Protective Effect And Potential Mechanisms Of Polydatin Against Experimental Liver Injury

ObjectiveLiver,as the largest and most important parenchymatous organ of human body,play a crucial role in synthesis,metabolism and detoxification of a variety of biochemical substance.Liver is fragile,it is vulnerable to attack from poison,drug and toxic metabolites.In recent years,with the increasing pressure on people’s lives,the serious pollution of our survival environment and frequent accidents of food and drug,the incidence of liver injury is rising year by year.Hence,it is urgent for us to prevent and cure liver injury,and it has also become a hotspot of research nowadays.Recently,traditional Chinese medicine has gradually gained attention among the world for its high efficiency in treating liver diseases with the advantage of multi-target and less side-effect therapy.Polygonum cuspidatum,which has been reported to exert heat-clearing,detoxifying,promoting blood flow and phlegm-resolving effects,is derived from the rhizome and radix of Polygonum cuspidatum Sieb.et Zucc.Polydatin,as the quality control components of polygonum cuspidatum,has been shown to exert various pharmacological effects,including anti-oxidation,anti-inflammation,anti-apoptosis and liver-protective effects.In this study,we aim to assess the antioxidant activity of PD by various in vitro antioxidant methods,and then study the potential protective effect and underlying mechanism via intracorporal treatments using D-gal-induced liver injury model and alcohol-induced liver injury model.Methods1.In vitro determination of antioxidant activity of PDIn this part,four analysis methods-DPPH,ABTS,·H and ·O2-free radical scavenging assays-were performed to assess the antioxidant capacity of PD.2.Protective effect of PD on D-gal-induced liver injury miceAll animals were randomly assigned to six groups(n=10):vehicle(vehicle),model(D-gal),positive(VE),PD(50,100 and 200 mg kg-1)groups.Except for the vehicle group,all the mice were injected subcutaneously with D-gal at a dose of 200 mg·kg-1 once a day for 8 weeks.Mice in the PD and VE groups were given corresponding doses of drugs by oral gavage at the same time.The mice in the vehicle group were subcutaneously and orally given normal saline(0.9%,w/v),in the same volumes.During the study,the general appearance,such as body weight,diet,mental state,hair,activities were observed.Twenty-four hours after the last administration,all the animals were anesthetized and killed.Blood samples were obtained for ALT and AST assay by automatic biological instrument.ELISA kits were usd for TNF-α,IL-1β and IL-6 assays.Liver was homogenized to obtain the homogenate,and then biochemical quantitative analysis were used for CAT,T-AOC,SOD,GSH-Px,MDA assay.The protein expression of Bcl-2,Bax and caspase-3 were determined by western blot in order to explore the potential mechanisms of polydatin in the treatment of D-gal-induced liver injury.3.Protective effect of PD on alcohol-induced liver injury ratsSixty male SD rats were divided into 6 groups randomly:vehicle group,model group,Alcohol+silymarin group(100 mg/kg,positive drug)and alcohol+PD groups(25,50,100 mg/kg).Except for vehicle and model group,rats in PD and silymarin group were given corresponding doses of drugs by oral gavage for 7 days,vehicle and model groups were given the same volume of saline.At day 8,except for vehicle group,rats in other groups were given alcohol(7 ml/kg,56%v/v)by gavage every 12 hours for 5 times.12 hours later after the last administration,rats from all groups were sacrificed humanely.Blood samples were obtained from abdominal aorta,then centrifuged to obtain serum for measurement of ALT,AST,ALP,LDH.The liver tissue was obtained and fixed in paraformaldehyde,H&E staining was used for histological examination,Oil red 0 staining was used for testing the severity of hepatic steatosis.The concentratio1s of cytokines including TNF-a,IL-6 and IL-1β in serum were evaluated by mouse ELISA kits.The level of TG,MDA,ROS,SOD,CAT and GSH-PX in liver were determined using commercially available assay kits.Western blot was used for evaluating the protein expression of CYP2E1,Nrf2,HO-1,TLR4,NF-κB p65 to further elucidate the possible molecular mechanisms of PD in treating alcohol-induced liver injury.Results1.In vitro determination of antioxidant activity of PDPD had excellent in vitro antioxidant abilities on DPPH,ABTS,·OH and·O2-radicals scavenging assays,which the IC50 was 87±2.10μg.mL-1,20 ±1.30 μg.mL-1,125 ± 3.50 μg.mL-1,181 ± 2.90 μg.mL-1 respectely.2.Protective effect of PD on D-gal-induced liver injury miceAccording to the observation of general appearance,we found that mice in model group exhibited symptoms of dim feather,shed hair,dullness,decreased activity.However,this situation in PD group had recovery to some extend.Compared to model group,50 mg/kg and 100 mg/kg of PD can markly attenuate the decrease of spleen and thymus indexes.However,there was no significant difference in kidney index although it was decreased either.We found that long-tern D-gal exposed led to an obvious increase in serum ALT and AST levels relative to the vehicle group,and pretreatment with PD suppressed this trend.H&E staining results indicated that mice in model group exhibited hepatocyte apoptosis and necrosis,inflammatory cell infiltration.Three doses of PD groups significantly improved D-gal-induced liver tissue damage.Compared with model group,PD treatment enhanced the content of T-AOC and the activities of CAT,GSH-Px,SOD,lower the content of MDA in liver.decreasing the expression of proinflammatory cytokines(TNF-α α IL-1β and IL-6).Western blot results indicate’d that PD significantly prevented D-gal-induced Bax and caspase-3 protein expression,up-regulated Bcl-2 protein expression in liver.3.Protective effect of PD on alcohol-induced liver injury ratThe results of liver functions assay showed that different doses of PD can remarkably reversed the excessive content of ALT,AST,ALP,LDH in a dose-dependent manner.H&E staining results showed that apparent hepatic parenchymal necrosis,inflammatory cell infiltration,disordered hepatic cords were observed in model group,oil red o staining results showed a large amount of lipid droplets were visualized in the livers of model group.Interestingly,PD treatment remarkably improved this situation.Relative to the model group,PD could also markedly suppress the content of TG,MDA,ROS and enhanced the activities of SOD,GSH-Px,and CAT in the liver.The protective effectof PD against oxidative stress was associated with down-regulated the expression of hepatic cytochrome P450 2E1(CYP2E1),and upregulated the expression of nuclear factorerythroid 2-related factor 2(Nrf2)and haem oxygenase-1(HO-1)PD pretreatment inhibited the release of proinflammatory cytokines(TNF-α,IL-1β and IL-6)via down-regulating toll-like receptor4(TLR4)and nuclear factor kappa B(NF-κB)p65.ConclusionOur in vitro study confirmed that PD exhibited strong antioxidant activity;A D-gal-induced liver injury mice model was established and demonstrated the protective effect of PD against D-gal-induced liver injury,the mechanism might be associated with decreasing the oxidative stress,inflammation and apoptosis caused by D-gal;Besides,an alcohol-induced liver injury model was also established and showed that short-term oral administration of PD was proved to exert a pronounced effect in elevating antioxidant enzyme activity to relieve ethanol-induced oxidative stress via the activation of Nrf2/HO-1 and CYP2E1 pathway and inhibiting pro-inflammatory cytokines expression to mitigate liver impairment through stimulation of TLR4/NF-κB pathway.