Effects And Mechanisms Of Sini Powder On Mice Model Of Non-alcoholic Steatohepatitis

Objective:To copy a mouse model of non-alcoholic steatohepatitis induced by MCD diet and a HepG2cell model of non-alcoholic steatohepatitis induced by palmitic acid according to a synthetical reference study and research the preventive effect and mechanism of Sini Powder in vitro and vivo respectively.Methods:1.C57BL/6mice were randomly divided into control group,model group, Probucol group,high dose of Sini Powder group and low dose of Sini Powder group. Mice were fed with MCD diet while probucol and Sini Powder were administered for4weeks,the serum and liver of mice were taken for detection. The content of ALT、AST and TG in serum and the content of TG、SOD、GSH and MDA in liver were detected; Histopathological changes of liver were observed by HE staning and lipid accumulation in liver were observed by Oil Red O staning; Changes of mitochondrial ATP enzyme、mitochondrial swelling and membrane potential in liver were measured; The content of TNFaand IL-22were assayed by Elisa.AdipoR2、PPARα、CPT-1、AOX、HO-1and REF-1mRNA expression levels of liver were detected by RT-PCR and Q-PCR.2.HepgG2cells were divided into blank control group,model group,5%serum of Sini Powder group and10%serum of Sini Powder group. HepG2cells were lipid-overloading induced by Palmitic Acid for24h, meanwhile different concentrations of serum with Sini Powder were used to culture cells. Cells were stained with Oil-Red-O to examine the amount of lipid accumulation in the cells. TG、ALT、AST、SOD and MDA in homogenates from cells were measured. The mRNA expression levels of AdipoR2、AMPK、PPARα、CPT1and AOX1were detected by Q-PCR. Results:1. The mice in model group were weight loss and impatient,accompanied by increased water volume、hydrouria and black stool, showing the symptoms of effulgent liver fire in TCM. The liver color of model mice were more yellow than control group,however, no increase in liver weight and liver index. Hepatic steatosis, a lot of inflammatory cell infiltration and hepatocyte swelling deformation were observed in liver by HE staining. Accumulation of lipid droplets in liver were observed by Oil Red O staining. Compared with control group, the content of TG in serum of model mice were reduced meanwhile increased in liver, ALT、AST and TNFawere significantly raised.2.Sini Powder can improve the color of liver. Hepatic steatosis, a lot of inflammatory cell infiltration and hepatocyte swelling deformation in high dose of Sini Powder group while more serve hepatic steatosis and inflammatory cell infiltration in low dose of Sini Powder group were observed by HE staining. Compared with model group, less lipid droplets in high dose of Sini Powder group, however, more lipid droplets in Probucol group and low dose of Sini Powder.In high dose of Sini Powder, levels of ALT、AST and TNFawere declined, the content of TG were increased in serum while reduced in liver.3. In high dose of Sini Powder, the content of SOD and GSH were raised while MDA was declined; ATP enzyme activity of mitochondrial were reduced to normal level;the content of TNFawas effectively reduced while IL-22was increased, but the effect of elevating IL-22level was not obvious; the gene expressions of AdipoR2, PPARa,CPT-1, AOX, HO-1and REF-1were increase significantly. Sensitivity to high concentrations of calcium solution and uptake of rhodamine123of mitochondrial were raised obviously in low dose of Sini Powder group.4. After induced by Palmitic acid for24h, intracellular lipid vacuoles in HepG2 cell line were seen by Oil Red O staining, indicating that the formation of the NAFLD lipid deposition in vitro model.5.After prevented by different concentrations of serum with Sini Powder for24h, intracellular lipid deposition was reduced, the content of TG, ALT,AST and MDA were lower significantly, and increase SOD content obviously. The gene expressions of AMPK, CPT-1and AOX were increased in10%serum of Sini Powder group, while the gene expression of AdipoR2was increased in5%serum of Sini Powder group.Conclusions:1.NASH model of mice can be induced by MCD diet for4weeks and it has similar clinical pathology.2. Sini Powder can prevent NASH by alleviating hepatic lipid deposition, improving liver function and protecting the liver cells.3. Sini Powder can enhance the capacity of fatty acid oxidation and antioxidant in liver by upregulating the expression of AdipoR2-PPARapathway and the gene expression of antioxidant. It is concluded that Sini Powder can be an important target for the treatment of NASH.4.Sini Powder can inhibit inflammation in NASH,and it may be one of mechanisms that related to NF-κB pathway.