Study On The Hepatoprotective Effect And The Underlying Mechanism Of Polydatin In High Fructose-induced Liver Iniury Of Metabolic Syndrome

Evidences from clinical and basic researches indicate that excessive fructose consumption induces metabolic syndrome,with non-alcoholic steatohepatitis(NAFLD),which aggravates the progression of liver fibrosis.Thus,it is necessary to strengthen researches on its pathological mechanism and drug intervention.Oxidative stress is one of the key factors in the pathological development of NAFLD.Nuclear factor erythroid 2-related factor 2(Nrf2)dissociates from the Kelch-like ECH-associated protein 1(Keap1)and eters the nucleus,initiating various antioxidant enzymes to defense oxidative stress.MicroRNA200a(miR-200a)regulates redox homeostasis.Targetscan 5.2 database predicts that miR-200 binds to the 3 ’non-coding region(UTR)of Keapl mRNA.Our previous study preliminarily clarifies that high fructose-produced reactive oxygen species(ROS)triggers thioredoxin-interacting protein(TXNIP)over-expression to activate NOD-like receptor protein 3(NLRP3)inflammasome,resulting in liver inflammation and lipid accumulation.Whether high fructose affects miR-200a expression and Keapl/Nrf2 pathway to ROS over-production,causing liver inflammation and lipid accumulation?In addition,activation of transformation growth factor β1(TGF-β1)/Smads signaling induces hepatocytes epithelial mesenchymal transition(EMT),which promotes the progression of liver fibrosis.Survivin induces cancer cell EMT through positive regulation of TGF-β1.Significant down-regulation of miR-200a is observed in carbon tetrachloride-induced mice fibrotic liver.MiR-203 is down-regulated in various fibrotic tissues.Targetscan 5.2 and starBase v2.0 database predict that both miR-200a and miR-203 bind to the 3’UTR of survivin mRNA.Zinc finger E-box binding homeobox 1(ZEB1)translocate nucleus to suppress the transcription of miR-200 family member and miR-203.Whether high fructose induces ZEB1 nuclear translocation to affect miR-200a and miR-203 expression,by which enhances survivin-controlled TGF-β1/Smads signaling activation in hepatocyte EMT and liver fibrosis?Polydatin is one of main compounds in Polygonum cuspidatum Siebold&Zucc Clinically,polygonum cuspidatum has excellent hepatoprotective effects.Pharmacological studies have shown that polydtain possesses hepatoprotection.However,the improvement of polydatin on high fructose-induced metabolism syndrome and liver injury as well as the possible molecular mechanism needs further investigation.1.The study on the protection of polydatin in fructose-induced metabolic syndrome and liver injuryThis thesis developed the Sprague-Dawley rat model of metabolic syndrome with the treatment of 7.5,15 and 30 mg/kg polydatin or 4 mg/kg pioglitazone(positive control)by intragastric gavage,respectively.Results of oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)confirmed that polydatin and pioglitazone could increase glucose tolerance and insulin sensitivity in this animal model.They decreased serum insulin,total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-c),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels,and reduces inflammatory cells infiltration,red lipid droplets in parallel with decrease in IL-1β,TNF-α,TG and TC levels in high fructose-fed rat livers.In addition,polydtain and pioglitazone significantly decreased serum hyaluronic acid,laminin and III procollogen levels,hepatic hyaluronic acid and hydroxyproline levels,and reduced perisinusoidal or portal/peripotal collagen deposition.These observations indicate that polydatin and pioglitazone effectively improved insulin resistance,hyperinsulinemia,dyslipidemia and systemic inflammation,and liver inflammation,lipid accumulation and fibrosis in this animal model.2.The mechanism study of polydatin on regulation of miR-200a-mediated Keapl/Nrf2 pathway in fructose-induced liver inflammation and lipid accumulationThis thesis first examined whether high fructose down-regulated miR-200a expression and up-regulate Keapl to inhibit Nrf2 antioxidant pathway with induction of ROS,resulting in liver inflammation and lipid accumulation,and the molecular mechanism by which polydatin ameliorates inflammation and lipid accumulation.Consistent with our previous studies,fructose-induced hepatic ROS-driven TXNIP overexpression activated NLRP3 inflammasome to promote production of IL-1β and interfere with lipid metabolism-related protein peroxisome proliferators-activated receptor α(PPAR-α),carnitine palmitoyl transferase 1(CPT-1),sterol regulatory element binging protein 1(SREBP-1)and stearoyl-CoA desaturase-1(SCD-1)expression,causing rat liver inflammation and lipid accumulation.Compared with control-vehicle group,miR-200a expression levels were significantly down-regulated,accompany with increase in Keap1 protein levels,decrease in nuclear Nrf2,glutathione transferase(GST),heme oxygenase 1(HO-1)and quinone oxidoreductase(NQO1)protein levels in high fructose-fed rat livers and in high fructose-exposed BRL-3A and HepG2 cells.Tese data indicated that high fructose-induced low-expression of miR-200a and impaired Keap1/Nrf2 pathway could be involved in rat liver oxidative stress.To confirm miR-200a targeting Keap1,dual luciferase reporter assay and Keapl siRNA transfection were carried out.In fact,miR-200a targeted the predicted site with 3’-UTR of Keap1 mRNA;miR-200a mimic decreased Keap1 protein levels(24 h).These results suggest that miR-200a targets Keap1 in high fructose-exposed BRL-3A cells.To explore the role of miR-200a targeting Keap1 in oxidative stress,inflammation and lipid accumulation,further studies confirmed that both miR-200a mimic,Keapl siRNA up-regulated Nrf2 nuclear,GST,HO-1 and GST(24 h)protein levels,abrogated over-production of ROS(24 h),over-expression of TXNIP,up-regulation of NLRP3,apoptosis-associated speck-like protein(ASC),Caspase-1 and IL-1β,deregulation of PPAR-α,CPT-1,SREBP-1 and SCD-1,with TG and TC accumulation(48 h)in BRL-3A cells cultured with fructose.tert-butylhydroquinone(tBHQ)pretreatment for 8 h succeeded in up-regulating nuclear Nrf2,GST,HO-1 and NQO1 protein levels(24 h),down-regulating TXNIP,NLRP3,ASC,Caspase-1,IL-1β,SREBP-1 and SCD-1 protein levels,up-regulating PPAR-a and CPT-1 protein levels,and decreasing TG and TC levels(48 h)in BRL-3A cells exposed to fructose.These data demonstrates that fructose decreases miR-200a expression to up-regulate Keap1 and inhibit Nrf2-antioxidant pathway with increase in ROS,and then activate TXNIP/NLRP3 inflammasome to promote IL-1β production and disturb lipid metabolism-relative protein expression,causing liver injury with oxidative stress,inflammation and lipid accumulation.Polydatin and pioglitazone significantly increased miR-200a expression levels,down-regulated Keapl protein levels,up-regulated nuclear Nrf2 and antioxidants protein levels,down-regulated TXNIP,NLRP3,ASC,Caspase-1,SREBP-1 and SCD-1 as well as up-regulated PPAR-α and CPT-1 protein levels in the liver of fructose-fed rats.In addition,polydatin and pioglitazone also exerted the similar effects in fructose-exposed BRL-3A and HepG2 cells.The results from miR-200a mimic transfection,Keap1 siRNA transfection,tBHQ pretreatment and so on in high fructose-stimulated BRL-3A cell models discover the new pathway of protection against fructose-induced metabolism syndrome-associated liver injury by polydatin and pioglitazone,by which polydatin and pioglitazone may increase miR-200a expression and repair the Keap1/Nrf2 pathway to reduce ROS production,and then block activation of TXNIP/NLRP3 inflammasomes to decrease IL-1β levels and regulate lipid metabolism-related proteins expression,thereby ameliorating fructose-induced liver oxidative stress,inflammation and lipid accumulation.3.The study on the pathway and mechanism of polydatin in ameliorating high fructose-induced liver fibrosisThis thesis explored whether ZEB1 nuclear translocation caused by high fructose decreased miR-200a and miR-203 expression to up-regulate survivin,and then activated TGF-β1/Smads signaling in hepatocyte EMT and liver fibrosis,and intervention mechanism of polydatin and pioglitazone on these pathological aspects.Compared with control-vehicle group,high fructose significantly increased nuclear ZEB1 but decreased cytoplasm ZEB1 protein levels,down-regulated miR-203 expression levels,increased survivin mRNA and protein levels;increased fibrotic regulator TGF-β1 levels,and up-regulation of TGF-β1 as well as its downstream p-Smad2,p-Smad3 and Smad4 protein levels,indicating that TGF-β1/Smads signaling be activated.Moreover,high fructose increased liver protein levels ofα-smooth muscle actin(α-SMA)and collagen 1(COL1A1),with down-regulation of Ca2+ dependent cell adhesion molecule E(E-cadherin)and up-regulation of fibroblast specific protein 1(FSP1),N-cadherin and vimentin with COL1A1 part co-location with COL1A1 in rat lives.Similar results were observed in fructose-exposed BRL-3A cells.These results suggested that high fructose caused rat liver fibrosis with EMT.To confirm the role of high fructose-induced ZEB1 nuclear translocation in hepatocyte EMT,this thesis first explored the relationship between ZEB1 nuclear translocation and low-expression miR-200a and miR-203.ZEB1 siRNA up-regulated miR-200a and miR-203 expression levels;miR-200a mimic significantly elicited miR-203 expression(4 h),but miR-203 mimic didn’t change miR-200a expression(12 h).These results suggested that high fructose induce ZEB1 nuclear translocation to down-regulate miR-200a,and then inhibit miR-203 expression.Second,further analyzed the possible targeting relationship between miR-200a,miR-203 and survivin in high frucyose-exposed BRL-3A cells to find that miR-200a mimic decreased but miR-200a inhibitor increased survivin protein levels(24 h);miR-203 targeted the predicted site with 3’-UTR of survivin mRNA;miR-203 mimic decreased survivin protein levels(24 h).Third,this thesis focused on the role of ZEB1 nuclear translocation to inhibit miR-200a and miR-203 expression in fibrosis.Further studies comfirmed that all of ZEB1 siRNA、miR-200a mimic、miR-203 mimic significantly decreased survivin,TGF-β1(24 h),p-Smad2,p-Smad3 protein levels and Smad4 nuclear translocation(48 h),and then up-regulated E-cadherin,down-regulated FSP1,N-cadherin,vimentin and COL1A1(48 h)in fructose-exposed BRL-3A cells.In addition,both survivin siRNA and TGF-β1 siRNA significantly abrogated fructose-induced up-regulation of survivin(24 h)as well as TGF-β1(24 h),p-Smad2,p-Smad3 and Smad4 nuclear translocation(48 h),which in turn increased E-cadherin,decreased FSP1,N-cadherin,vimentin and COL1A1(48 h)in BRL-3A cells.These data indicate that fructose may induce ZEB1 nuclear translocation to reduce miR-200a and miR-203 expression,causing survivin-mediated TGF-(31/Smads signaling activation in hepatocyte EMT and liver fibrosis.Polydatin and pioglitazone significantly decreased hepatic ZEB1 nuclear but increased cytoplasm ZEB1 protein levels,up-regulated miR-203 expression levels,decreased survivin mRNA and protein levels,reduced TGF-(31 levels,decreased TGF-β1,p-Smad2/3 and Smad4 protein levels,increased E-cadheirn with reduction FSP1,N-cadherin,vimentin,COL1A1 and α-SMA protein levels in high fructose-induced metabolic syndrome animal model.In high fructose-exposed BRL-3A cells model,polydatin and pioglitazone also exerted the similar effects.The results from ZEB1 siRNA,miR-200a mimic,miR-200a inhibitor,miR-203 mimic,surivin siRNA or TGF-β1 siRNA transfection in high fructose-stimulated BRL-3A cell models discovered the new molecular mechanisms of improvement fructose-induced liver fibrosis by polydatin and pioglitazone,by which polydatin and pioglitazone may suppress ZEB1 nuclear translocation to enhance miR-200a and miR-203 expression,and then down-regulate survivin to inhibit TGF-β1/Smads signaling activation and reverse hepatocyte EMT,alleviating fructose-induced liver fibrosis.In conclusion,fructose-induced miR-200a low-expression increased Keap1 to inhibit Nrf2 antioxidant pathway,and then caused ROS-driven TXNIP to promote NLRP3 inflammasome activation and lipid metabolism-related protein dysregulation,resulting in liver oxidative stress,inflammation and lipid deposition.;fructose induced ZEB1 nuclear translocation to decrease miR-200a expression,further repress miR-203 expression,and then target survivin to activate TGF-β1/Smads signaling,developing hepatocyte EMT in liver fibrosis.Polydatin increased miR-200a expression to decrease Keapl and activate Nrf2 antioxidant pathway,and then blocked ROS-driven TXNIP to suppress NLRP3 inflammasome activation and regulated lipid metabolism-related proteins,protecting against fructose-induced inflammation and lipid deposition;repressed ZEB1 nuclear translocation to increase miR-200a and miR-203 expression,and then suppress survivin-controlled TGF-β1/Smads signaling activation and reverse hepatocyte EMT,alleviating fructose-induced liver fibrosis These results illustrate the new ways of high fructose-induced liver oxidative stress,inflammation,lipid accumulation and fibrosis,as well as reveal the new molecular mechanisms of the hepatoprotection by polydatin.These findings provided experiment basis for polydatin used in the treatment and prevention of metabolic syndrome and metabolic syndrome-associated liver injury in the clinic.