Effect Of FZHY Recipe On Hepatic Expression Of IKK-β In Mice With Nutritional Fibrosing Steatohepatitis

Objective: Non-alcoholic fatty liver disease (NAFLD) is a complexspectrum of diseases, ranging from asymptomatic steatosis to non-alcoholicsteatohepatitis (NASH), fibrosing steatohepatitis, cirrhosis and hepatocellularcarcinoma. Fibrosing steatohepatitis, through which NASH develops intocirrhosis, is the key stage in the spectrum of NAFLD. Up to now, thepathogenesis of nutritional fibrosing steatohepatitis is still an open question,and definitive therapy for these patients are lacking. Hence, searching for theeffective antifibrotic methods is important to reverse nutritional fibrosingsteatohepatitis. In the light of Chinese medicine theory of hepatic fibrosis thatdeficiency of health energy and blood stasis, Fuzheng Huayu recipe (FZHY)was produced. It consists of six Chinese medicinal herbs, namely SemenPersicae, Radix Salvia Miltiorrhizae, Gynostemma Pentaphyllammak,Cordyceps, Pollen Pini and Fructus Schisandrae Chinensis. We havedemonstrated that FZHY improved nutritional fibrosing steatohepatitis in miceby lowering TGF-β related genes level. Inhibitor κB kinase-β (IKK-β), as theupstream regulator of the nuclear factor-kappa B (NF-κB) signaling pathway,which mediates NF-κB activation in response to a number of different stimuli.IKK-β/NF-κB signaling controls the expression of genes involved ininflammation and immunity. However, it, whether IKK-β/NF-κB signalingpathway plays also a key role in nutritional fibrosing steatohepatitis, is notclear. In this study, experimental hepatic fibrosis models were established byfeeding male C57BL/6J mice with high fat, methionine-choline deficient(MCD) diet, and the mice were intervened with FZHY or/and hemeoxygenase-1(HO-1) chemical inducer (hemin). The aim of this study was toexplore the potential roles of IKK-β, monocyte chemoattractant protein-1(MCP-1), matrix metalloproteinase-2(MMP-2) and connective tissue growthfactor (CTGF) and effects of FZHY on these genes regulation in nutritional fibrosing steatohepatitis induced by MCD diet.Methods: C57BL/6J mice were fed with methionine-choline deficient(MCD) diet for8weeks to induce nutritional fibrosing steatohepatitis. FZHY,hemin or the combination of FZHY and hemin were administered to mice,respectively. Control animals were fed with choline-methionine supplementeddiet (Control group). Experimental animal hair, activities and body weightwere observed. Hepatic steatosis, liver inflammation and fibrosis wereobserved under HE staining, and liver fibrosis by Masson staining. Theexpression of IKK-β, MCP-1, MMP-2and CTGF mRNA and protein wereanalyzed by RT-PCR, Western blot and immunohistochemistry, respectively.Results:1The general situation of experimental animals: Mice in control grouplooked energetic, swift with shiny hair and with weight increased gradually,while the mice fed with MCD diet looked exhausted and lost weightapparently. Mice administered with FZHY, hemin or the combination of heminand FZHY presented better situation compared to MCD feeding mice.2Liver histopathology: The liver histology is normal in the control. Theliver sections from mice fed an MCD diet exhibited severe macrosteatosis,spot or focal hepatocyte necrosis and inflammatory infiltration, portal fibrosisand fibrous septum (NAS7~8S3~4). The extent of steatohepatitis and fibrosisin liver of FZHY and hemin treated group is dramatically decreased(NAS5~6S1~2). Co-administration of hemin and FZHY had a furtherimproved effect on hepatic inflammation and fibrosis (NAS4~5S0~1).3The hepatic mRNA and protein expression of pro-inflammation andpro-fibrosis factors: As compared with the control mice, the mRNA andprotein expression of IKK-β were up-regulated in MCD fed mice, which wassignificantly blunted by given FZHY and hemin, companied withdown-regulation of the pro-inflammation and pro-fibrosis factors MCP-1,MMP-2and CTGF. The combination of FZHY and hemin led to a furthereffect on preventing these factors expression and liver fibrosis. Conclusions：1IKK-β, MCP-1, MMP-2and CTGF played a crucial role in the processof nutritional fibrosing steatohepatitis established by feeding mice with MCDdiet for8weeks.2FZHY recipe could prevent nutritional fibrosing steatohepatitisthrough modulation of expression of IKK-β and related genes.3Co-administration of FZHY and hemin played a synergistic effect onameliorating nutritional fibrosing steatohepatitis by mediating keyinflammatory and fibrogenesis related genes.