大黄灵仙方对胆道炎症模型大鼠胆管组织miRNA-30b表达及炎症-纤维化相关因子的影响

Effect of Dahuang Lingxian Formula(大黄灵仙方)on miRNA-30b Expression and Inflammation-fibrosis Related Factors in Biliary Duct Tissue of Cholecystitis Model Rats

目的探讨大黄灵仙方减轻胆道炎症防治胆石症的可能作用机制。方法将50只SD大鼠随机分为空白组、模型组、大黄灵仙组、大黄灵仙+空白抑制剂组、大黄灵仙+抑制剂组,每组10只。除空白组外,其余各组均采用脂多糖(LPS)诱导构建大鼠胆道炎症动物模型。大黄灵仙组、大黄灵仙+空白抑制剂组、大黄灵仙+抑制剂组三组大鼠制备胆道炎症模型前3天开始中药灌胃,给药剂量为320 mg/(kg·d),空白组、模型组大鼠给予2 ml/100 g蒸馏水灌胃,每天2次,连续干预6天。采用HE染色观察各组胆管病理形态改变,免疫组化法检测胆管核因子κB(NF-κB)蛋白表达,ELISA法检测大鼠血清白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)表达水平,实时PCR法检测胆管组织中微小核糖核酸-30b(miRNA-30b)及转化生长因子β1(TGF-β1)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)和骨形态发生蛋白2(BMP2)mRNA表达,Western blot法检测TGF-β1、NLRP3以及BMP2蛋白表达量。结果与空白组比较,模型组大鼠胆管结构异常,汇管区内可见大量淋巴细胞、浆细胞浸润以及胆小管扩张,NF-κB蛋白阳性表达增加,出现核浸润,血清炎症因子IL-1β、TNF-α表达明显升高,胆管组织中TGF-β1、NLRP3、BMP2 mRNA以及蛋白表达量显著增加,miRNA-30b表达水平明显下降(P<0.01)。与模型组比较,大黄灵仙组和大黄灵仙+空白抑制剂组胆管病理形态均改善,炎性细胞浸润减少,NF-κB阳性表达降低,同时核浸润减轻,血清炎症因子IL-1β、TNF-α表达水平下降,胆管组织中TGF-β1、NLRP3、BMP2 mRNA以及蛋白表达量均降低,miRNA-30b表达水平均明显升高(P<0.05或P<0.01),而大黄灵仙+抑制剂组各指标改善不明显(P>0.05)。大黄灵仙组和大黄灵仙+空白抑制剂组各指标差异亦无统计学意义(P>0.05)。结论大黄灵仙方可能通过调控miRNA-30b及炎症-纤维化相关因子表达,起到延缓胆石症病程进展的作用。

Objective To explore the possible mechanism of reducing cholecystitis and preventing cholelithiasis by Dahuang Lingxian Formula(大黄灵仙方,DLF).Methods Fifty SD rats were randomly divided into blank group,model group,DLF group,DLF+blank inhibitor group,and DLF+inhibitor group,with 10 rats in each group.The rat model of cholecystitis was established by lipopolysaccharide(LPS)induction in all the groups except for the blank group.Rats in DLF group,DLF+blank inhibitor group and DLF+inhibitor group received intragastric administration of 320 mg/(kg·d)of DLF 3 days before the preparation of cholecystitis model,while those in blank group and model group were given 2 ml/100 g of distilled water by gastric,twice a day,for 6 consecutive days.Hematoxylin-eosin(HE)staining was used to observe the histopathologic changes of bile duct tissues in each group.The expression of nuclear factorκB(NF-κB)protein in bile duct tissues was detected by immunohistochemistry.The expression levels of interleukin1β(IL-1β)and tumor necrosis factorα(TNF-α)in serum of rats were detected by enzym-linked immunosorbent assay(ELISA).The mRNA expression levels of miRNA-30b,transforming growth factorβ1(TGF-β1),nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and bone morphogenetic protein 2(BMP2)in bile duct tissues were detected by real-time PCR,and the protein expression levels of TGF-β1,NLRP3 and BMP2 were detected by Western blot.Results Compared to those in the blank group,the structure of the bile duct in the model group was abnormal,and a large number of lymphocytes,plasma cell infiltration and bile canaliculi dilation were seen in the portal area;the positive expression of NF-κB protein increased;there was nuclear infiltration;the expressions of serum inflammatory factors IL-1βand TNF-α,as well as the mRNA and protein expressions of TGF-β1,NLRP3 and BMP2 in bile duct tissue significantly increased,while the expression level of miRNA-30b significantly decreased(P<0.01).Compared to those in the model group,the pathological morphology of the bile ducts in the DLF group and DLF+blank inhibitor group was improved,and the infiltration of inflammatory cells was reduced,with decreased positive expression of NF-κB and nuclear infiltration;expression levels of serum inflammatory factors IL-1βand TNF-α,and the mRNA and protein expressions of TGF-β1,NLRP3 and BMP2 in bile duct tissue decreased,while the expression level of miRNA-30b significantly increased(P<0.05 or P<0.01).Compared to the model group,those indicators in the DLF+inhibitor group was not significantly improved(P>0.05).There was no significant difference in the indicators between the DLF group and the DLF+blank inhibitor group(P>0.05).Conclusion DLF may play a role in delaying the progression of cholelithiasis by regulating the expression of miRNA-30b and inflammation-fibrosis related factors.