肿瘤坏死因子α诱导蛋白8样分子1在小鼠急性肝损伤中的作用及机制

Role and potential mechanisms of tumor necrosis factor alpha-inducible protein 8-like molecule 1 in acute liver injury in mice

目的探讨肿瘤坏死因子α诱导蛋白8样分子1(TNFAIP8L1)在小鼠急性肝损伤中的作用及机制。方法选择C57BL/6J雄性野生型(WT)小鼠和C57BL/6J雌性TNFAIP8L1^(+/-)小鼠杂交繁殖的第2代TNFAIP8L1^(+/-)小鼠和WT小鼠,进一步自交繁殖出第3代雄性TNFAIP8L1^(-/-)小鼠和第3代WT雄性小鼠。分别取5只正常第3代雄性WT小鼠和5只正常第3代雄性TNFAIP8L1^(-/-)小鼠,检测比较2种正常小鼠的血清丙氨酸氨基转移酶(ALT)水平,苏木精-伊红(HE)染色观察2种正常小鼠肝组织中炎症细胞浸润及细胞坏死情况,采用流式细胞术检测2种正常小鼠肝组织髓系细胞亚群中性粒细胞(Neu)、嗜酸性粒细胞(EOS)、树突状细胞(DC)、骨髓来源的巨噬细胞(BMDMs)、骨髓来源的单核细胞(BMNCs)所占百分比。另取5只第3代雄性WT小鼠和4只第3代雄性TNFAIP8L1^(-/-)小鼠,采用脂多糖(LPS)/D-半乳糖胺(D-Gal)诱导制备急性肝损伤小鼠模型,24 h后采取上述方法检测比较2种急性肝损伤小鼠血清ALT水平,观察2种急性肝损伤小鼠肝组织中炎症细胞浸润及细胞坏死情况,并检测2种急性肝损伤小鼠肝组织髓系细胞亚群Neu、EOS、DC、BMDMs、BMNCs所占百分比。结果正常WT小鼠和TNFAIP8L1^(-/-)组小鼠肝组织中髓系细胞亚群Neu、EOS、DC、BMDMs、BMNCs百分比及血清ALT水平比较差异无统计学意义(P>0.05)。正常WT小鼠和TNFAIP8L1^(-/-)小鼠的肝组织HE染色结果均显示肝小叶结构完整清晰,肝细胞形态正常、排列整齐,无明显炎症细胞浸润及细胞坏死。急性肝损伤24 h后,TNFAIP8L1^(-/-)小鼠肝组织髓系细胞亚群中Neu、BMNCs百分比及血清ALT水平显著高于WT小鼠(P<0.05);2组小鼠肝组织髓系细胞亚群EOS、DC、BMDMs百分比比较差异无统计学意义(P>0.05)。急性肝损伤WT小鼠肝组织中肝小叶结构模糊,肝细胞肿胀、散在空泡样脂肪变性,有少量炎症细胞浸润。急性肝损伤TNFAIP8L1^(-/-)小鼠肝组织中肝小叶结构几乎不存在,肝细胞损伤严重且大量坏死,并有大量炎症细胞浸润。结论TNFAIP8L1基因缺陷不影响小鼠肝组织中髓系细胞的发育和小鼠肝脏稳态。TNFAIP8L1在急性肝损伤的发生发展过程中起抑制作用。TNFAIP8L1基因缺陷加重LPS/D-Gal诱导的急性肝损伤,有可能是通过增加Neu和BMNCs浸润而招募其他类型的免疫细胞浸润入肝组织,从而加重肝细胞的坏死。

Objective To investigate the role and potential mechanisms of tumor necrosis factor alpha-inducible protein 8-like molecule 1(TNFAIP8L1)in acute liver injury in mice.Methods The second generation of C57BL/6J male wild-type(WT)mice and the C57BL/6J female TNFAIP8L1^(+/-)mice and WT mice were selected to further self-breed the third generation of male TNFAIP8L1^(-/-)mice and the third generation of WT male mice.Five normal third-generation male WT mice and five normal third-generation male TNFAIP8L1^(-/-)mice were selected.The serum alanine aminotransferase(ALT)levels of the two types of normal mice were measured and compared.The infiltration of inflammatory cells and cell necrosis in the liver tissues of the two types of normal mice were observed after hematoxylin&eosin(HE)staining.Flow cytometry was used to detect the percentages of neutrophils(Neu),eosinophils(EOS),dendritic cells(DC),bone marrow-derived macrophages(BMDMs),and bone marrow-derived mononuclear cell(BMNCs)in the liver myeloid cell subsets of the two types of normal mice.Another 5 third-generation male WT mice and 4 third-generation male TNFAIP8L1^(-/-)mice were selected to induce acute liver injury mouse models using lipopolysaccharide(LPS)/D-galactosamine(D-Gal).After 24 hours,the serum ALT levels of the two types of acute liver injury mice were detected and compared,the infiltration of inflammatory cells and cell necrosis in the liver tissues of the two types of acute liver injury mice were observed,and the percentages of Neu,EOS,DC,BMDMs and BMNCs in the liver myeloid cell subsets of the two types of acute liver injury mice were measured by using the above methods.Results There was no significant difference in the percentages of Neu,EOS,DC,BMDMs and BMNCs,and serum ALT levels in the livermyeloid cell subsets of normal WT mice and TNFAIP8L1^(-/-)mice(P>0.05).HE staining results of liver tissues in normal WT mice and TNFAIP8L1^(-/-)mice showed that hepatic lobules were structurally complete and clear,hepatocytes were morphologically normal and arranged neatly,and there was no obvious inflammatory cell infiltration or cell necrosis.Twenty-four hours after acute liver injury,the percentages of Neu and BMNCs in the liver myeloid cell subsets and the serum ALT levels in the liver tissues of TNFAIP8L1^(-/-)mice were significantly higher than those of WT mice(P<0.05);there was no significant difference in the percent ages of EOS,DC and BMDMs in the liver myeloid cell subsets of mice between the two groups(P>0.05).In the liver tissues of WT mice with acute liver injury,hepatic lobules were structurally blurred,hepatocytes were swollen with scattered vacuolated steatosis,and a small amount of inflammatory cells were infiltrated.In the liver tissues of TNFAIP8L1^(-/-)mice with acute liver injury,hepatic lobules were structurally non-existent,and hepatocytes were severely damaged and extensively necrotic,with a large amount of inflammatory cell infiltration.Conclusion The deficiency of the TNFAIP8L1 gene in mice does not affect the development of liver myeloid cells and the homeostasis of the liver.TNFAIP8L1 plays an inhibitory role in the occurrence and development of acute liver injury.TNFAIP8L1 gene deficiency aggravates LPS/D-Gal-induced acute liver injury,possibly by increasing Neu and BMNCs infiltration and recruiting other types of immune cells to infiltrate liver tissues,thereby exacerbating liver cell necrosis.