华支睾吸虫感染大鼠肝脏miR-122表达及其与炎症细胞因子的相关性

The expression of miR-122 in the liver of rats infected with Clonorchis sinensis and its correlation with inflammatory cytokines

目的观察华支睾吸虫感染大鼠肝脏组织微小RNA(miR)-122的表达变化及其与血清炎症细胞因子表达水平的相关性。方法选择24只SPF级Wistar雄性大鼠,采用随机数字表法按体质量(100~120 g)分为对照组(100μl生理盐水灌胃)、感染4周组(100个华支睾吸虫囊蚴灌胃)、感染8周组(100个华支睾吸虫囊蚴灌胃),每组8只。感染第3周开始收集大鼠粪便,采用生理盐水直接涂片进行华支睾吸虫感染Wistar大鼠动物模型鉴定;感染4、8周组大鼠分别于感染4、8周后处死,对照组大鼠分别于感染4、8周后各处死4只,采集各组大鼠心脏血及左叶肝脏组织。采用苏木精-伊红(HE)染色光镜下观察肝脏病理损伤情况,实时荧光定量PCR检测肝脏miR-122表达水平,Luminex 200液相悬浮芯片检测血清炎症细胞因子[肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)]表达水平;利用Pearson相关分析miR-122与炎症细胞因子的相关性。结果光镜下,对照组大鼠肝细胞形态正常,未见炎症细胞浸润;感染4周组大鼠肝脏组织汇管区周围有淋巴细胞、嗜酸性粒细胞等炎症细胞浸润;感染8周组大鼠肝细胞排列紊乱、出现不同程度肿胀,细胞质疏松浅染,部分肝细胞呈水样变性,肝脏组织内可见胆管扩张、胆管壁增厚。对照组和感染4、8周组大鼠肝脏miR-122(1.00±0.32、2.57±0.60、3.63±1.63),血清TNF-α[(0.14±0.06)、(0.43±0.09)、(0.61±0.10)ng/ml]、IL-6表达水平[(0.03±0.01)、(1.06±0.24)、(1.48±0.33)ng/ml]比较,差异均有统计学意义(F=13.36、69.99、82.23,均P<0.001);IL-1β表达水平比较差异无统计学意义(F=2.15,P=0.141)。Pearson相关分析结果显示,肝脏miR-122与血清TNF-α和IL-6表达水平均呈正相关(r=0.67、0.80,均P<0.001)。结论华支睾吸虫感染可引起宿主肝脏miR-122表达升高,且miR-122与血清炎症细胞因子TNF-α、IL-6表达水平密切相关。

Objective To observe the expression changes of microRNA(miR)-122 in liver tissue of rats infected with Clonorchis sinensis and its correlation with expression level of inflammatory cytokines.Methods Totally 24 SPF grade Wistar male rats were selected and randomly divided into a control group(100μl physiological saline gavage),a 4-week infection group(100 Clonorchis sinensis metacercariae gavage),and an 8-week infection group(100 Clonorchis sinensis metacercariae gavage)based on body weight(100-120 g)using a random number table method,with 8 rats in each group.Starting from the third week of infection,rat feces were collected and directly smeared with physiological saline for identification of Wistar rat animal models infected with Clonorchis sinensis.After 4 and 8 weeks of infection,the rats in the 4-and 8-week infection groups were euthanized,while 4 rats in the control group were euthanized,respectively.The heart blood and left lobe liver tissue and serum samples were collected from each group of rats.Using hematoxylin-eosin(HE)staining to observe liver pathological damage under the light microscope,real-time fluorescence quantitative PCR to detect the expression level of miR-122 in liver tissue,and Luminex 200 liquid suspension chip to detect the expression levels of serum inflammatory cytokines[tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)].The correlation between miR-122 and inflammatory cytokines was analyzed using Pearson correlation.Results Under the light microscope,the morphology of hepatocytes in control group was normal,and no inflammatory cell infiltration was observed.There was inflammatory cells such as lymphocyte,eosinophil and other inflammatory cell infiltration around the portal area in the 4-week infection group.The hepatocytes of the 8-week infected rats were arranged in a disordered manner,with varying degrees of swelling,loose and lightly stained cytoplasm,and some hepatocytes showed watery degeneration;additionally,bile duct dilation and thickening of the bile duct wall were observed in the liver tissue.There were statistically significant differences of liver miR-122(1.00±0.32,2.57±0.60,3.63±1.63),serum TNF-α[(0.14±0.06),(0.43±0.09),(0.61±0.10)ng/ml],and IL-6 expression levels[(0.03±0.01),(1.06±0.24),(1.48±0.33)ng/ml]in control group,4-and 8-week infection groups(F=13.36,69.99,82.23,P<0.001).There was no statistically significant difference in expression level of IL-1β between different groups(F=2.15,P=0.141).The Pearson correlation analysis showed that the expression level of miR-122 was positively correlated with the expression levels of inflammatory cytokines TNF-α and IL-6(r=0.67,0.80,P<0.001).Conclusion Clonorchis sinensis infection can increase the expression of miR-122 in the host liver tissue,and the miR-122 is closely related to the expression levels of inflammatory cytokines TNF-α and IL-6.