白细胞介素-12对干燥综合征小鼠肝脏的损伤作用及其机制

目的 探讨白细胞介素-12（IL-12）对干燥综合征（SS）小鼠肝脏的损伤作用及其机制。方法 选取12周龄C57BL/6小鼠、非肥胖型糖尿病（NOD）SS模型小鼠、IL-12基因敲除NOD小鼠各5只,分别纳入正常对照组、SS模型组和IL-12 KO NOD组。各组小鼠取外周静脉血分离提取外周血单个核细胞（PBMC）和血清,处死小鼠解剖获取颌下腺组织和肝脏组织。（1）取正常对照组和SS模型组小鼠肝脏组织制备石蜡切片,HE染色观察肝脏肝小叶结构,MASSON染色观察肝脏纤维化情况。（2）取正常对照组和SS模型组小鼠肝脏组织、颌下腺组织的石蜡切片免疫组织化学染色观察比较IL-12阳性细胞光密度（OD）值的改变。（3）采用实时荧光定量聚合酶链反应（qRT-PCR）检测正常对照组、SS模型组小鼠PBMC和肝脏组织IL-12 mRNA的相对表达量,以及正常对照组、SS模型组、IL-12 KO NOD模型组小鼠肝脏组织纤维连接蛋白（FN）、转化生长因子-β（TGF-β）、Ⅰ型胶原（Col1）mRNA的相对表达量。（4）取正常对照组、SS模型组、IL-12 KO NOD模型组小鼠外周血血清和肝脏组织,酶联免疫吸附试验（ELISA）检测铁死亡相关蛋白谷胱甘肽过氧化物酶4（GPX4）、溶质载体家族7成员11（SLC7A11）、转铁蛋白（TRF）、转铁蛋白受体（TFR）蛋白的表达情况。结果 （1）HE染色可见正常对照组小鼠肝小叶结构基本正常,而SS模型组小鼠肝脏的肝小叶、肝索结构紊乱,肝细胞胞浆内有空泡出现。MASSON染色可见SS模型组小鼠肝脏组织中见弥漫不规则的染为蓝色的胶原纤维,正常对照组小鼠肝组织中染为蓝色的胶原纤维则少见。（2）正常对照组小鼠颌下腺和肝脏IL-12阳性细胞OD值分别为0.08±0.01、0.03±0.01,高于SS模型组的8.74±0.78、2.58±0.07,差异均有统计学意义（t=24.82、80.64,P值均<0.001）。（3）正常对照组小鼠肝脏组织及PBMC中IL-12 mRNA表达分别为1.07±0.15、1.03±0.14,均低于SS模型组的3.00±0.50、3.01±0.57,差异均有统计学意义（t=8.27、7.54,P值均<0.001）。与正常对照组比,SS模型组FN、TGF-β mRNA的相对表达量均增高,差异均有统计学意义（P值均<0.001）;与SS模型组比较,IL-12 KO NOD组FN、TGF-β mRNA的相对表达量均降低,差异均有统计学意义（P值均<0.05）;3组间Col1 mRNA的相对表达量差异无统计学意义（P=0.243）。（4）与正常对照组比较,SS模型组血清和肝脏组织中SLC7A11、GPX4、TRF蛋白相对表达量均明显降低,TFR蛋白相对表达量均增高,差异均有统计学意义（P值均<0.05）。与SS模型组比较,IL-12 KO NOD组血清和肝脏组织中SLC7A11、GPX4、TRF的相对表达量均增加,血清TFR蛋白的相对表达量降低,差异均有统计学意义（P值均<0.05）;而肝脏组织中TFR蛋白的相对表达量差异无统计学意义（P>0.05）。结论 IL-12表达增高对SS小鼠肝脏组织的损伤起到了促进作用,其机制可能与增高的IL-12诱导肝脏细胞铁死亡有关。

The roles and mechanisms of interlekin-12 in the liver injury of mice with Sjogren's syndrome

Objective This study aimed to investigate the damage effect of interleukin-12 (IL-12) on the liver of mice with Sjogren's syndrome (SS). Methods Five of the 12-week-old C57BL/6, NOD, and IL-12 knockout NOD mice were selected. The C57BL/6 mice were the normal control group. The NOD mice comprised the SS model mice for the SS group. The IL-12 knockout NOD mice were included in the IL-12 KO NOD group. The peripheral blood of vein from each group of mice was isolated to peripheral blood mononuclear cells (PBMC) and serum. Mice were dissected for submandibular gland tissue and liver tissue. (1) Paraffin sections were prepared from normal control group and SS group mice treated in liver tissue. Liver lobular structure and inflammatory cell infiltration were observed by HE staining. Liver fibrosis index was observed by MASSON staining. (2) Immunohistochemical staining of IL-12-positive cells was carried out for samples that were obtained from liver tissue and submandibular gland tissue of normal control and SS group mice. The paraffin sections of liver tissues and submandibular glands from the normal control and SS group mice were obtained for immunohistochemical staining to observe the change in the optical density (OD) values of IL-12 positive cells. (3) Real-time quantitative fluorescence polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of IL-12 mRNA in PBMC and liver tissues of mice in the normal control and SS group. The relative mRNA expression levels of fibronectin (FN), transforming growth factor-β (TGF-β), and collagen type Ⅰ (Col1) were obtained in the liver of mice in the normal control, SS, and IL-12 KO NOD group. (4) The expressions of iron-death-related proteins glutathione peroxidase 4（GPX4）, solute carrier family 7 members 11（SLC7A11）, transferrin（TRF）, and transferrin receptor（TFR） were detected by enzyme linked immunosorbent assay (ELISA) in the peripheral blood serum and liver tissues of mice in the normal control, SS, and IL-12 KO NOD group. Results (1) HE staining results revealed that SS control group mice had essentially normal hepatic lobular structure. However, the hepatic lobule and hepatic cord structure in the liver of SS group mice were disordered, and vacuoles appeared in the cytoplasm of hepatocytes. MASSON staining results showed diffuse irregular collagen fibers in the liver tissue of SS group mice, but this condition was rarely observed in normal control group mice. (2) Immunohistochemical staining of liver and submandibular glands in normal control and SS group mice showed that the OD values of IL-12 positive cells in the normal control group mice were 0.08±0.01, 0.03±0.01, which were higher than those in the SS group with values of 8.74±0.78 and 2.58±0.07, respectively. The difference was significant (t=24.82, 80.64, all P values <0.001). (3) Comparison of IL-12 mRNA in mouse PBMC and liver tissues. The expression levels of IL-12 mRNA in the liver tissue and PBMC of mice in normal control group were 1.07±0.15 and 1.03±0.14, respectively, which were significantly lower than those in SS group with values of 3.00±0.50 and 3.01±0.57, respectively (t=8.27, 7.54, all P values <0.001). Based on the comparison of liver fibrosis indexes in mice, compared with normal control group, the relative expression levels of FN and TGF-β mRNA were significantly increased in the SS group (all P values <0.001). In comparison with the SS group, the relative expression levels of FN and TGF-β mRNA in the IL-12 KO NOD group significantly decreased (all P values <0.05). No significant difference was observed in the relative expression level of Col1 mRNA among the three groups (P=0.243). (4) Comparison of iron-death-related proteins in the serum and liver of mice. In comparison with the normal control group, the relative expression levels of SLC7A11, GPX4, and TRF in the serum and liver of SS group significantly decreased, while the relative expression levels of TFR proteins significantly increased (all P values <0.05). In comparison with the SS group, the relative expression levels of SLC7A11, GPX4, and TRF in the serum and liver of IL-12 KO NOD group significantly increased, whereas those of serum TFR protein significantly decreased (all P values <0.05). No significant difference was observed in the relative expression level of TFR protein in liver tissues (P>0.05). Conclusion The increased expression of IL-12 promoted the damage of liver tissue in SS mice, and the mechanism may be related to the iron death of liver cells induced by the increased expression of IL-12.