利福平在胆汁淤积模型中对多药耐药相关蛋白2表达的影响

目的：探究利福平对肝内胆汁淤积模型大鼠肝组织中多药耐药相关蛋白2表达的影响。方法：通过使用α-萘异硫氰酸酯（ANIT）诱导大鼠肝内胆汁淤积后，给予大鼠利福平和熊去氧胆酸治疗，其分组为空白对照组（NC组）、模型对照组（M组）、利福平治疗组（R组）、熊去氧胆酸治疗组（U组）、利福平+熊去氧胆酸治疗组（R+U组）。通过对各组大鼠血清中肝损伤相关指标进行检测、大鼠胆汁流速对比、肝组织HE染色，观察大鼠的肝脏损伤情况，再使用RT-PCR和Western-blot对大鼠肝脏组织中多药耐药相关蛋白2（MRP2）的mRNA表达水平及蛋白表达水平进行检测。同时将大鼠的肝脏组织进行体外培养24 h后使用MTT法和PI单染流式细胞术检测其细胞活性及细胞周期。结果：M组相对于NC组血清中肝损伤的相关指标谷丙转氨酶（ALT）、碱性磷酸酶（ALP）、谷草转氨酶（AST）、总胆红素（TBIL）、γ-谷氨酰转移酶（γ-GGT）明显升高（P<0.05），同时治疗组各组与M组相比ALT、ALP、AST、TBIL、γ-GGT显著降低（P<0.05）；各组大鼠胆汁流速进行比较，M组 < R组 < U组 < R+U组 < NC组（P<0.05）；从大鼠的肝脏组织HE染色结果显示，M组大鼠肝细胞受损严重，肝细胞发生肿胀且出现中性粒细胞浸润现象，在给予治疗后，治疗组大鼠的肝细胞有不同程度的改善，细胞肿胀和浸润现象消失。在使用RT-PCR和Western-blot对各组大鼠肝脏组织中MRP2进行检测后发现，M组大鼠相对于NC组肝脏组织中MRP2 mRNA表达水平和MRP2蛋白表达水平明显下降，在经过利福平和熊去氧胆酸治疗后，R组、U组及R+U组与M组进行比较，肝脏组织中MRP2 mRNA表达水平和MRP2蛋白表达水平显著上升（P<0.05）。使用MTT法和PI单染流式细胞术对体外培养的各组大鼠肝脏组织细胞活力检测后发现，M组大鼠细胞存活率相对于NC组明显下降（P<0.05），细胞大量停滞于G₀/G₁期，G₂/M期细胞基本消失，治疗组大鼠肝脏细胞与M组相比存活率上升，G₀/G₁期细胞比例下降（P<0.05）。结论：利福平对于ANIT诱导的大鼠肝内胆汁淤积有一定的治疗效果，同时会上调肝脏组织中MRP2的mRNA表达水平及蛋白表达水平。

Effect of rifampicin on expression of multidrug resistance related protein 2 in cholestasis model

OBJECTIVE To investigate the effect of rifampicin on the expression of multidrug resistance related protein 2 in liver tissues of intrahepatic cholestasis rats.METHODS After cholestasis was induced by α-naphthalene isothiocyanate (ANIT),rats were treated with rifampicin and ursodeoxycholic acid.They were divided into blank control group (NC group),model control group (M group),rifampicin treatment group (R group),ursodeoxycholic acid treatment group (U group) and rifampicin+ursodeoxycholic acid treatment group (R+U group).The liver injury of rats was observed by detecting the liver injury-related indicators in the serum of rats in each group,comparing the bile flow rate of rats,and HE staining of liver tissues,and then the mRNA expression level and protein expression level of multidrug resistance-associated protein 2 (MRP2) in the liver tissues of rats were detected by RT-PCR and Western-blot.At the same time,the liver tissues of rats were cultured in vitro for 24 h and then the cell viability and cell cycle of rat liver tissue cells were detected using MTT assay and PI single-staining flow cytometry.RESULTS Alanine aminotransferase (ALT),alkaline phosphatase (ALP),aspartate aminotransferase (AST),total bilirubin (TBIL) and γ-glutamyl transferase (γ-GGT) were significantly increased in group M compared with group NC (P<0.05),and ALT,ALP,AST,TBIL and γ-GGT were significantly decreased in each group compared with group M (P<0.05).After RT-PCR and Western-blot were used to detect MRP2 in the liver tissues of rats in each group,it was found that the MRP2 mRNA expression level and MRP2 protein expression level in the liver tissues of rats in group M were significantly decreased compared with those in the NC group,and after rifampicin and ursodeoxycholic acid treatment,the MRP2 mRNA expression level and MRP2 protein expression level in the liver tissues of rats in group R and U+U were significantly increased (P<0.05).After the viability of liver tissue cells of rats in each group cultured in vitro was detected using MTT assay and PI single-staining flow cytometry,it was found that the cell survival rate of rats in group M was significantly decreased compared with the NC group (P<0.05),a large number of cells were arrested in G0/G1 phase,and cells in G2/M phase basically disappeared,and the survival rate of liver cells in the treatment group increased compared with group M,and the proportion of cells in G0/G1 phase decreased (P<0.05).CONCLUSION Rifampicin has certain therapeutic effect on intrahepatic cholestasis induced by ANIT in rats,and can up-regulate MRP2 mRNA and protein expression levels in liver tissues.