巨噬细胞极化参与肝脏I/R。将C57 BL/6小鼠随机分为5组：假手术组、I/R组、氯膦酸盐组、Clo + BMDM组和Clo + LV MCPIP BMDM组。建立大鼠肝脏I/R模型，对肝脏标本进行组织病理学和免疫组化检查，免疫荧光双标法检测MCPIP在肝脏中的定位。分离原代肝细胞，模拟体外缺氧/复氧（H/R）模型。提取骨髓来源的巨噬细胞（BMDM）并进行慢病毒转导以敲低MCPIP表达。将MCPIP缺失或未缺失的BMDM暴露于H/R上清液，并通过流式细胞术测量极化状态。RT-PCR和Western blot分析。与Sham组相比，I/R组肝功能和Suzuki评分均下降，而氯膦酸二钠组肝功能和Suzuki评分均下降。在I/R组regnase-1表达的增加随着氯膦酸盐脂质体的预处理而减弱。随后的双重免疫荧光染色确定regnase-1在肝脏巨噬细胞中的定位。在Clo + BMDM组中，随着BMDM的过继转移，氯膦酸盐组中的损伤病变逐渐加重，并且在Clo + LV MCPIP BMDM组中，随着MCPIP敲减的BMDM的输注，损伤病变进一步加重。通过RT-PCR检测M1和M2标记基因的表达，表明MCPIP敲除倾向于有利于M1转化。随后，离体流式细胞术检测显示，在H/R上清液刺激下，LV-MCPIP BMDM比BMDM具有更高的M1/M2比率。最后，我们发现MCPIP通过NF-κB、C/EBPβ和PPARγ信号通路参与肝I/R时巨噬细胞M1/M2极化。我们的研究证实regnase-1通过调节巨噬细胞极化在肝脏I/R中起关键作用，因此可能提供潜在的治疗靶点。
Regnase-1 (MCPIP) has been identified as an anti-inflammatory agent, but little is known about its influence on liver ischemia/reperfusion (I/R) injury. Macrophages can evolve biphasic responses and differentiate into remarkable polarizations, contributing greatly to the uncontrolled inflammatory cascades during liver I/R injury. Therefore, the aim of this study was to explore whether regnase-1 participated in liver I/R via manipulating macrophage polarization. C57BL/6 mice were randomly divided into five groups: Sham, I/R, Clodronate, Clo + BMDM, and Clo + LV MCPIP BMDM. A liver I/R model was established, and histopathological and immunostaining examinations were performed for the liver specimens; double immunofluorescence staining was used to localize MCPIP in the liver. Primary hepatocytes were isolated to simulate a hypoxia and reoxygenation (H/R) model in vitro. Bone marrow-derived macrophages (BMDM) were extracted and subjected to lentiviral transduction to knockdown MCPIP expression. BMDM with or without MCPIP deletion were exposed to H/R supernatants, and the polarized states were measured by flow cytometry. RT-PCR analysis and Western blot were also conducted. Compared to those in the Sham group, liver functions and Suzuki’s scores were deteriorated in the I/R group, which were reversed in the Clodronate group. The increased expression of regnase-1 in the I/R group diminished with pretreatment of clodronate liposomes. Subsequent double immunofluorescence staining established the localization of regnase-1 in macrophages in the liver. The insulted lesions in the Clodronate group became progressively aggravated with adoptive transfer of BMDM in the Clo + BMDM group, and they were further exacerbated with the transfusion of BMDM with MCPIP knockdown in the Clo + LV MCPIP BMDM group. Gene expressions of M1 and M2 markers were detected by RT-PCR, suggesting that MCPIP knockdown tended to favor the M1 transformation. Subsequently, ex vivo flow cytometrical detection showed that, upon stimulation by H/R supernatants, LV-MCPIP BMDM posed a higher ratio of M1/M2 than BMDM. Finally, we found that MCPIP participated in macrophage M1/M2 polarization through the NF-κB, C/EBPβ, and PPARγ signaling pathways during liver I/R. Our study confirms that regnase-1 plays a critical role in liver I/R via regulation of macrophage polarization and, thus, might offer a potential therapeutic target.