重组白细胞介素-22通过抑制炎症因子表达减缓肝纤维化的实验研究

Experimental study of recombinant interleukin-22 in reducing hepatic fibrosis by inhibiting the expression of inflammatory factors

目的 观察重组白细胞介素(IL)-22通过抑制炎症因子表达对肝纤维化小鼠的影响。方法 选取16只6周龄雄性BABL/c小鼠,均腹腔注射20%四氯化碳造模,2次/周,在造模后第8周,将小鼠随机分成重组IL-22组(n=8)和对照组(n=8)。重组IL-22组和对照组小鼠分别腹腔注射0.3μg/g重组IL-22和等体积0.5%牛血清白蛋白(以磷酸盐缓冲液为溶剂,pH 7.0),1次/d,直到第10周处死小鼠。通过苏木精-伊红(HE)和Masson染色了解两组小鼠的肝脏病理改变,根据Ishak评分系统评估两组小鼠的肝纤维化程度;利用免疫组织化学法检测肝脏组织中α-平滑肌肌动蛋白(α-SMA)、IL-22的表达;采用流式细胞仪检测两组小鼠脾脏Th22细胞数的变化;采用实时荧光定量PCR法检测肝脏组织IL-22R1、IL-10R2、IL-22、IL-17A、IL-6和肿瘤坏死因子(TNF)-α mRNA的表达,采用ELISA法检测血浆中IL-22、 IL-17A、IL-6和TNF-α蛋白表达水平。结果 (1)HE和Masson染色结果显示,重组IL-22组小鼠肝脏组织的炎症、坏死和纤维化程度较对照组小鼠明显减轻,Ishak评分较对照组小鼠下降(P<0.05)。(2)重组IL-22组小鼠肝脏组织α-SMA的蛋白表达水平,以及肝脏组织和血浆中IL-22的表达水平均明显低于对照组(均P<0.05)。(3)重组IL-22组小鼠脾脏组织Th22细胞百分比和肝脏组织IL-22、IL-22R1、IL-10R2的mRNA表达水平均低于对照组(均P<0.05)。(4)重组IL-22组小鼠肝脏组织IL-17A、IL-6和TNF-α的mRNA表达水平,以及血浆IL-22、IL-17A、IL-6和TNF-α的蛋白表达水平均低于对照组(均P<0.05)。结论 重组IL-22可减缓肝纤维化,其作用机制可能与其减弱肝星状细胞的活性和下调炎症因子的水平有关。

Objective To observe the effect of recombinant interleukin(IL)-22 on hepatic fibrosis mice by inhibiting the expression of inflammatory factors.Methods Sixteen 6-week-old male BABL/c mice were selected and intraperitoneally injected with 20% carbon tetrachloride for modeling,twice a week.At the 8th week after modeling,the mice were randomly divided into a recombinant IL-22 group(n=8) and a control group(n=8).Mice in the recombinant IL-22 group and the control group were intraperitoneally injected with 0.3 μg/g recombinant IL-22 and epuivoluminal 0.5% BSA(phosphate buffered salina as solvent,pH 7.0) once a day,respectively,until the mice were euthanized at the 10th week.Hematoxylin-eosin(HE) and Masson staining were used to observe the pathological changes of liver in the two groups of mice,and the degree of hepatic fibrosis was evaluated according to Ishak scoring system.The expressions of α-smooth muscle actin(α-SMA) and IL-22 in liver tissue were detected by streptavidin-perosidas.Flow cytometer was used to detect the changes of Th22 cells number in spleen tissue of two groups.The expressions of IL-22R1,IL-10R2,IL-22,IL-17A,IL-6 and tumor necrosis factor(TNF)-α mRNA in liver tissue were detected by real-time fluorescence quantitative-PCR,and the expression levels of IL-22,IL-17A,IL-6 and TNF-α protein in plasma were detected by ELISA.Results(1) The results of HE and Masson staining showed that the degrees of inflammation,necrosis and fibrosis in the liver tissue of the recombinant IL-22 group mice were significantly less than those in the control group,and the Ishak score was lower than that in the control group(P<0.05).(2) In the recombinant IL-22 group,the protein expression level of α-SMA in liver tissue,and the expression level of IL-22 in liver tissue and plasma were significantly lower than those in control group(all P<0.05).(3) The percentage of Th22 cells in spleen tissue and the mRNA expression levels of IL-22,IL-22R1 and IL-10R2 in liver tissue of the recombinant IL-22 group mice were lower than those in the control group(all P<0.05).(4) The mRNA expression levels of IL-17A,IL-6 and TNF-α in liver tissue and the protein expression levels of IL-22,IL-17A,IL-6 and TNF-α in plasma of the recombinant IL-22 group mice were lower than those in the control group(all P<0.05).Conclusion Recombinant IL-22 can alleviate hepatic fibrosis,and its mechanism may be related to reducing the activity of hepatic stellate cells and down-regulating the levels of inflammatory factors.