Fuzheng Huayu Recipe Prevents Nutritional Fibrosing Steatohepatitis In Mice

Non-alcoholic fatty liver disease (NAFLD) is one of the chronic liverdiseases which is strongly linked with metabolic syndrome including obesity,dyslipidemia, type2diabetes, hypertension and so on. The prevalence ofNAFLD is estimated to be about20%in the western world, while it isincreasing rapidly in China and assumes the younger age tendency. Someindividuals can progress to hepatocacinoma eventually after reaching thefibrotic stage and subsequently cirrhosis threatening human health seriously.Fibrosis is a reversible healing response to chronic injuries, hence, searchingfor the effective antifibrotic methods is of great importance to the treatment ofNAFLD.Fuzheng Huayu recipe (FZHY), a compound of Chinese herbal medicinefor hepatic fibrosis, consists of six Chinese medicinal herbs, namely SemenPersicae, Radix Salvia Miltiorrhizae, Gynostemma Pentaphyllammak,Cordyceps, Pollen Pini, Fructus Schisandrae Chinensis. Based onpharmacological experiment repeatedly,it was produced in the light ofChinese medicine theory that deficiency of healthy energy and blood stasis arethe basic pathological changes of hepatic fibrosis. The previous clinical trialsshowed that FZHY could significantly improve clinical symptoms, liverfunction, reverse hepatic fibrosis and decrease portal pressure in patients withchronic hepatitis B and liver cirrhosis. However, the effect and mechnism ofFZHY on fibrosing steatohepatitis remains unknown.In this study, we investigated the protective role and molecular basis ofFZHY in the evolution of nutritional fibrosing steatohepatitis induced by highfat, methionine and choline deficient (MCD) diet in mice, which will providetheoretical evidence for the therapy of nutritional fibrosing steatohepatitis andthe development of traditional Chinese medicine. Part1Effect of FZHY recipe on general condition, serum ALT, AST,histopathologal changes in mice fed with MCD dietObjective: To explore the protective role of FZHY recipe in anti-fibrosis,and whether the co-administration of FZHY and hemin showed a synergeticeffect on anti-fibrosis, which could lay the foundations for antifibrotictreatment.Methods: C57BL/6J mice were fed with methionine-choline deficient(MCD) diet for8weeks to induce fibrosing steatohepatitis. Control animalswere fed with choline-methionine supplemented diet (control group). FZHYand/or heme oxygenase-1(HO-1) chemical inducer (hemin) wereadministered to mice, respectively. The effect of FZHY was assessed bycomparing the severity of hepatic injury, levels of hepatic lipid peroxides, andthe expression of oxidative stress, and inflammatory related genes. Serumalanine aminotransferase (ALT) and aspartate aminotransferase (AST) weremeasured by enzymic kinetic method using an automatic biochemical analyzer.Hematoxylin and eosin (HE) stained and Masson trichromatism stainedparaffin-embedded liver sections were scored for hepatic steatosis,inflammation and fibrosis according to the guidelines for diagnosis andtreatment of non-alcoholic fatty liver diseases and histological scoring systemfor NAFLD.Results:1Behavior alterationsAll the experimental mice survived. Mice in control group lookedenergetic, swift with shiny hair and without weight loss, while mice fed withMCD diet lost weight significantly, looked exhausted. There was nosignificance in comparing the general condition of mice between hemin andFZHY group with unbalanced hair, weight loss, less ingestion intake. Whilemice administered with hemin and FZHY presented worse general conditioncompared to mice fed the control diet.2Histopathology examinationThe liver sections from mice fed an MCD diet alone exhibited disordered lobule structure, severe macrosteatosis, spot or focal hepatocyte necrosis andinflammatory infiltration, portal fibrosis and fibrous septum (NAS7~8S2~3).However, mice treated with FZHY in the presence or absence of hemin couldnotably ameliorate hepatic steatosis, necrotic inflammation and improved liverfibrosis (NAS5~6S1~2). Co-administration of FZHY and hemin had a furtherimproved effect on hepatic inflammation and fibrosis (NAS1~2S0~1).3Serum levels of ALT and AST in experimental miceSerum ALT (222.00±6.96U/L) and AST (316.00±14.2U/L) of mice fedwith MCD diet significantly hegher than that of mice fed the control diet(30.60±5.86U/L,32.00±5.57U/L)(P<0.001, P<0.001). A significant decreaseof ALT and AST levels was noted after hemin (146.80±9.20U/L,134.00±11.03U/L) or FZHY (106.20±8.44U/L,108.60±10.48U/L) treatmentcompared to mice fed with MCD diet (P<0.01, P<0.01). An additive effectwas observed in the mice treated with FZHY and hemin, the ALT and ASTlevels in which (71.25±12.76U/L,55.25±8.50U/L) were markedly decreasedthan those of mice treated with FZHY (P<0.001, P<0.001) or hemin (P<0.01,P<0.01) respectively.Conclusions:1FZHY treatment could improve the general condition, reduce the serum ALT,AST levels, ameliorate hepatic steatosis, necrotic inflammation and improvedliver fibrosis.2Co-administration of FZHY and hemin showed a synergetic effect onanti-fibrosis in mice with nutritional fibrosing steatohepatitis induced by MCDdiet.Part2Effect of FZHY recipe on hepatic expression of lipid peroxidationmediator, oxidative stress related genes and inflammatory genes innutritional fibrosing steatohepatitis in miceObjective: To explore the effect and mechnism of FZHY recipe onhepatic expression of lipid peroxidation mediator, oxidative stress relatedgenes and inflammatory genes in nutritional fibrosing steatohepatitis in mice.Methods: Grouping and intervention methods in accordance with part1. Malondialdehyde (MDA) content was measured by Thiobarbituric acidreactive substances. The hepatic mRNA and protein expressions ofanti-oxidative gene HO-1and pro-oxidative stress gene cytochrome P4502E1(CYP2E1) were deteimined by quantitative real-time PCR. The mRNAexpression of inflammatory related genes TNF-α and IL-6was detected byquantitative real-time PCR.Results:1Effect of FZHY on hepatic oxidative stressThe levels of MDA in mice fed with MCD and control diet were in linewith part1. FZHY (3.88±0.79nmol/mg) or hemin (3.58±0.71nmol/mg)lowered hepatic oxidative stress as demonstrated by TBARS assay comparedwith MCD fed mice (P<0.05, P<0.05), while the combination of FZHY andhemin (3.17±0.77nmol/mg) showed a better effect on suppressing MDAconcentrations than FZHY (P<0.01) or hemin (P<0.05) treatment.2Effect of FZHY on the expression of lipid peroxidation mediator CYP2E1The mRNA and protein expressions of CYP2E1elevated significantly inMCD feeding mice. After FZHY or hemin treatment for8weeks, the mRNAand protein expressions of CYP2E1were down-regulated by compared withthat of mice fed MCD diet. However, Co-administration of hemin and FZHYfurther reduced the hepatic expression of CYP2E1.3Effect of FZHY on the expression of anti-oxidative stress factor HO-1The mRNA and protein expressions of HO-1in MCD feeding miceincreased significantly compared with mice fed with control diet. After hemintreatment the mRNA and protein expressions of HO-1were higher than that ofMCD fed mice. While FZHY treatment could down-regulated the HO-1expression. Co-administration of FZHY and hemin showed higher HO-1expression than FZHY and lower than hemin treatment.4Effect of FZHY on the mRNA expression of inflammatory ralated genesTNF-α and IL-6Relative to control mice, hepatic TNF-α and IL-6were up-regulated inMCD diet-fed mice, which was significantly blunted by treatment with FZHY or hemin. The combination of FZHY and hemin led to a further effect onpreventing the TNF-α and IL-6mRNA expression compared withMCD+hemin groupConclusions:1Oxidative stress and lipid peroxidation existed in the liver of nutritionalsteatohepatitis induced by MCD diet, which might lead to fibrogenesis.2FZHY treatment significantly reduce expression of HO-1and CYP2E1, thusprevent or ameliorate the hepatic injury triggered by excessive lipidperoxidation products.3Administration of FZHY and/or hemin significantely reduced the hepaticexpression of TNF-α and IL-6, consequently, alleviated or prevented fibrosingsteatohepatitis by decreasing liver inflammation.4Co-administration of FZHY and hemin showed a synergetic effect on liverinjury through inhibiting oxidative stress by mediating key oxidative stressrelated factors CYP2E1and HO-1, inflammatory ralated genes TNF-α andIL-6, which is beneficial for the treatment of fibrosing steatohepatitis.Part3Effect of FZHY recipe on hepatic expression of pro-fibrosis genesin nutritional fibrosing steatohepatitis in miceObjective: To explore the role and the molecular mechanism of FZHYrecipe in nutritional fibrosing steatohepatitis in mice.Methods:Grouping and intervention methods in accordance with part1.Hepatic mRNA and protein expressions of alpha-smooth muscle actin(α-SMA), transforming growth factor-β1(TGF-β1), collagen type I (Col-1)and Col-3were assayed by quantitative real-time PCR and Western blotrespectively.Results:1Effect of FZHY recipe on hepatic expression of α-SMA, TGF-β1In MCD feeding mice, mRNA and pritein expressions of α-SMA,TGF-β1had a marked elevation. However, administration of FZHY and/orhemin could reduce hepatic expressions of the two genes. The combinationof hemin and FZHY showed an additive effect on suppressing the above genes' expressions.2Effect of FZHY recipe on hepatic expression of Col-1and Col-3The mRNA and protein expressions of Col-1and Col-3were induced byMCD diet. After hemin or FZHY treatment the expression of Col-1andCol-3was significantly down-regulated in the livers of mice, especially inthe livers of mice treated with the combination of hemin and FZHY.Conclusions:1FZHY recipe supressed hepatic expression of pro-fibrosis genes such asα-SMA, TGF-β1, Col-1, Col-3in MCD diet induced fibrosing steatohepatitis,especially the combination of FZHY and hemin showed a synergetic effecton inhibiting hepatic expression of pro-fibrosis genes.2Compound of Chinese herb medicine FZHY plays a protective role inameliorating nutritional fibrosing steatohepatitis.