Research Of Protective Effects And Mechanisms Of Polydatin On Nonalcoholic Fatty Liver Disease

BackgroundNonalcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by excessive fat storing in liver and hepatic cellular degeneration,without alcohol over ingestion. NAFLD consists of a spectrum of liver disease, ranging from simple steatosis to non-alcoholic steatohepatitis(NASH),fibrosis,eirrhosis.When weight of fat in liver exceed 5% of the liver or 1/3 above of liver cells have steatosis in liver biopsy,the liver is called fatty liver. Non-alcoholic fatty liver disease account for 80% of all the fatty liver.Morbidity of NAFLD is different in different countries, about 10%～24% in western country,but in Asian and Pacific regions the number is 12%-24%. In obesity incidence in patients the morbidity is 57.5%～74%. Incidence of children is about 2.6 %, while in those overweight the numbe can reach 52.8%.With the improvement of the standard of living, the change of the eating habits,more and more people are suffering nonalcoholic fatty liver(NAFLD) in china. Data shows in China, NAFLD prevalence has reached 15.4%,11.7% respectively in Shanghai and guangdong provinces. It appears that obesity is concerned with the cause of NAFLD. And there is a tendency of young. NAFLD is the most medium cause of liver dysfunction, accounting for 90%.The spectrum of NAFLD ranges from simple fatty liver (hepatic steatosis), with benign prognosis, to a potentially progressive form, nonalcoholic steatohepatitis (NASH), which may lead to liver fibrosis and cirrhosis, resulting in increased morbidity and mortality. WHO estimated that at least two million patients will develop cirrhosis due to hepatic steatosis in the years to come. Longitudinal cohort studies have demonstrated that those patients with cirrhosis have a similar risk to develop hepatocellular carcinoma as those with other causes of cirrhosis. All features of the metabolic syndrome, including obesity, type 2 diabetes, arterial hypertension, and hyperlipidemia (in the form of elevated triglyceride [TG] levels) are associated with NAFLD/NASH. NAFLD has become the third most important indication for liver transplantation. Total mortality of NAFLD significantly higher than normal. Prevention NAFLD has become a global attention as a medical and social problems.Pathogenesis of NAFLD is not very clear until now, "two hits" theory is approved by most medical scientists."first hit"is insulin resistance(IR).NAFLD almost exist insulin resistance. Insulin resistance lead liver cells to fatty degeneration through increasing lipolysis, in the meantime, sensitiity of damage factor inside and outside is rose."second hit"is lipid peroxidation caused by exceed lipid peroxide(LPO) and some cytokine. the "second hit"leads liver cells to inflammation,necrosis, fibrosis. Recent research suggests that PPAR-αand SREBP-1c is the main regulating factor to balance lipid synthesis and decomposition. Signaling pathways of PPAR-αand SREBP-1c closely linked to two hits. their regulation mechanisms become the hot spot being researched by most medical scientists. At the present time,there is no special drug to cure NAFLD, physicians focus on developing drug can decreace liver lipidosages by regulating signaling pathways of PPAR-αor/and SREBP-1c, especially from Chinese herbal medicine. There is no NAFLD in chinse tradional medicine, NAFLD is equivalent to Chinese "obesity""hypochondriac pain""jaundicet".According to TCM theory, intake excessive energy caused dampness-heat stagnating in spleen, dampness-heat in middle jiao, then qiji is blocked by dampness and heat, with the passing of time blood stasis is induced. To sum up,dampness, heat and blood stasis are the key for NAFLD.Giant Knotweed Rhizome,as a common herb,have the function of clearing heat, promoting diuresis,removing blood stasis. It can deal with most key Pathological factors,for that, Giant Knotweed Rhizome is used to treat obesity,hypochondriac pain or jaundicet.Polydatin is one of the main active ingredients Giant Knotweed Rhizome.Data shows that polydatin can decreace fat accumulation in liver, protective liver cell, etc. The experiment aims to observe the systematic effect of polydatin on NAFLD,and make further reseach the mechanism. The conclusion of this experiment provide more scientific basis for using Giant Knotweed Rhizome in clinic,and the author expect to explore a drug for treating NAFLD from Chinese herbal medicineObjectivesUsing the model of NAFLD recognized by the world and linking to the evolution of modern research of NAFLD,we observed the body weight,liver wet weight,the liver index,hepatic tissue pathology,Liver function,blood lipid,FFA,oxidative stress,TNF-αand hepatic tissue expression of PPAR-a, SREBP-lc and LXR-a protein and mRNA by the technique of pathology, biochemistry and molecular biology in the research.The aim of this investigation was to evaluate the efficacy and the poteintial mechanisms of the polydatin on the experiment nonalcoholic fatty liver disease(NAFLD) induced by high fat forage. MethodsForty-four clean graded male SD(Sprague Dawley) rats,weight 200±10g,were fed 1W normally,then were randomly divided into 2 groups,namely normal control group(A,n=8),model group(M,n=36), The normal control group had being fed with ordinary forage,while the rest with high fat forage(88% ordinary forage+10% lard+ 2% cholesterin) for 8w. After 8W,two rats were randomly choosed from each group,we observed samples of liver tissue sections that stained with HE method by light microscope to confirm NAFLD model rats replicated successfully.Then,the model group were randomly divided into 5 groups,namely blank model group(B,n=7),PD groups:low dosage,medium dosage,high dosage (C1,C2,C3, n=7, respectively) and Fenofibrate Tablets group(D,n=7). The forage of the normal control group don't change, the rest group were fed with high fat forage unceasingly. Since then, we poured PD Suspension liquid into stomach of rats of C1,C2,C3 groups,160mg/kg/d,80mg/kg/d,40mg/kg/d qd separately;the rats of D group are poured suspension of Fenofibrate tablet into stomach, 100mg/kg/d,qd;the rats of A and B group are poured isotonic nachloride into stomach,1 ml/100g.d,qd. At the end of twelve week of the experiment,all rats were fasted for 12h,then were killed after being narcotized with chloraldurat to leave over serum and hepatic tissue rapidly according to routine, which is fasted by formalin,packed with paraffin, sectioed,stained with HE,evaluated the degree of hepatic steatosis and inflammation under the optical microscope. Then reckoned liver index,to detect the content of serum ALT,AST,TC,TG,LDL,HDL,FPG,FIN,TNF-α. We leave over hepatic tissue to make 10% liver homogenate at 4℃for detecting content of T-AOC,SOD,GST,MDA, detected the protein expression of PPAR-α,LXR-α,SREBP-1c by Western blotting. The expression levels of PPAR-amRNA,LXR-amRNA,SREBP-1cmRNA were determined by real time quantitative RT-PCR. The data were analyzed with SPSS 13.0:Measurement data is expressed as the mean±SD(x±s). Differences between two group were analysed using unpaired Student'd-test,and on-way ANOVA for multiple comparisons. During ANOVA, data were tested via homogeneity test for variance first, if variance is homogenous, data were analyzed by on-way ANOVA, and groups were compared by LSD; if not, data would be analyzed by Dunnett T3 method after data tested via welch for vatiance.Rank data were analyzed by Kruskal-Wallis non-Parametric test. P<0.05. was considered significant.ResultsThe experiment I is to study the effect of PD on NAFLD.1.According to the hepatic tissue pathology and the state of hepatic function of rats,we succeeded in replicating the models of rats with NAFLD.2.Compared with the blank model group,the body weight and the liver index of PD high dosage (C1) group decrease significantly(P<0.01).During the experiment, weight of PD high dosage group grows slower, liver volume decreases.Liver wet weight and liver index decreased significantly (P<0.01). Hepatic pathology revealed, high polygoni cuspidati glycosides in rats, the fat deposition in liver cells reduced significantly (P< 0.01). Liver cell morphology, arrangement are improved significantly.3. Compared with the blank model group,the serum ALT of the PD high-dosage group and PD medium dosage group decreased significantly (P<0.01, P<0.05), and the effect of improving liver function is superior than fenofibrate group (P<0.05). High, medium polygoni cuspidati nucleoside dosage group of serum TG reduce rat (P< 0.01, P<0.05). Serum TC,LDL-c of the PD high dosage group and PD medium dosage group reduced significantly (P<0.05, P<0.01), at the same time HDL-C increased (P<0.01). FFA of Each PD group reduced markedly (P<0.01). The second part:Research of partly mechanisms of polydatin on nonalcoholic fatty liver disease1.Compared with the normal control group, T-AOC, SOD, GST of the blank model rats significantly reduced (P<0.01), the liver tissue T-AOC, SOD, GST of PD high and medium dosage group all are heighten(P<0.01), while reducing liver tissue MDA compared with blank model group(P<0.01). Liver anti-oxydation ability of PD high dosage group is more effective than fenofibrate group (P<0.01).2.Compared with the normal control group, FBG FIN of the blank model rats are notably higher (P<0.01), the IRI increased, and ISI are lower (P<0.01, P<0.05). Compared with blank model group, FBG,FIN,IRI of PD high dosage group,medium dosage group and fenofibrate reduced (P<0.01, P<0.05), at the same time there ISI heighten (P<0.01, P<0.05), including effect of PD high dosage is better than fenofibrate group (P<0.01, P<0.05). While each index of PD low dosage is no differences with blank model group (P>0.05). PD High and medium is more effective than PD low dosage group (P<0.01).3.Compared with the normal control group, serum TNF-αof the blank model rats increased significantly (P<0.01).Compared with blank model group, PD high and medium dosage group and fenofibrate group significantly reduced (P<0.01), PD high dosage group is obviously superior to medium and low dosage groups (P<0.01, P<0.01),but compared with fenofibrate group,there are no difference(P>0.05). PD low dosage group can not reduce serum TNF-αcontent, there are no significant differences from the blank model group (P>0.05).4.Compared with the normal control group, The expression of SREBP-1c,LXR-αprotein of the blank model rats increased significantly (P<0.01). while the expression of PPAR-a protein decresced. Compared with blank model group, the expression of SREBP-1c,LXR-αprotein PD high and medium dosage group significantly reduced (P<0.01), the two group are more effective than fenofibrate and PD low group(P<0.01). But the expression of PPAR-a protein was higher than the blank model group(P<0.01), The expression of PPAR-a protein of PD groups are no differences from the blank model group (P>0.05).Conclusions1.Polydatin can decrease significantly the body weight, improve hepatomegaly, reduce liver wet weight and the liver index. The experiment hints:to reduce the abdomen fat may be one of the mechanism of polydatin in the treatment of nonalcoholic fatty liver.2.Polydatin can reduce serum TG, TC,LDL-C and ALT,while increase HDL-C, and it shows that polydatin can correct blood disorder and keep the liver cells intact. According to the pathological polydatin can reduce the fat deposition in the hepatocyte of NAFLD rats,when there are in the stage of simple fatty liver (hepatic steatosis), thus reduce fat cause Swelling of liver cells and hypoxia and inflammation.The possible mechanism for polydatin to reverse liver fat deposits, improve liver function and protect liver cells is reducing free fatty acids flowing into the liver cells, to reduce excess synthesis of liver triglycerides and its toxicity.3.Polydatin can enhance the anti-oxidization ability, reduce lipid peroxidation. Polydatin reduce liver cell free fatty acids flowing into the liver cells, reduce oxidatie load of mitochondrial, thereby improve (3-oxidation, reduce Ros and free radicals.4.Polydatin can reduce TNF-a in blood, and improve insulin resistance. Polydatin reduces lipoclasis, and FFA released from fat tissue, through which Polydatin reduces the at deposits of liver cells.5.Polydatin can cut lipid synthesis factor SREBP-lc, LXR-a protein and gene expression,through decreacing expression of SREBP-lc, polydatin reduce lipid synthesis, which may be an key mechanism for polydatin to treat NAFLD.