The Role Of Mrp3 Efflux Transporter In Bile Acid Transport

Objective:Cholestasis is a pathophysiological process caused by bile secretion or excretion disorders,which is characterized by excessive accumulation of bile components such as bile acids in the liver and systemic circulation.Cholestasis will progress to liver fibrosis and even cirrhosis in the long-term persistence.In recent years,the occurrence of cholestasis is thought to be closely related to the abnormal expression of bile acid transporters.As one of the bile acid transporters on the basolateral membrane of the cell,the physiological function of multidrug resistance-associated protein 3(Mrp3)is to trasnport bile acid from cells into the blood,which plays an important role in maintaining the bile acid homeostasis.When cholestasis occurs,the protein expression of Mrp3 is significantly up-regulated,which increases the secretion of bile acids and reduces the accumulation of bile acids in the liver,an important way for liver detoxification.However,the effect and mechanism of Mrp3 on bile acid transport in normal physiological state and cholestasis have not been well studied.Therefore,we used Mrp3 knockout mice(Mrp3-/mice)as animal model to investigate the changes of bile acid profile and content in normal physiological state and cholestasis,and to explore the role and mechanism of Mrp3 in the transport of bile acids.Methods1、The role of Mrp3 in bile acid transport under the normal physiological conditions1)Blood and feces of Mrp3-/-mice and wild type mice(WT),C57BL/6J mice were collected at 12-weeks,16 weeks and 20weekss,respectively.Urine was collected at 20 weeks.Liver,gallbladder,duodenum,jejunum,ileum,colon and kidney samples of mice were collected after dissected.2)Fresh liver tissue was fixed with 4%paraformaldehyde for 48 h to prepare paraffin sections,and liver pathological examination was performed with hematoxylin-eosin(HE)staining to examine whether the Mrp3 deficiency has damage the liver under normal physiological condition.3)Ultra-high performance liquid chromatography-mass spectrometry(UHPLC-MS/MS)was used to simultaneously quantify the 24 major bile acids in the biological samples.4)The mRNA level of bile acid synthase(cholesterol 7α-hydroxylase Cyp7a1),bile acid uptake protein(Na+-dependent taurocholic cotransporting polypeptide(Ntcp)and organic anion transporting polypeptide lal(Oatp1a1)),bile acid efflux transporter(Mrp3,Multi drug resistance-associated protein 2(Mrp2),multidrug resistance-associated protein 4(Mrp4),P-glycoprotein(P-gp),organic solute transporter-α/β(Ostα/β),bile salt export pump(Bsep),organic anion transporter 2b 1(Oatp2b1)),the relative nuclear receptors(farnesoid X receptor(Fxr),pregnane X receptor(Pxr),constitutive androstane receptor(Car))and cell transcription factor tumor suppressor protein P53(p53)in the liver of Mrp3-/-mice and WT mice under normal physiological condition were mearsured by RT-PCR.2、The role of Mrp3 in bile acid transport in LCA-induced cholestasis1)9-10 week old male and female Mrp3-/-mice and WT mice were used to induce cholestasis using LCA.Mice in each group of cholestasis(LCA group)were injected intraperitoneally(i.p.)of 125 mg/kg/d LCA for 5 days.The control group(CO group)was intraperitoneally injected with the corresponding volume of corn oil.2)The fresh liver tissue of LCA group and CO group mice were collected and fixed with 4%paraformaldehyde for 48 h,and then analyzed by HE staining to observe the damage of liver tissue.3)UHPLC-MS/MS was used to quantify the bile acid profile in each tissues of LCA group and CO group.4)RT-PCR was used to mearsure the mRNA level of bile acid synthase,bile acid uptake protein,bile acid efflux protein,the relative nuclear receptors and cell transcription factor in liver of LCA group and CO group.Results:1、The role of Mrp3 in bile acid transport under the normal physiological conditions1)Liver HE staining results showed no difference in liver pathology between Mrp3-/mice and WT mice under normal physiological condition,There was slight inflammation in the liver of Mrp3-/-mice2)Under normal physiological condition,the liver total bile acid and ileal total bile acid of Mrp3-/-mice were significantly increased compared with WT mice(P<0.05).There was a slight change in bile acid content in other biological samples(the total bile acid did not change more than 2 flods).3)Under normal physiological condition,the mRNA level of Bsep in the liver of male Mrp3-/-mice was significantly higher than that in male WT mice(P<0.05).Compared with the female WT mice,the mRNA level of Mrp2 in the liver of female Mrp3-/-mice increased significantly(P<0.05).2、The role of Mrp3 in bile acid transport in LCA-induced cholestasis1)HE staining results showed that after WT and Mrp3-/-mice were induced by LCA,the liver was extensvely necrotic with inflammatory cell infiltration and neutrophil infiltration,and the contour of hepatic lobule was unclear.Also there were no significant changes in liver pathology of WT cholestasis mice and Mrp3 deficient cholestasis mice.2)The content of serum total bile acid and liver total bile acid in LCA-induced cholestasis mice were significantly higher than those in control mice(P<0.05).Serum free bile acids,and gallbladder free bile acids and total bile acids in the female Mrp3-/-LCA group were significantly higher than the WT-LCA female group(P<0.05).3)Compared with the corresponding control mice,the Mrp2 mRNA level in LCA-induced cholestasis mice was significantly lower(P<0.05),but the mRNA level of Ostβ was significantly increased(P<0.05).Compared to the corresponding control group,the level of Cyp7a1 mRNA in Mrp3-/-cholestasis mice was significantly lower(P<0.05),but the Cyp7a1 mRNA level in WT cholestasis mice and WT control mice was no significantly different.Conclusion:Under normal physiological condition,there was no obvious liver damage in Mrp3-/mice and only slight inflammation,indicated that the bile acid accumulated in the liver of Mrp3-/-mice was not sufficient to cause liver damage,possibly due to up-regulated Mrp2 and Bsep mRNA level.In LCA-induced cholestasis,there was no significant difference in liver pathological examination between Mrp3-/-cholestasis mice and WT cholestasis mice.And the total bile acids in the enterohepatic circulation between Mrp3-/-cholestasis mice and WT cholestasis mice were no difference.However,compared with the corresponding control group,the Cyp7a1 mRNA level of Mrp3-/-cholestasis mice was significantly decreased,while the Cyp7a1 mRNA level of WT cholestasis mice did not change.