The Prel Iminary Study On The Mechanism Of Idiosyncratic Liver Injury Induced By Polygonum Multiflorum Thunb.

ObjectiveBased on the idiosyncratic characteristic of clinical liver injury of Polygonum multiflorum Thunb.(HSW),an immune-induced animal model was established.On this basis,the characteristics of liver injury caused by Polygonum multiflorum Thunb.were comprehensively evaluated,and its serum metabolic markers were excavated.Thus,its potential mechanism was discussed preliminarily,which could provide some reference for the clinical safe use of Polygonum multiflorum Thunb.in the future.Methods1.Establish an animal model of liver injury induced by HSW,which is more similar to the clinical characteristics of liver injury.After 3 days of adaptive feeding,SD rats were randomly divided into six groups:blank group,LPS group,HSW low-dose group(L-HSW)and LPS+HSW-low dose group(L-HSW+LPS),HSW high-dose group(H-HSW)and LPS+HSW high-dose group(H-HSW+LPS),with 6 rats in each group.The rats in LPS group,L-HSW+LPS group and H-HSW+LPS group were injected with 2 mg/kg LPS via tail vein on the first day of the experiment,while other groups were injected with the same volume of saline solution according to their body weight.Two hours later,rats in L-HSW group and L-HSW+LPS group were given 2 g/kg HSW extract,H-HSW group and H-HSW+LPS group were given 10 g/kg HSW extract,and other groups were given the same volume of water.The animal model of idiosyncratic liver injury induced by HSW was established by intragastric administration for 7 days.2.Based on the animal model in vivo,the effect of HSW on liver injury was evaluated.After establishing the animal model of idiosyncratic liver injury of HSW,rat serum and liver samples were collected.We observed the body weight,behavior and compared the changes of liver index of rats in each group.Then,the changes of AST,ALT and TBA in serum samples were detected by these test kits,to evaluate the effect of HSW on liver function,and the expression of cytokines including iNOS,IL-6,COX-2 and HMGB-1 in serum were detected by ELISA assay.The paraffin sections of liver tissue stained with hematoxylin-eosin were observed under microscope,and the pathological changes of liver were evaluated from the structure of hepatocytes.Combined with the above indexes,the idiosyncratic injury effect of HSW on liver was comprehensively evaluated in the rat model induced by LPS.3.Study on the hepatocyte injury effect induced by HSW based on L02 cell in vitro.L02 cell line,that is human normal hepatocyte,was used as cell model in vitro.MTT assay was used to detect the effect of HSW,LPS and the combination of both on L02 cell proliferation.Flow cytometry was used to observe the apoptosis of L02 cells.Comprehensively,the effect of HSW on proliferation and apoptosis of L02 cell was evaluated through results above.4.Study on serum metabonomics of rats based on the in vivo model of liver injury induced by HSW.Based on the animal model of idiosyncratic liver injury induced by HSW established above,the serum differential metabolites and related metabolic pathways of rats between each group were analyzed and identified by metabonomics method.Meanwhile,the potential marker metabolites and mechanism of idiosyncratic liver injury induced by HSW were excavated.Results1.After injection of LPS,the rats had poor mental state,slow response,reduced intake of food and water,and loose stools,but gradually recovered as time went by.The body weight of rats in LPS group and HSW group increased naturally,there was no significant increase in liver index and liver biochemical indexes including AST,ALT and TBA in LPS group and HSW group.Nevertheless,after injection with LPS and then intragastric administration of HSW for 7 days,the mental state of rats in the LPS+HSW group continued to be depressed,ate and drank less,remained having loose stools,and could not recover by themselves.In addition,the liver index and the AST,ALT and TBA contents of rats in the LPS+HSW group increased significantly compared with the blank group.HE staining pathological sections showed that there were no obvious pathological changes in the liver between blank group and HSW group,and there were a few inflammatory cell infiltrations in LPS group,but no liver injury.While it is showed in the pathological section that there were a large number of inflammatory cell and neutrophil infiltration,destruction of hepatocyte structure,obvious vacuolar lesion and steatosis,as well as a small amount of necrotic area could be seen in LPS+HSW group.2.By detecting the expression of cytokines in serum,it was found that the expression of early inflammatory factors including iNOS,IL-6 and COX-2 increased significantly in LPS group and LPS+HSW group,indicating that LPS successfully induced inflammatory response in rats.It can simulate the immune stress state of human body when idiosyncratic drug liver injury occurs in clinic.The expression of HMGB-1,a late inflammatory mediator,increased significantly likewise,suggesting the prolongation of immune stress response in vivo.It could be speculated that the increase level of HMGB-1 can continuously activate the production and release of early inflammatory factors,as well as further promote the liver injury of HSW.3.HSW increased the cell survival rate at normal dose(15-120 μg/mL),which showed a promoting effect on the growth of L02 cells,and at ultra high dose(250-2000 μg/mL),the cell survival rate decreased with the increase of concentration,and significantly inhibited the proliferation of L02 cells.At the dose of 250 μg/mL,L02 cells began to appear in apoptosis,and the apoptosis rate increased in a dose-dependent manner.LPS had no significant effect on the survival rate of L02 cells at the dose of 2-5 μg/mL,which showed that low dose LPS had no inhibitory effect on the growth of L02 cells,and no apoptosis of L02 cells was found by flow cytometry assay.While at the dose of 10-15 μg/mL,LPS decreased the survival rate of L02 cells and showed obvious cytotoxicity.The effect of HSW on hepatocytes was not affected by the addition of LPS.4.Through the analysis of serum metabonomics,it was found that under the pre-stimulation of LPS,the serum metabolic profile changed significantly after administration of HSW.Compared with the blank group,LPS group and H-HSW group had little effect on the changes of serum metabolic profile.By comparison with the blank group,a total of 32 serum potential metabolic markers related to idiosyncratic liver injury induced by HSW were screened,including arachidonic acid metabolites such as KODE,HPODE,Prostaglandin B2,HETE,rostaglandin E1 and HEPE,and phospholipid metabolites such as PA(41:3),LysoPE(20:3),PE(42:7),PC(32::0),LysoPA(18:0)and LysoPA(16:0).It involves lipid-related metabolic pathways including arachidonic acid metabolic pathway,phosphatidylinositol signal pathway and glycerophospholipid metabolism.By comparing with LPS group,31 serum potential metabolic markers related to idiosyncratic liver injury of HSW were screened,including arachidonic acid metabolites such as Prostaglandin El,Leukotriene D5,Leukotriene B5,N-Acetyl-leukotriene E4,and phospholipid metabolites such as PGP(34:4),PS(34:5),PGP(a-13:0/18:2),LysoPC(14:1),LysoPA(20:4),PIP(16:0/20:1),PE(38:6),PGP(40:7),as well as cholicacid,Glycochenodeoxycholic acid,Glycocholic acid,Docosapentaenoic acid(22n-3)and other metabolites related to bile acid synthesis.It involves lipid-related metabolic pathways including glycerophospholipid metabolism pathway,primary bile acid biosynthesis and arachidonic acid metabolism.Conclusion1.In the body state of immune stress,HSW causes liver injury no matter in the clinical dose commonly used or slightly higher than the clinical dose.And its liver injury is an idiosyncratic liver injury based on inflammatory stress.2.LPS can activate the immune system and inflammatory cascade signal pathway,which can release a large number of inflammatory factors in the early stage of inflammation,but does not cause pathological damage to the liver.As for HMGB-1,on the one hand,it released in the late stage of inflammatory reaction,which can serve as a catalyst for the continuous production and release of early inflammatory factors,to promote the process of idiosyncratic liver injury caused by HSW;on the other hand,it can play a warning role to reflect the degree of hepatocyte injury.3.HSW at normal dose can promote the proliferation of L02 cells,and apoptosis can be induced only at ultra-high dose.The effect of HSW on hepatocyte apoptosis shows that it does damage hepatocytes in a dose-dependent manner.4.LPS does not induce immune stress reaction through hepatic parenchyma cells,and its response cells may be hepatic kupffer cells.5.Disturbance of lipid metabolism has occured in the process of idiosyncratic liver injury of HSW.