白细胞介素17通过促进巨噬细胞M1型极化调控小鼠非酒精性脂肪性肝病组织炎症反应的机制

Interleukin-17-mediated inflammation promotes nonalcohofic fatty fiver disease in mice with regulation of Ml-type macrophage polarization

目的研究白细胞介素17(IL-17)促进巨噬细胞M1型极化加剧小鼠非酒精性脂肪性肝病(NAFLD)组织炎症反应的机制,为NAFLD的发生和发展机制研究提供参考。方法高脂饮食构建NAFLD小鼠,将小鼠分为对照组、模型组、IL-17组、抗IL-17组。苏木素-伊红染色检测小鼠肝脏病理,化学比色法检测小鼠外周血丙氨酸氨基转移酶和天冬氨酸氨基转移酶的表达水平,流式细胞术检测小鼠肝组织浸润M1型巨噬细胞和M2型巨噬细胞比例,其中F4/80-PE和CD11C-FITC标记M1型巨噬细胞,F4/80-PE和CD206-APC标记M2型巨噬细胞,免疫组织化学检测CD168在肝组织的表达,酶联免疫吸附试验和RT-QPCR检测M1型巨噬细胞标志性分子诱导型一氧化氮合成酶(iNOS)、肿瘤坏死因子(TNF)α、IL-6的蛋白和mRNA水平,Western blot检测JAK-STAT信号通路以及MCP-1的表达水平。采用单因素方差分析和t检验进行统计学分析。结果高脂饮食成功构建小鼠非酒精性脂肪性肝病模型,IL-17可以提高小鼠肝组织中M1型巨噬细胞的比例,下调M2型巨噬细胞比例(P<0.05):正常组小鼠肝组织中M1型巨噬细胞比例为7.9%±1.1%、M2细胞比例为19.2%±1.8%,模型组中M1型巨噬细胞比例为17.3%±2.5%,M2型巨噬细胞比例为15.0%±2.1%,而IL-17组中M1型细胞比例33.8%±4.2%相比模型组显著增高,而M2型巨噬细胞比例为7.8%±1.0%,相比模型组显著降低。肝组织中M1型巨噬细胞的标志性分子iNOS、IL-12、TNFα、IL-6的蛋白水平和mRNA水平相比模型组、对照组以及抗IL-17组显著增高(P<0.05)。IL-17组小鼠肝组织中JAK1、STAT1以及单核细胞趋化蛋白1的表达水平,肝组织中CD168表达增高(P<0.05),外周血丙氨酸氨基转移酶和天冬氨酸氨基转移酶水平相比其他三组显著增高(P<0.05)。结论IL-17可以促进巨噬细胞M1型极化加重NAFLD小鼠肝组织炎症反应,促进NAFLD的进展。

ObjectiveTo study the mechanism of interleukin (IL)-17 in mice with non-alcoholic fatty liver disease for promoting M1-type macrophage polarization to exacerbate liver inflammation, and to provide references for the mechanism of NAFLD occurrence and development.MethodsA mouse model of NAFLD was constructed by high-fat diet. Mice were divided into control group, model group, IL-17 group, and anti IL-17 group. Histopathological changes of the liver were observed by HE staining. The serum levels of ALT and AST in peripheral blood of mice was detected by chemical colorimetry. Macrophages labeled with F4/80-PE, CD11C-FITC was designated as M1-type macrophages, those labeled with F4/80-PE, and CD206-APC was designated as M2-type macrophages. The proportion of M1 and M2 macrophages infiltrated into the liver tissues of mice were measured by flow cytometry. CD168 expression level of liver tissues was detected using immunohistochemistry. Protein and mRNA levels of the marker molecules (iNOS, TNF-alpha and IL-6) of M1 macrophages were detected using ELISA and RT-Q PCR. Western blot was used to detect the protein expression of JAK-STAT signal pathway and the expression level of MCP-1. Data were analyzed using one-way ANOVA and t-test.ResultsHigh-fat diet NAFLD mice model was successfully constructed. IL-17 had increased the proportion of M1 macrophages in mice liver tissues and decreased the proportion of M2 macrophages (P<0.05). The proportion of M1 and M2 macrophages in the liver tissues of normal mice was 7.9%±1.1% and 19.2%±1.8%. The proportion of M1 and M2 macrophages in the model group was 17.3% ± 2.5% and 15.0%±2.1. The proportion of M1 macrophages (33.8%±4.2%) in IL-17 group was higher than model group, while the proportion of M2 macrophages (7.8%+1.0%) in IL-17 group was lower than model group. Protein and mRNA marker levels of M1 macrophage (iNOS, IL-12, TNFα and IL-6) in liver tissues were significantly higher than model group, control group, and anti-IL-17 group (P<0.05). The expression levels of JAK1, STAT1, MCP-1, and CD168 in mice liver tissues of IL-17 group had increased (P<0.05). The levels of aspartate and alanine aminotransferases in peripheral blood of mice in IL-17 group were significantly higher than other three groups (P<0.05).ConclusionIL-17 can promote M1-type macrophage polarization, and exacerbates the liver inflammatory response to accelerate the progression of NAFLD in mice.