Exploring The Effect Of Enhancing Liver-targeting Of Rein By Vinegar-baked Radix Bupleuri Based On HNF1α/HNF4α

Background and the aim: Previous study showed that vinegar-baked Radix Bupleuri(VBRB)enhanced the liver-targeting effect of rhein,oxymatrine and resveratrol by affecting drug transporters and metabolism enzymes.However,the regulating passway is not clear.Therefore,in this paper,an acute hepatitis model companied with deficient HNF1α/HNF4α expression was established by lipopolysaccharide(LPS),and the pharmacological effect of VBRB on rhein and its upper and down stream regulating targets were studied,thereafter,the liver targeting enhancing effect of different parts of VBRB was analyzed to give some clues for the active constituents of liver targeting enhancing effect of VBRB.Method: 1)Therapeutic effect and mechanism of VBRB combined with Rhein on acute liver injury model.Six days after the prophylactic administration,plasma and tissue samples were collected after intraperitoneal injection of lipopolysaccharide(LPS)in 4.5 h.To comprehensively evaluate the effect of VBRB combined with rhein on acute liver injury,the liver index,ALT,AST,SOD and related inflammatory factors(IL-6,IL-1β,TNF-α,INF-γ)was measured.The expression of HNF1α/HNF4α in the liver of each group,and expression of protein levels of Pgp,Mrp2,OAT2 and OATP2 were determined.2)Effects of different parts of VBRB on the in vivo distribution of rhein.Rhein combined with VBRB polysaccharide,n-butanol part of VBRB,and small molecule part of VBRB were administered to rats,respectively.There were two ways,one is to collect plasma and tissue samples at different time points through a single administration,and the other is to collect plasma and tissue samples at peak time after administration for 6 days.The content of rhein in the samples was determined;and the contents of HNF1α/HNF4α and ATPase,drug transporters in liver tissues were determined.Results: 1)Therapeutic effect and mechanism of VBRB combined with Rhein on acute liver injury model.Compared with the normal control,the levels of AST and ALT in plasma were significantly increased,HNF1α/HNF4α in liver were decreased,the expressions of Pgp and Mrp2 transporter were significantly increased,and the expressions of OATP2 and OAT2 were decreased in the model group.Compared with the model group,the Rhein + VBRB(low dose)could significantly reduce the content of AST and ALT in plasma and increase the activity of SOD and ATPase in the liver.Compared with the Rhein group,the low dose of VBRB increased the concentration of rhein in the liver,and promoted the expression of HNF1α/HNF4α;meanwhile inhibited the expression of Pgp and Mrp2,and increased protein expression of OATP2 and OAT2.2)Effects of different parts of VBRB on the in vivo distribution of rhein.In single administration experiment,all three parts of VBRB increased the distribution of rein;in multiple doses administration experinment,micromolecular hydrophilic extract(MHE)and polysaccharides increased liver concentration of rhein significantly.In the single administration experiment,MHE and polysaccharides of the VBRB increased the Cmax of rhein,and all three parts delayed the elimination of rhein.MHE,polysaccharides and n-butanol parts increased the relative uptake ratio(RUE)by 93%,60% and 50% respectively;and increased relative targeting efficient ratio(RTE)by 49%,49% and 30% respectively.Besides MHE had the tendency of increasing the acitivity of phase II metabolism enzyme,all three parts had the tendency of increasing the activity of HNF1α/4α.In the multidose experiment,MHE and polysaccharides parts increased liver/blood ratio by 39% and 33%,respectively;all three parts of VBRB increased the expression of OAT2 and OATP2 as well as HNF1α/4α,and meanwhile inhibited the expression of Mrp2.Conclusion: In this paper,the synergistic effect of VBRB was demonstrated by using LPS-induced acute hepatitis model,and this synergistic effect is related to the enhancement of liver targeting of rhein.Mechanism studies showed that VBRB promoted the expression of animal liver HNF1α/HNF4α,affected the drug transporters Mrp2,Pgp,OAT2 and OATP2,and achieved the liver targeting enhancing effect.All the studied parts of VBRB had the effect of increasing rhein concentration in liver,and were the active parts of VBRB.