Mechanism of Yinzhihuang Injection in Regulating MDR3 in Treatment of Hepatic Cholestasis Based on FXR

LI Qi; SHE Shi-feng; CAO Min; LAN Shao-yang; WANG Jia-chuan; LI Hui; TAN Mei-ao

doi:10.13422/j.cnki.syfjx.2017130145

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Mechanism of Yinzhihuang Injection in Regulating MDR3 in Treatment of Hepatic Cholestasis Based on FXR

更新时间：2024-01-04

- Mechanism of Yinzhihuang Injection in Regulating MDR3 in Treatment of Hepatic Cholestasis Based on FXR
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 23, Issue 13, Pages: 145-152(2017)
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- DOI：10.13422/j.cnki.syfjx.2017130145
  CLC： R285
- Published：2017
- 稿件说明：
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LI Qi, SHE Shi-feng, CAO Min, et al. Mechanism of Yinzhihuang Injection in Regulating MDR3 in Treatment of Hepatic Cholestasis Based on FXR[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(13): 145-152.
DOI：

LI Qi, SHE Shi-feng, CAO Min, et al. Mechanism of Yinzhihuang Injection in Regulating MDR3 in Treatment of Hepatic Cholestasis Based on FXR[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(13): 145-152. DOI： 10.13422/j.cnki.syfjx.2017130145.

摘要

目的：通过研究茵栀黄注射液（Yinzhihuang injection，YZH）对人正常肝细胞系HL-7702细胞的法尼酯X受体（farnesoid X receptor，FXR），多药耐药相关蛋白3（multidrug resistance associated protein 3，MDR3）基因表达的影响，探讨茵栀黄注射液调控肝脏胆汁酸代谢的作用靶点及机制，为茵栀黄注射液治疗肝内胆汁淤积的临床应用提供实验依据。方法：不同浓度茵栀黄注射液干预HL-7702，采用细胞活性检测试剂盒（cell counting kit-8，CCK-8）探索茵栀黄注射液对HL-7702的活性影响，结合实时荧光定量聚合酶链式反应（Real-time PCR）法确定茵栀黄注射液的理想干预浓度和时间；用GW4064（FXR激动剂）促进FXR基因的表达，分为茵栀黄注射液组，GW4064组，GW4064+茵栀黄注射液组，正常组，DMSO组；小干扰RNA（small interfering RNA，siRNA）沉默FXR基因，分为siRNA组，siRNA+茵栀黄注射液组，正常组，阴性组。Real-time PCR法、蛋白质免疫印迹（Western blot）法分别检测FXR，MDR3 mRNA及蛋白质的相对表达量，免疫荧光（IF）检测FXR蛋白的表达。结果：研究结果表明茵栀黄注射液对HL-7702的理想干预浓度1.08%，理想干预时间为15.47 h；茵栀黄注射液对HL-7702活性影响呈剂量依赖性；与正常组比较，GW4064组促进FXR，MDR3 mRNA和蛋白的表达（P<0.01）；与正常组比较，siRNA组抑制FXR，MDR3 mRNA和蛋白的表达（P<0.01）；与GW4064组比较，GW4064组+茵栀黄注射液组促进FXR，MDR3 mRNA和蛋白的表达（P<0.01）；与siRNA组比较，siRNA+茵栀黄注射液组促进FXR，MDR3 mRNA和蛋白的表达（P<0.01）。结论：茵栀黄注射液促进FXR，MDR3 mRNA和蛋白的表达，茵栀黄注射液通过FXR促进MDR3 mRNA和蛋白的表达来调控肝脏胆汁酸的代谢，本实验为其临床应用于治疗肝内胆汁淤积提供实验依据。

Abstract

Objective: To study gene expressions of farnesoid X receptor(FXR) and multidrug resistance associated protein3(MDR3) in human normal hepatocytes HL-7702 cell through the effect of Yinzhihuang injection (YZH)

in order to explore the target and mechanism of YZH injection in regulating hepatic bile acid metabolism

and provide an experimental basis for the clinical application of YZH in the treatment of intrahepatic cholestasis. Method: HL-7702 was treated with different concentrations of YZH. The effect of YZH on the activity of HL-7702 was explored by cell counting kit-8 (CCK-8). Real-time PCR was used to determine YZH's ideal intervention concentration and time. The gene expression of FXR was up-regulated by GW4064 (agonist of FXR)

and divided into YZH group

GW4064 group

GW4064+YZH group

normal group and DMSO control group. The gene expression of FXR was silenced by small interfering RNA (siRNA) and divided into siRNA group

siRNA+YZH group

normal group and negative group. Relative mRNA and protein expressions of FXR and MDR3 were detected by real-time quantitative PCR and Western blot

and the protein expression of FXR was detected by immunofluorescence(IF). Result: The ideal intervention concentration of YZH on HL-7702 was 1.08%

the ideal intervention time was 15.47 h

and YZH had an effect on the activity of HL-7702 in a dose-dependent manner. Compared with the normal group

GW4064 group promoted the mRNA and protein expressions of FXR and MDR3(P<0.01). Compared with the normal group

siRNA group inhibited FXR and MDR3 mRNA and protein expressions (P<0.01). Compared with GW4064 group

GW4064 group + YZH group promoted the mRNA and protein expressions of FXR and MDR3 (P<0.01). Compared with siRNA group

siRNA + YZH group promoted the mRNA and protein expressions of FXR and MDR3(P<0.01). Conclusion: YZH promotes FXR and MDR3 mRNA and protein expressions. YZH regulates hepatic bile acid metabolism by promoting the expression of MDR3 mRNA and protein with FXR. This study provides an experimental basis for its clinical application in the treatment of intrahepatic cholestasis.

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