| Liver fibrosis is a pathological process characterized by excessive deposition of extracellular matrix(ECM)due to various pathogenic factors in liver.It is also the crucial step in the conversion development of different kinds of chronic liver diseases to liver cirrhosis.Thus,it is very important for the prevention and treatment of liver cirrhosis to eagerly control and attenuate the development of liver fibrosis.It has been approved that transformation-epithelial membrane(EMT)in hepatocytes driven by transforming growth factor-β1(TGF-β1)/Smads signaling pathway,can promote the occurrence and development of liver fibrosis.MicroRNA-34a(miR-34a)enhances p53 activity by inhibiting the expression of Sirtuin 1(Sirtl),indicating that miR-34a/Sirtl/p53 signaling may participate in the regulation of liver fibrosis.Clinical studies confirm that excessive intake of dietary fructose worsen liver fibrosis in patients with nonalcoholic fatty liver disease(NAFLD).Animal studies also observe that high fructose diet induces hepatic lipid deposition,accompanying with liver fibrosis.These observations suggest that excessive intake of fructose is closely related to the occurrence and development of liver fibrosis.Previous results from our research group confirmed that 10%fructose solution-fed rats caused metabolic syndrome and liver dysfunction,as well as liver inflammation and lipid accumulation.However,whether high fructose affect miR-34a/Sirt1/p53 and TGF-β1/Smads signaling pathway to trigger EMT in hepatocytes and liver fibrosis still need to be explored.Pterostilbene belonging to polyphenolic compounds,is an active ingredient mainly existed in natural plants.It is reported that pterostilbene alleviates dimethylnitrosamine-induced liver fibrosis in rats,possibly being associated with the suppression of TGF-β1/Smads signaling pathway.Our previous studies preliminarily confirmed that pterostilbene alleviated high fructose-induced pathological features of metabolic syndrome,improved hepatic oxidative stress and lipid deposition in rats.But,the effects on liver fibrosis and the molecular mechanism are worthy to further study.Therefore,this thesis explored whether high fructose could induce EMT in hepatocytes and liver fibrosis in a rat model of fructose-induced metabolic syndrome as well as fructose-exposed hepatocytes through altering miR-34a/Sirtl/p53 and TGF-β1/Smads signaling pathway?On the basis,this thesis focused on whether pterostilbene improved high fructose-induced EMT in hepatocytes and liver fibrosis by regulating miR-34a/Sirtl/p53 and TGF-β1/Smads signal pathway?This thesis established a rat model of liver fibrosis induced by high fructose routinely.Rats were fed with 10%fructose solution for continuous 16 weeks.At Week 7,high fructose-fed rats were received 10,20 and 40 mg/kg pterostilbene,as well as 5 mg/kg allopurinol(positive control)by intragastric gavage for next 10 weeks,respectively.Compared to normal group,decreased insulin sensitivity was confirmed by oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)in this animal model,in parallel with increased body weight,serum total cholesterol(TC)and triglyceride(TG)levels,indicating insulin resistance and dyslipidemia occurred in high fructose-fed rats,which were restored by pterostilbene or allopurinol.In this animal model,increased serum glutamic-oxaloacetic transaminase(AST)and glutamic-pyruvic transaminase(ALT)levels,hepatic TC,TG,interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)levels,red lipid droplets(oil red staining analysis)and inflammatory cells infiltration(hematoxylin-eosin(HE)staining analysis)were observed,demonstrating liver inflammation,lipid deposition and dysfunction.Furthermore,hyaluronic acid and hydroxyproline levels,collagen deposition(masson staining analysis),fluorescence of immunohistochemical a-smooth muscle actin(a-SMA)and fibroblast specific protein 1(FSP1),a-SMA,Collagen 1 and FSP1 protein levels were increased in the liver of high fructose-fed rats,and E-Cadherin protein levels were decreased,indicating that high fructose-fed rats happened to liver fibrosis and EMT in hepatocytes.Pterostilbene and allopurinol were found to restore these disturbances.These results suggested that pterostilbene protected liver function by normalizing hepatic inflammation,lipid deposition,and fibrosis in high fructose-fed rats.To further confirm the effects of high fructose and pterostilbene on EMT in hepatocytes,this thesis developed hepatocyte model(rat liver cell line,BRL-3A;human hepatocellular carcinoma cell line,HepG2)stimulated by high fructose(5 mM),and co-treated with pterostilbene(10 μM)and allopurinol(100 μM)respectively.Compared to normal group,increased protein levels of FSP1 and decreased protein levels of E-Cadherin were significantly observed in BRL-3A and HepG2 cells exposed to high fructose for 72 h,showing that high fructose induced hepatocytes to develope EMT.Pterostilbene and allopurinol recovered protein levels of FSP1 and E-Cadherin in these cell models,indicating that pterostilbene may weaken high fructose-induced EMT in hepatocytes.To detect whether high fructose and pterostilbene impacted TGF-β1/Smads signaling pathway in rat liver and hepatocytes,relevant protein levels were tested in this thesis.Compared to normal group,TGF-β1/Smads was activated because of increased TGF-β1,phosphorylated Smad2,phosphorylated Smad3 and Smad4 protein levels in the liver of high fructose-fed rats and-exposed hepatocytes(BRL-3A,HepG2:48 h).Compared with high fructose model group,pterostilbene and allopurinol down-regulated TGF-β1,phosphorylated Smad2,phosphorylated Smad3 and Smad4 protein levels in these animal and cell models,demonstrating that pterostilbene may alleviate high fructose-induced liver fibrosis and EMT in hepatocytes by suppressing TGF-β1/Smads signaling pathway.To investigate if fructose changes miR-34a/Sirtl/p53 signaling in rat liver and hepatocytes,this thesis analyzed miR-34a expression levels,Sirtl and acetylated p53 protein levels in high fructose-fed rat liver and-exposed hepatocytes.Compared to normal group,high fructose increased miR-34a expression levels,down-regulated Sirtl protein levels and up-regulated acetylated p53 protein levels in the liver of rats.It also elevated miR-34a expression levels(BRL-3A:12 h;HepG2:24 h),decreased Sirtl protein levels(BRL-3A:12 h;HepG2:24 h),increased acetylated p53 protein levels(BRL-3A:12 h;HepG2:24 h).Compared with high fructose model group,pterostilbene and allopurinol could reverse fructose-induced these phenomenons in these animal and cell models,indicating that pterostilbene may inhibit fructose-induced the activation of liver miR-34a/Sirtl/p53 signaling.To further verify possible target relationship between miR-34a/Sirtl/p53 and TGF-β1/Smads signaling pathway,next,this thesis transfected hepatocytes with miR-34a inhibitor or p53 siRNA,respectively.Compared to negative control-transfected hepatocytes under high fructose condition,miR-34a inhibitor raised Sirtl protein levels(BRL-3A:12 h;HepG2:24 h),down-regulated acetylated p53 protein levels(BRL-3A:12 h;HepG2:24 h)in hepatocytes stimulated by high fuctose.While p53 siRNA did not alter miR-34a expression levels(BRL-3A:12 h;HepG2:24 h)and Sirtl protein levels(BRL-3A:12 h;HepG2:24 h)in fructose-exposed hepatocytes.However,both miR-34a inhibitor and p53 siRNA down-regulated protein levels of TGF-β1(BRL-3A:48 h;HepG2:48 h),phosphorylated-Smad2(BRL-3A:48 h;HepG2:48 h),phosphorylated-Smad3(BRL-3A:48 h;HepG2:48 h),Smad4(BRL-3A:48 h;HepG2:48 h)as well as FSP1(BRL-3A:72 h;HepG2:72 h)in high fructose-exposed hepatocytes.These results indicate that high fructose may induce miR-34a/Sirtl/p53 signaling to activate TGF-β1/Smads signaling pathway,causing hepatocytes to MET.In high fructose-stimulated hepatocyte model transfected with miR-34a inhibitor or p53 siRNA,compared to negative control-transfected hepatocytes under high fructose condition,high Sirtl protein levels,as well as low acetylated p53,TGF-β1,phosphorylated Smad2,phosphorylated Smad3,Smad4 and FSP1 protein levels were observed in hepatocytes treated with pterostilbene and allopurinol These results indicate that pterostilbene may reduce high fructose-induced EMT in hepatocytes possibly by regulating miR-34a/Sirtl/p53 signaling to suppress TGF-β1/Smads signaling activation.Summary:high fructose may induce miR-34a/Sirtl/p53 signaling to activate TGF-β1/Smads signaling pathway,then lead to rat liver fibrosis with EMT in hepatocytes.Pterostilbene may regulate miR-34a/Sirtl/p53 signaling to inhibit TGF-β1/Smads signaling pathway,resulting in the improvement of high fructose-induced liver fibrosis and hepatocyte EMT.This thesis found the new way of high fructose-induced liver fibrosis,and initially revealed the molecular mechanism by which pterostilbene improved fructose-induced liver fibrosis with metabolism syndrome.This thesis may provide the experimental basis for its effective prevention and treatment of liver fibrosis with metabolism syndrome in clinical... |
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