The Character And Partial Possible Mechanism Of Mice Liver Inflammation And Fibrosis Induced By Vitamin D

Background And AimThe vitamin D deficiency is a global public health problem. It was estimated about 1 billion population in a state of vitamin D deficiency or inadequate in the world.Beside general population and many patients who suffering from chronic diseases both were observed with vitamin D deficiency. In recent years, many clinical studies have found that vitamin D levels in patients with chronic liver disease was significantly lower than normal control group. The reason may be associated with less sunlight, inadequate intake of vitamin D in the diet, liver damage. Patients with lower vitamin D have worse liver function, poorer prognosis.Traditional researches suggest vitamin D is mainly involved in calcium phosphate, bone metabolism, bone growth,but it was found that VDR widely exists in the brain, heart, skin, prostate, breast, and hepatocyte and many immune cells according to recent researches. Recent researches confirmed that vitamin D is associated with a variety of pathophysiological process, such as inflammation, regulating immune function,cell proliferation, tumor occurrence and development, participation in fibrosis, etc.Currently the relationship between vitamin D deficiency and chronic liver disease is mainly concentrated in the clinical epidemiology. Some study focused on function of vitamin D for cell proliferation, apoptosis, cytokine secretion, etc. The study based on animal model of vitamin D deficiency on liver inflammation and fibrosis is less.Could the vitamin D deficiency induce the liver inflammation and fibrosis?And how? these complex problems have been not resolved. Therefore, the purpose of this study is to construct an animal model of liver inflammation and fibrosis induced by vitamin D deficiency, and to research the some possible mechanism.We hoping to provide the experimental basis for the clinical effects of vitamin D deficiency on the liver.Methods4 weeks old male Balb/c mice were randomly divided into 3 groups according to treatment ways:Normal group, Vitamin D deficiency(VDD),and carbon tetrachloride group(CCL4). Group Normal fed a standard feed,normal light environment and normal circadian.Group VDD was fed the standard purification of vitamin D deficient diet, cage with shadeing and ventilation; Group CCL4 was fed the standard diet, normal light environment. The feeding ways of Group Normal and VDD group were invariant to the end of the experiment. Group CCL4 keep above way in 1-4 weeks, give CCL4 intraperitoneal injection in 5-9 week, lml/100g body weight/time,2 times/week, keep for 5 weeks, the CCL4 concentration were 2%,5%,10%,20%,30% week after week. During the experiment, mice were monitored daily on activity, reaction, hair color changes and recorded.At 9 weeks, all mice were sacrificed after anesthesia, taken blood about 300-600 μL and separated serum, frozen at-80℃. A portion of the liver tissue fixed in 10% neutral Formaldehyde Solution, paraffin embedding, slicing, staining with H & E and Sirius red.The remaining liver tissue freezed rapidly in liquid nitrogen and preservation in-80℃ refrigerator.To construct a mice model with liver inflammation and fibrosis induced by vitamin D deficiency. In this experiment,target index at 9 weeks including serum 25 (OH) D levels, ALT,AST,H & E,Sirius red staining, expression of some gene:CD14, TLR-4, IL-1β, IL-6, TNF-α, IL-10, IL-13, HO-1, MCP-1, α-SMA, Coll, ColⅢ, TGF-β, MMP13, TIMP-1, MMP9. The enzyme activit of MMP-2 and MMP-9 were assessed by gelatin zymography.To research the effects of vitamin D deficiency on the apoptosis of hepatocyte in mice. Damage even death of hepatocyte are the basis of liver inflammation and fibrosis. Apoptosis play an important role in maintaining the banlance of cell according to self-destruct mechanism. Recent literature suggests effects of vitamin D on normal liver cells and hepatoma cell apoptosis are different, vitamin D can promote apoptosis of hepatocellular carcinoma cells and inhibit the apoptosis of normal liver cells.Therefore it is believed that vitamin D has potential therapeutic effect on hepatocellular carcinoma.We hypothesized that vitamin D deficiency may contribute to apoptosis of hepatocyte in mice, can cause liver damage, then induced liver inflammation and fibrosis. The purpose of the study is to investigate the some possible mechanisms that hepatocyte apoptosis induced by vitamin D deficiency in mice.Outcome measures included:TUNEL staining of liver tissue, Fas, Fas-L, and Caspase3 Immunohistochemistry staining of liver tissue, The mRNA expression of Bcl-2, Bcl-XL, Bax, TGF-a, TGF-β1 1 and Caspase3 with Real time-PCR.ResultsAfter 9 weeks of experiment,we found group VDD mice become obscure fur and less active,serum 25 (OH) D levels significantly were lower than that of group Normal. ALT and AST levels significantly higher than group Normal (P<0.01).H & E staining shows obvious inflammation and necrosis in group CCL4, group VDD displayed obvious fatty degeneration and necrosis. The Knodell score of inflammation in group CCL4 and VDD were significantly higher than that of group Normal (P<0.05).The sirus red staining shows fiber area in group VDD and group CCL4 t were bigger than that of group Normal.The enzyme activity of MMP9 of group VDD was lower than that of group Normal and MMP2 was higher than that of group Normal; Real time-PCR test results show the mRNA expression of CD 14, TLR-4, IL-1 β, IL-6, TNF-α increased in group VDD. The mRNA expression of IL-10, IL-13, HO-1 decreased. The mRNA expression of α-SMA, Col I, Col Ⅲ, TGF-β, MMP13, TIMP-1 increased. The mRNA expression of MMP9 decreased.The results of TUNEL staining:group Normal were weaker than group VDD and group CCL4.Positive precipitates were mainly located inside the cell membrane. The apoptotic rate of group Normal, CCL4, VDD separately were:28.32±12.26%、47.09±6.31%、42.40±17.69%, of which there was a significant difference between group Normal and CCL4 (Z=-4.099, p<0.001), No significant difference between other group(P>0.05).Immunohistochemistry staining and semi quantitative analysis of Fas, Fas-L,Caspase3:The expression of Fas and Fas-L mainly located in hepatocytes, some in bile duct epithelial cells and lymphocytes.Caspase3 is mainly expressed in hepatocellular cytoplasm. Fas staining in group CCL4 and VDD were stronger than that in group Normal. The positive indices of group Normal, CCL4, VDD were as follows:35.00±1.56%、144.43±55.00%、61.27±9.69%.It were found significant differences between group Normal and CCL4 (Z=-2.898,P=0.004<0.01). The positive indices of Fas-L in group Normal,CCL4,VDD separately were:81.67±25、04%、118.91±56.09%、 88.49±24.22%. The positive indices of Caspase3 in group Normal,CCL4,VDD separately were:27.70±3.72%,172.85±33.90%, 105.32±52.95%. The mRNA expression of Bcl-2, Bcl-XL, TGF-α were decreased in group VDD than that of group Normal, and mRNA expression of Bax, Caspase3, TGF-β1 increased.ConclusionsSuccessfully build a early liver fibrosis and chronic inflammation mice models induced by vitamin D deficiency.Vitamin d deficiency can promote the gene expression of inflammatory molecules in Balb/c mice, inhibit expression of some anti-inflammatory cytokine. Vitamin D deficiency promotes activation of hepatic stellate cell and increase mRNA expression of type Ⅰ and type Ⅲ collagen.MMP9 activity decreased.The results induced early liver fibrosis with inflammation.Vitamin d deficiency can contribute to apoptosis of hepatocytes of Balb/c mice, and cause liver damage, promote early liver inflammation and fibrosis.Mechanism of hepatocytes apoptosis induced by vitamin D deficiency implicated in the increase mRNA expression of Fas, Fas-L,Bax, Caspase3, TGF-β1 and decrease mRNA expression of Bcl-2,BCL-XL, and TGF-α.