The Effect Of Emodin On FXR/BSEP Signaling Pathway In Cholestasis

Objective: Cholestatic liver disease(CSLD)is a common clinical liver disease.It refers to bile formation,secretion and/or excretion disorders caused by various causes inside and outside the liver,so that bile flow can not flow into the intestine and deposit in the hepatobiliary system.The pathological state,its etiology is complex,the pathogenesis needs to be further clarified,and there is still insufficient effective treatment.The nuclear receptors farnesoid X receptor(FXR)is currently considered to be a key target for the treatment of cholestasis.Emodin is the main active ingredient in Chinese medicine rhubarb.It has been proven by domestic and foreign scholars to have liver protection and anti-inflammatory,anti-viral,anti-bacterial,immune regulation,promotion of gastrointestinal motility,anti-oxidation,scavenging free radicals,improving microcirculation,etc.Many pharmacological effects.In this study,we investigated the intervention of emodin by FXR/BSEP signaling pathway on cholestasis by in vitro and in vivo experiments,and hope to provide a new strategy for the treatment of cholestasis.Methods: 1、 The human liver cell line LO2 was cultured,and the normal group,the UDCA group,the DXM group,the emodin low concentration group,the emodin medium concentration group,and the emodin high concentration group were respectively set.After emodin intervention,cells were harvested,q RT-PCR was used to determine the expression level of FXR/BSEP signaling pathway-related mRNA in LO2 cells,and the expression level of FXR/BSEP signaling pathway-related protein in LO2 cells was determined by western-blot method.2、 Synthesis of FXR si RNA sequences for down-regulation of FXR molecules in LO2 cells.To make FXR gene overexpression in vitro,we transfected LO2 cells with corresponding FXR lentiviral vectors.When LO2 cells were transfected for 48 h and 72 h,GFP was determined by fluorescence microscope respectively.To confirm the transfection effect,we measured the mRNA expression of FXR,BSEP,and protein levels of FXR1,FXR2,BSEP,at 72 h after transfection.3、 We examined the effect of Emodin on LO2 cells after FXR down-regulated or overexpression.After being transfected with lentiviral vectors for 72 h,LO2 cells were challenged with Emodin as previous before harvest.After 24 h,the cells were harvested,and the mRNA and protein levels of molecules related to FXR/BSEP signal pathway were measured.4、 We use we use two-step collagenase digestion method to extract and cultivate rat primary hepatocytes,and we used immunofluorescence to detect CK-18 protein in cells to identify whether the extracted cells were rat primary hepatocytes.Immunofluorescence self-excitation,keratin CK-18 immunofluorescence assay was used to verify the purity and trypan blue exclusion test to verify viability of hepatocytes.After judging the viability and purity of the cells were more than 95%,the rat primary hepatocytes were obtained and cultured in vitro.Primary hepatocytes were treated with Emodin,than harvested after 24 h.The mRNA and protein expression of molecules related to FXR/BSEP signal pathway were measured by Qpcr and western-blot method.5、 Forty-two male Sprague-Dawley(SD)rats aging 3 weeks were divided averagely and randomly into normal group,model group,UDCA group,DXM group,and three Emodin treatment groups at doses of 20 mg/kg,40 mg/kg,and 80mg/kg,respectively.The experiment lasted for 8 days.The animals in the last six groups received ANIT at a dose of 2ml/100 g body weight(250mg ANIT in100ml peanut oil)via oral gavage on the 5th day.On the 1st to 6th day,each group was given normal saline or the corresponding concentration drugs respectively.On the 8th day,the end of the experiment,blood samples and liver tissnes were collected from each rat and stored at-80 °C.The biochemical parameters of rat serum were measured by ELISA.HE staining was applied to confirmed the degree of histopathological changes in liver.The distribution and expression levels of FXR1,FXR2 and BSEP in liver tissues were detected by IHC method.The mRNA and protein levels of molecules related to FXR/BSEP signal pathway were detected by q RT-PCR and western-blot.Results: 1、In LO2 cells,the mRNA expressions of BSEP and FXR were significantly elevated in the emodin group when compared with the control group.The protein expressions of BSEP,FXR1 and FXR2 were also significantly increased in the emodin groups.2、After FXR was down-regulated by si RNA,the mRNA expressions of BSEP,FXR and the protein expressions of BSEP,FXR1 and FXR2 of the si RNA group were significantly lowered when compared with the control group.While compared with the FXR-si RNA group,the mRNA expressions of BSEP and FXR were significantly elevated in the emodin groups,the protein expressions of BSEP,FXR1 and FXR2 were also significantly elevated in the emodin groups.3、After FXR over-expression by lentivirus transfection,the mRNA expressions of BSEP and FXR were significantly increased in the lentivirus-up group,and the protein expressions of BSEP,FXR1 and FXR2 were also significantly elevated in the lentivirus-up group when compared with the control group.Compared with the lentivirus-up group,the mRNA expressions of BSEP and FXR were significantly elevated in the emodin groups,while the protein expressions of BSEP,FXR1 and FXR2 were also significantly elevated in the emodin groups. 4、In rat primary hepatocytes,compared with the untreated cells,consistent with the results of the LO2 hepatocyte cell line.the emodin group showed significantly elevated mRNA levels of FXR and BSEP.The protein expression of FXR1,FXR2 and BSEP were also significantly increased.5、In SD rats with cholestatic,compared with the control group,the mRNA levels of BSEP and FXR of the model group were decreased apparently.The mRNA expression of BSEP and FXR was affected by emodin treatments when compared with model group.The protein expressions of BSEP,FXR1 and FXR2 were significantly decreased in the model group when compared with the control group.While compared with the model group,the protein expressions of BSEP,FXR1 and FXR2 were significantly elevated in the emodin groups.Emodin also had a notable effect on rat pathological manifestation,BSEP positive staining in liver tissues and the test of liver function.Conclusion: 1、 In LO2 cell line and rat primary hepatocytes,Emodin can increase FXR-BSEP signaling pathway-related molecules.2、 In SD rats,Emodin could relieve the histopathologic changes in the liver and exert anti-cholestasis properties through stimulating FXR-BSEP signaling pathway.