The Experimental Study On The Molecular Mechanism Of Monascus On Preventing NAFLD Rat Models Induced By High-fatty Diet

BackgroundNon-alcoholic fatty liver disease(NAFLD) is the most frequent cause of abnormal liver function tests in China,with an estimated prevalence of 14-20%.It is caused by triglyceride(TG) accumulation within the liver and can either be a benign self-limiting state or a condition associated with steatohepatitis,which may progress to end-stage liver disease requiring liver transplantation.The most common disorder associated with NAFLD is genetic susceptibility,insulin resistance,intestinal endotoxemia,PPARα,an association which holds true in both lean and obese subjects. Insulin resistance is in turn a key element in the pathogenesis of NAFLD,However, the changes of IE and PPARαare "second-hit".Overexpressing lipoprotein lipase (LPL) in the liver in mice results in fatty liver and liver-specific insulin resistance.There is no similar title of NAFLD in traditional Chinese medicine.But there is similar manifestation of NAFLD in Internal Classic of Huang Di,such as'hepatic frllness',`liver swelling','hypochondriac pain',and so on.The disease located in the liver,which has a close relationship with gallbladder,spleen,stomach,kidney.The causes of NAFLD are overeating high-fat diet,excessive drinking,suffered from dampness-heat,imbalance of emotional,etc.These factors lead to Phlegm and Blood Stagnation in the liver.The effective components of Monascus are Lovastatin,which has an exact effect in anti-inflammatory and lipid-lowering.The studying team speculate that the Monascus maybe has an effect of anti-NAFLD.So,as a matter of fact,to discuss the mechanism of Monascus is an innovation in studying Chinese herb. Based on the theory of TCM and the research progress of modern medicine,We use high-fat diet to induce an experimental of NAFLD in rats.Each model is intervened by different dose of Monascus for eight weeks.the changes of blood lipid,hepatic biochemical,insulinre sistance,TNF-α,IL-6,IL-10,PPARαin rats are detected by HE stains,automatic biochemistry analyzer,RT-PCR,Western-blot,ELISA.To discuss the mechanism of Monascus is to improving the research level of TCM.ObjectiveTo observe the effect of monascus on insulin resistance,hepatic biochemical, blood lipid and the expression of TNF-α,IL-6,IL-10,PPARαin an experimental rat model of NAFLD induced by high-fat diet.The purpose is to discuss the pathogenic mechanism of NAFLD and the intervention effect of monascus.MethodAfter one week adaptive feed 48 SD rats were randomly divided into 6 groups with 8 rats in each group:(1)Normal group,(2)Blank group,(3)gynostemae group, (4)low-dose intervention group,(5)medium-dose intervention group,(6)high-dose intervention group.Group 1 was fed by common diet for 8 weeks,the other group were fed by high-fat diet for 8 weeks.Meanwhile,except normal group,the other group were treated by intragastric administration respectively using normal saline,gynostemae and different dose of Monascus once a day.In the ninth week,all the rats are sacrificed.The levels of blood lipid,hepatic biochemical,insulinre sistance,TNF-α,IL-6,IL-10,PPARαin rats are detected by HE stains,automatic biochemistry analyzer, RT-PCR,Western-blot,ELISA.Statistical analysis:All the data are represented as mean+SE;the statistical analysis was carried out using SPSS 11.5 software followed by One-way ANOVA-test and p-values less than 0.05 were considered as statistical significance.Result1.To study the influence of monascus on symptoms,physical signs in NAFLD rat model.The normal group expressed weigh increased,nose humid and clean,normal stool,no death in experiment.However,Rats in blank group displayed lassitude,no appetite,grew slowly,fur withered,retarded,and so on.Compared with blank group,there is an improvement in the expression of behavior,weight and nutrition in each intervention group(P=0.487).Especial in high-dosage monascus.2.The influence ofmonascus on liver morphology in NAFLD rat modelAfter the animal experiment gross manifestations such as the changes of liver histomorphology,the successful rate of NAFLD is 100 percent.The liver lobular is characterized by fatty degeneration,monocytes infiltration,lymphocyte infiltration and spotty necrosis in blank group.However,there is little fatty degeneration and a few inflammatory cells infiltration in gynostemae group and high-dose Monascus group.The changes in medium-dose and low-dose Monascus groups lie between the blank group and the high-dose group.3.The effects of monascus on serum hepatic function and lipid in NAFLD rat modelThe blank group shown an high levels of ALT and AST from the changes in serum hepatic function.This means that there is a pathology manifestation of hepatic cells apoptosis in NAFLD.After treatment with monascus or gynostemae,the levels of ALT and AST are on the decline.Compared with the intervention group,there is a significant difference in the levels of ALT,AST(P=0.000).There is no significant difference between Monascus and gynostemae group(P=0.337).There is an obvious lipid disorder in the blank group.Such changes are characterized by a rise of TG or LDL and fall of HDL.Compared with normal groups, there is a significant difference in blank groups(P=0.001).After treatment with monascus the changes of blood lipid are not obvious.There is no significant difference between Monascus group and Blank group(P=0.256).There is a significant difference in the expression of CHOL and HDL-C between high-dosage monascus group and gynostemae group(P=0.013,P=0.006).4.The influence of monascus on liver fibrosis in NAFLD rat modelFrom the changes of laboratory examination of liver cirrhosis,We can see clearly that the expression of LN,HA,PCⅢ,CⅣare on the increased in blank group.Except for low-dosage monascusmonasucs,there are no significant changes of above indexes in the other intervention group.There is no significant difference between blank group and high-dosage,medium-dosage,low-dosage monascusgroup and gynostemae(P=0.057).The monasucs has no effect on NAFLD.5.The effects of monascus on insulin resistance in NAFLD rat modelThe difference in the changes of blood sugar is great before the experiment began.The BS in blank is higher than that in normal group(P=0.030).This means there is a dysmetabloism of blood sugar in NAFLD rat models.Sixty minutes after the experiment,the changes of blood sugar show a homeostasis(5.3mmol/l),which means the formation of glucose clamp,is success.The changes of BS are still in an state of equilibrium.There is an similar change of blood sugar in high-fat diet group(including blank group and intervention group) and normal group.After the the glucose clamp experiment,we found that the GIR in high-fatty diet group is lower than that in the ordinary diet group.This means there is an insulin resistance in high-fat diet group. After treatments with monascus,the levels of insulin are on the decline.The monascus has an exact effect of decrease insulin.6.The influence of monascus on intestinal endotoxemia in NAFLD rat model Judgement from the changes in intestinal endotoxemia,The levels in the TNF-α,IL-6 and IL-10 are as follows.In blank group,the expression of above indexes are respectively 50.05±3.74,34.35±2.36 and 20.20±5.79;In the gynostemae group,the levels of above indexes are respectively 34.99±7.99,30.79±6.50 and 20.61±7.44;In the Monascus intervention group,the expression of above indexes are respectively 49.40±6.61,34.89±7.27 and 27.65±4.28;44.90±7.27,30.75±1.81 and 25.18±9.82; 35.68±7.29,28.61±9.51 and 24.00±11.15.Compared with blank group,the difference is obvious(P=0.000) in the changes of TNF-αin different intervention group.There is no significant difference between the blank group and other intervention group in the changes of IL-6 and IL-10(P=0.879,P=0.326).The expression of TNF-α,IL-6 and IL-10 mRNA in the liver are as follows.In normal group,the expression of above indexes are 0.51±0.50,0.29±0.16 and 0.55±0.07;In blank group are 1.21±0.55,0.53±0.18 and 0.32±0.16;In gynostema group are 0.54±0.24,0.41±0.22 and 0.40±0.13;In different monascus group are 0.63±0.28,0.65±0.50 and 0.73±0.39;0.29±0.27,0.50±0.18 and 0.50±0.14; 0.32±0.12,0.33±0.12 and 0.47±0.23.There is no significant difference between blank group and other groups in the changes of TNF-α,IL-6 and IL-10 mRNA(P=0.143, P=0.130,P=0.399).7.The influence of monascus on PPAR-alpha in NAFLD rat modelFrom the qualitative and quantitative expression of PPARαand protein in the liver,we can see clearly the sign that the PPARαmRNA in each group is 1.13±0.01,0.56±0.01,1.04±0.01,1.03±0.01,1.06±0.02,1.07±0.02 respectively.The PPARαprotein is 1.01±0.04,0.76±0.02,1.42±0.04,1.36±0.02,1.42±0.06,1.43±0.07.There is a significant difference between blank group and other groups(P=0.000).But there is no obvious difference between monasucs and gynostemae group(P=0.965).Conclusion1.NAFLD rat model was induced for eight weeks high-fatty diet.This model is similar to human NAFLD.The pathology was characterized by hyperlipidemia and hepatocytic macrovesicular steatosis,lobular inflammatory cell infiltration and necrosis.The metabolism is characterized by insulin-resistance,hyperinsulinemia, hyperlipoidemia and high levels in serum transaminase.This model is easy to establish,which provide a way to study the mechanism of NAFLD and screen a medicine on treating NAFLD. 2.The monascus maybe exert an effect on treating NAFLD.The mechanisms are as follows.①The monascus can improve the common condition in NAFLD rat model:For example,improving mentally condition,the consumption of food,nutrition.②The monascus can improve liver adipose degeneration and reduce the hepatic cells apoptosis and inflammatory cell infiltration according to the pathology manifestation of liver.③The monascus can decline the levels of serum ALT and AST in NAFLD rat model.The monascus restrain the changes of NASH by alleviate the inflammatory reaction.However,the monascus has no obvious effect on regulating the changes of blood lipid.④The monascus can block the "first attack"by declining the insulin resistance and hyperinsulinemia and increasing the sensitivity of insulin.⑤The monasucs can improve IE and alleviate selectivity IR by decreasing the levels of TNF-αin NAFLD rat model.So,as a matter of fact;the monascus can block the onset of NAFLD.⑥The monascus can exert an effect on alleviating fatty liver by up-regulating the expression of PPARαmRNA and protein in NAFLD rat model.Furthermore,the monascus can accelerate adiposeβ-oxidation in order to protect liver.ALL in all,The monascus may exert an exact effect on alleviating NAFLD by improving insulin r(?)sistance,inhibiting the expression of TNF-αas well as inducing the levels of mRNA and proteins of PPARαand maintaining the balance of lipid metabolism.The high-dose of monascus worth to be recommended.