| ObjectiveThe effects of Radix Polygoni multiflori processed with Black bean and emodin combined with daidzein on the expression of hepatic intestinal transporters and metabolic enzymes were investigated.The Ussing Chamber technique was used to study the infiltration of daidzein through the intestinal mucosa,and the toxicity attenuation mechanism of black soybean juice on Radix Polygoni multiflori was investigated.Methods1.Effects of polygonum multiflorum,processed polygonum multiflorum on rat hepatic intestinal transporters and metabolic enzymes.Rats were randomly divided into 6 groups(6 rats in each group):Control(CON)group,Polygonum multiflorum(PM)group,Processeed Polygonum multiflorum(PPM)group,Polygonum multiflorum +black bean(PM + BB)group,Processeed Polygonum multiflorum + black bean(PPM + BB)group,Polygonum multiflorum Processeed with black bean(PMBB)group.After 14 days of continuous intragastric administration,the liver and jejunum tissues were harvested for relative quantification of the relative transporter and metabolizing enzyme mRNA expression in rat by RT-qPCR and proteins expression in rat liver and intestine were detected by western blot.The quantitative genes and proteins of liver tissues including P-gp,Mrp2,Mrp3,Mrp4,Oatp1,Oatp2,Bsep,Ntcp,CYP3A1,CYP3A2 and UGT1A1.The quantitative genes and proteins of jejunum tissue include Mrp2,Mrp3 and Mrp4.2.Effects of Emodin on rat hepatic intestinal transporters and metabolic enzymes.Rats were randomly divided into control group,low emodin concentration(20mg/kg/d)group,medium concentration(40mg/kg/d)group,and high concentration(80mg/ kg/d)group.After 30 days of continuous administration,Serum biochemical indicators were tested,and RT-qPCR method was used to determine the expression of P-gp,Mrp2,Mrp3,Mrp4,Oatp1,Oatp2,Bsep,Ntcp,and UGT1A1 mRNA in livers of treatment groups,and the protein was detected by Western blot;RT-qPCR was used to determine the expression of P-gp,Mrp2,Mrp3,and Mrp4 mRNAs in each intestinal segment,and protein expression changes were determined by Western blot.3.Effects of emodin with different concentrations of daidzein on rat hepatic intestinal transporters and metabolic enzymes.Emodin(80 mg/kg/d)was combined with different concentrations of daidzein(4,8,16 mg/kg/d).The treatment group were randomly divided into emodin(EM)group,emodin + low concentration daidzein(EM +LD)group,emodin + medium concentration daidzein(EM + MD)group,emodin + high concentration daidzein(EM + HD)group.After 30 days of emodin and daidzein administration,the serum,liver and small intestine were collected and the serum biochemical indexes were measured.Meanwhile,the mRNA expressions of P-gp,Mrp3,Ntcp and UGT1A1 in rat liver were quantified by RT-qPCR and protein detected by western blot,Rat duodenum,jejunal transporter Mrp2 mRNA and protein relative quantification.3.Effect of P-gp,Mrp2,Mrp3 Inhibitors on Daidzein Intestine Abolism in Rat.Using the previous experimental model established by our research team,we investigated whether daidzein is the substrate of P-gp,Mrp2,Mrp3,respectively.In the rat intestinal mucosa plus inhibitor Verapamil,MK-571,Benzbromarone to detect daidzein concentration in the different Intestine sections absorption and secretion direction of the apparent permeability coefficient and efflux transport rate changes,Compared to the control group,to deduce whether daidzein P-gp,Mrp2 or Mrp3 substrate.Results1.Effects of polygonum multiflorum,processed polygonum multiflorum on rat hepatic intestinal transporters and metabolic enzymes.(1)There was no significant difference in mRNA expression of Mrp2,Mrp4,Oatp1,Oatp2,Bsep,Ntcp,CYP3A1,and CYP3A2 in each group compared with CON group.The expression and protein of liver transport protein p-gp and Mrp3 mRNA in PM group rats The level of UGT1A1 mRNA and protein were significantly down-regulated(P<0.01).Compared with PM group,PM and BB group,PPM+BB group,PMBB group p-gp mRNA and protein levels were significantly down-regulated(P<0.01);PM+BB group,PPM+BB group,PMBB group Mrp3 mRNA and protein levels Significantly down-regulated(P<0.01);PM + BB group,PPM + BB group,PMBB group UGT1A1 mRNA expression were significantly upregulated(P<0.01),and bean-made polygonum multicolor UGT1A1 protein expression was significantly upregulated(P<0.01).(2)Compared with CON group,the expression of Mrp4 mRNA in the jejunum segment of each group had no significant change.The expression and protein level of Mrp2 mRNA in the jejunum segment of PM group rats were significantly down-regulated(P<0.01).The expression of Mrp3 mRNA and the protein level Significantly upregulated(P< 0.01).Compared with PM group,PMBB group Mrp2 mRNA and protein expression were significantly upregulated(P<0.05);PPM+BB group and PMBB group Mrp3 mRNA expression were significantly downregulated(P<0.05),PPM group,PPM+BB group,PMBB group protein levels Significantly upregulated(P<0.05).2.Effects of Emodin on rat hepatic intestinal transporters and metabolic enzymes.(1)Compared with the CON group,there was no significant change in the levels of ALT,AST,TP,ALB,LAP,ADA,and GLB in different treatment groups.The serum TBIL,DBIL,TBA,and ALP in the emodin medium and high concentration groups were all significant.Significantly increased(P<0.05).(2)There was no significant difference in expression of Mrp2,Mrp4,Oatp1,Oatp2,and Bsep mRNAs in the treatment groups compared with the CON group.The p-gp and Mrp3 mRNA expression and protein levels were significantly up-regulated in the emodin middle and high concentration groups.(P<0.01),Ntcp and UGT1A1 mRNA expression and protein levels were significantly down-regulated(P<0.01).No significant changes in expression of p-gp,Mrp3,Ntcp,and UGT1A1 were observed in the low emodin group.(3)Compared with CON group,the expression of P-gp,Mrp3 and Mrp4 mRNA in treatment groups was not significantly changed in the intestinal segments;the mRNA and protein expression of Mrp2 in the duodenum and jejunum segments of emodin high-concentration group were significantly down-regulated,and emodin There was no significant change in the low and middle concentrations.3.Effects of emodin with different concentrations of daidzein on hepatic intestinal transporters and metabolic enzymes.(1)Compared with the CON group,there was no significant change in ALT,AST,TP,ALB,LAP,ADA,and GLB levels in the drug administration group,and serum ALP,TBIL,DBIL,and TBA levels in the EM group rats increased significantly(P<0.05),significantly decreased with different concentrations of daidzein.Compared with EM group,the levels of EM+LD group,EM+MD group,EM+HD group TBIL and ALP decreased significantly(P<0.05);the levels of EM+MD group and EM+HD group DBIL decreased significantly(P<0.05).The EM+HD group TBA levels decreased significantly(P<0.05).(2)Compared with the CON group,there was no significant difference in mRNA expression of EM group,EM+LD group,EM+MD group,EM+HD group P-gp and Ntcp,and the mRNA and protein level of liver transport protein Mrp3 in EM group rats.Significantly increased(P<0.01).Compared with EM group,the mRNA and protein levels of EM+MD group and EM+HD group Mrp3 were significantly increased(P<0.05).There was no significant change in EM+LD group.Compared with the CON group,the mRNA and protein levels of liver transporter protein UGT1A1 in EM group rats were significantly decreased(P<0.01),and significantly increased with the combination of different concentrations of daidzein.Compared with EM group,the mRNA and protein levels of UGT1A1 in EM+MD group and EM+HD group were significantly increased(P<0.05),but there was no significant change in EM+LD group.(3)Compared with the CON group,there was no significant difference in the level of Mrp2 mRNA in the dorsal intestine transporter between the treated group and the control group.The mRNA and protein levels of Mrp2 in the jejunum of the EM group rats were significantly higher(P<0.05)compared with the EM group.The mRNA and protein expression of group Mrp2 in EM+MD group and EM+HD group were significantly increased(P<0.05),but there was no significant change in EM+LD group.4.Effect of P-gp,Mrp2,Mrp3 Inhibitors on Daidzein Intestine Abolism in Rat.(1)Compared with the control group,after addition of verapamil,a P-gp inhibitor,the efflux rates of daidzein at different concentrations increased through the jejunum segment.Colonic segment efflux rate increased,while the high concentration of daidzein via the ileum and colon efflux rate decreased,but no statistically significant.(2)Compared with the control group,after the Mrp2 inhibitor mk571 was added,the efflux rates of daidzein at different concentrations through the duodenum segment decreased significantly(P<0.05).Compared with the control group,after adding Mrp2 inhibitor mk571,the concentration of daidzein decreased significantly(P<0.05)through the jejunum segment.Compared with the control group,after adding Mrp2 inhibitor mk571,the different concentrations of daidzein decreased significantly(P<0.01)through the ileum.(3)Compared with the control group,after adding the Mrp3 inhibitor benzbromarone,there was no significant difference in the efflux rates of daidzein in colon,jejunum,ileum.ConclusionThe mechanism of attenuating Polygonum multiflorum by black soybean juice may be related to the inhibition of the up-regulation of P-gp,Mrp3 and Mrp3 expression in the liver,the down-regulation of UGT1A1 in the liver and the expression of Mrp2 in the intestine.Cholestasis decreased after combined use of emodin and daidzein,liver injury was decreased,which may be related to the inhibition of the upregulation of Mrp3 and UGT1A1 expression in the liver and the down-regulation of Mrp2 expression in the jejunum segment.Daidzein may be the substrate of Mrp2;the mechanism of Polysaccharide of Polygonum multiflorum Thunb by black soybean juicei may be related to the influence of daidzein on the expression of Mrp3 and UGT1A1 in the liver and Mrp2 in the intestine. |
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