舒肝宁注射液调控FXR-MRP2/BSEP通路保肝、降酶及退黄机制研究

Study on the mechanism of Shuganning Injection in liver protection，enzyme reduction and jaundice regression via regulating the FXR-MRP2/BSEP pathway

关键词:  舒肝宁注射液  脓毒性肝内胆汁淤积  保肝  降酶  退黄  FXR-MRP2/BSEP通路

Key words:  Shuganning Injection  septic intrahepatic cholestasis  liver protection  enzyme reduction  jaundice regression  FXR-MRP2/BSEP pathway

摘要:

[目的] 考察舒肝宁注射液（SGN）对脓毒性肝内胆汁淤积（IC）大鼠的保肝降酶退黄作用及机制。[方法] 将36只SPF级雄性SD大鼠随机分为空白（Control）组、模型（LPS）组、熊去氧胆酸（UDCA）组、SGN低剂量（SGN-L）组、SGN中剂量（SGN-M）组和SGN高剂量（SGN-H）组，每组6只。通过腹腔注射脂多糖（LPS，5 mg/kg）建立脓毒性IC模型，Control组和LPS组尾静脉注射10%葡萄糖注射液[6 mL/（kg·d）]，UDCA组腹腔注射UDCA[25 mg/（kg·d）]，SGN-L、SGN-M、SGN-H组尾静脉分别注射0.625、1.350、2.700 mL/（kg·d）的SGN，连续给药5 d。收集大鼠胆汁并测定流速，生物化学法检测大鼠胆汁中总胆汁酸（TBA）、总胆红素（TBIL）、直接胆红素（DBIL）、还原性谷胱甘肽（GSH）含量和血清中天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）活性以及TBA、丙二醛（MDA）、GSH、TBIL含量，酶联免疫吸附（ELISA）法测定大鼠血清中白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）含量。测定大鼠肝脏系数，苏木精-伊红（HE）染色观察其病理学变化，免疫荧光法（IF）测定多药耐药相关蛋白2（MRP2）的表达水平，逆转录聚合酶链式反应（RT-PCR）和蛋白免疫印迹（Western blot）法测定MRP2、胆盐输出泵（BSEP）、法尼醇X受体（FXR）的基因和蛋白表达水平。[结果] 与LPS组比较，SGN各给药组肝组织结构、细胞形态和肝索排列情况得到改善，炎性细胞浸润、肝细胞变性和坏死程度减轻，胆汁中GSH、TBA、TBIL、DBIL含量升高，大鼠血清中AST、ALT、ALP活性增强，TBA、MDA、TBIL、IL-6、IL-1β含量降低，GSH含量升高，肝组织中MRP2荧光强度增强（P<0.05或P<0.01）；SGN-H组大鼠肝脏系数降低且胆汁流速加快，血清中TNF-α含量降低，肝组织中MRP2、BSEP、FXR基因和蛋白表达水平升高（P<0.05或P<0.01）。[结论] SGN能够改善LPS诱导的脓毒性IC大鼠胆汁淤积程度和肝功能，减轻炎症，发挥保肝降酶退黄作用，其机制可能是激活肝细胞的FXR受体，上调MRP2、BSEP基因及蛋白表达，从而促进胆汁酸排出。

Abstract:

[Objective] To investigate the hepatoprotective，enzyme-lowering and jaundice-relieving effects of Shuganning Injection（SGN） in rats with septic intrahepatic cholestasis（IC），and to explore its underlying mechanisms. [Methods] Thirty-six male SD rats of SPF grade were randomly assigned to the following groups：Control（blank），LPS（model），Ursodeoxycholic Acid（UDCA），SGN-L，SGN-M，and SGN-H，with six rats per group.A septic IC model was established via intraperitoneal injection of LPS（5 mg/kg）. The Control and LPS groups received tail vein injections of 10% glucose solution[6 mL/（kg·d）]，while the UDCA group received intraperitoneal injections of UDCA[25 mg/（kg·d）]. The SGN-L，SGN-M，and SGN-H groups received tail vein injections of SGN at 0.625，1.350，and 2.700 mL/（kg·d），respectively，administered continuously for 5 days.Collect rat bile and determine its flow rate. Biochemically measure total bile acids（TBA），total bilirubin（TBIL），direct bilirubin（DBIL），and reduced glutathione（GSH） in rat bile，alongside serum aspartate aminotransferase（AST），alanine aminotransferase（ALT），alkaline phosphatase（ALP） activity，and TBA，malondialdehyde（MDA），GSH，TBIL levels in serum. Enzyme-linked immunosorbent assay（ELISA） was employed to determine interleukin-6（IL-6），interleukin-1β（IL-1β），and tumour necrosis factor-α（TNF-α） concentrations in rat serum.Measurement of hepatic coefficients in rats，observation of pathological alterations via haematoxylin-eosin（HE） staining，determination of MRP2 expression levels by immunofluorescence（IF），and assessment of MRP2，BSEP，and FXR gene and protein expression levels via reverse transcription polymerase chain reaction（RT-PCR） and Western blot analysis. [Results] Compared with the LPS group，all SGN treatment groups exhibited improved hepatic tissue architecture，cellular morphology，and hepatic cord arrangement. Inflammatory cell infiltration，hepatocyte degeneration，and necrosis were reduced. Bile levels of GSH，TBA，TBIL，and DBIL increased，while serum AST，ALT，and ALP activities rose in rats. TBA，MDA，TBIL，IL-6，and IL-1β levels decreased，while GSH levels increased. MRP2 fluorescence intensity in liver tissue was enhanced（P<0.05 or P<0.01）. In the SGN-H group，the hepatic coefficient decreased and bile flow accelerated，serum TNF-α levels decreased，and MRP2，BSEP，and FXR gene and protein expression levels increased in liver tissue（P<0.05 or P<0.01）. [Conclusion] SGN ameliorates the severity of cholestasis and liver function in LPS-induced septic IC rats，alleviates inflammation，and exerts hepatoprotective，enzyme-lowering，and jaundice-reducing effects. Its mechanism may involve activating the FXR receptor in hepatocytes，thereby upregulating MRP2 and BSEP gene and protein expression to promote bile acid excretion.