| Objective Established NAFLD model of rats. To test the changes of inflammatory factors IL-1β, IL-18, NLRP3, Caspase-1 of serum and liver tissues on different time points. To analyse the changes of E. coli and Lactobacillus in the intestinal fecal in order to investigate the influence of dietary factors of NAFLD and study the pathogenesis of NAFLD.Method Choose six-week male SD rats healthy and clean weighing 140-180 g. After one week adaptive feeding, the rats were divided into two groups randomly: control group and model group, the control group was fed with normal diet, the model group was fed with high-fat diet. To observe the model were successful or not according to serum biochemical indexes and liver pathology results after 8 weeks. Then the model group was divided into four groups randomly, namely: high-fat group, high-protein group,high-carbohydrate group and normal group. These rats were randomly selected to be killed after feeding for 12 and 16 weeks. To test the expression level of serum IL-1β,IL-18 by ELISA. The level of IL-1β, IL-18, NLRP3, Caspase-1 m RNA of liver was assayed by q RT-PCR. The method of Western blot was used to detect the protein level of NLRP3 and Caspase-1 of liver. To extract the bacterial DNA to test the change of the expression level of E. coli and Lactobacillus by q RT-PCR.Results1. The result of Model The body weight, liver weight and liver index of rats in 4 or 8 weeks in modelgroup were significantly higher than concurrent control group. The level of ALT,AST,TC,TG in 4 or 8 weeks were significantly higher and HDL-C was significantly lower in model group than that in concurrent control group. HE staining and electron microscopy results in 8 weeks showed lipid deposition in liver tissue of rats adds in model group which suggests long-term high-fat diet induce hepatic steatosis. The result of the experiment suggested that the NAFLD rats model was established by feeding high-fat diet for 8 weeks successfully.2. The result was intervened by different dietary1) Result of serum biochemical parameters Compared with concurrent control group, the level of serum ALT, AST in every diet intervention group was elevated, that HDL-C was reduced. The level of serum TC, TG were higher in high-fat group, high-carbohydrate group and normal group than that in concurrent control group, while the level was lower in high-protein group, the difference was statistically significant(P<0.05). Compared with concurrent normal group, the level of serum ALT, AST in high-fat group, high-protein group and high-carbohydrate group was higher, and the level of HDL-C was lower; the level of serum TC, TG were higher in high-fat group and high-carbohydrate group than that in concurrent normal group,while the level was lower in high-protein group, the difference was statistically significant(P<0.05).2)The concentrstion level of serum IL-1β, IL-18 Compared with concurrent control group and normal group, the expression level of serum IL-1β, IL-18 in high-fat group, high-protein group and high-carbohydrate group were increased, the difference was statistically significant(P<0.05).3)The transcriptional level of liver IL-1β, IL-18, NLRP3, Caspase-1 m RNA Compared with concurrent control group and normal group, the transcriptional level of IL-1β, IL-18, NLRP3, Caspase-1 m RNA in high-fat group, high-protein group and high-carbohydrate group were increased. Compared with concurrent control group,the transcriptional level of IL-1β, IL-18, NLRP3 m RNA were increased, while the levelof Caspase-1 m RNA was decreased. the difference was statistically significant(P<0.05).4) The protein level of NLRP3 and Caspase-1 in liver The result showed the expression of protein of NLRP3 and Caspase-1 in control group and normal group were lower relatively, while the expression of protein of NLRP3 in normal group was higher than that in control group. The difference was statistically significant(P<0.05). The expression of protein of Caspase-1 in normal group was lower than control group, and the difference was statistically significant(P>0.05). Compared with control group and normal group, the expression of protein of NLRP3 and Caspase-1were increased in high-fat group, high-protein group and high-carbohydrate group. It tended to be upward obviously, the difference was statistically significant(P<0.05).5) Result of liver histopathology HE staining under light microscope showed the structure of hepatic lobular was undivided in the control group, there was large and round nucleus in central of cell, the cells of group grew with the steady cytoplasm. Hepatic lobular was still visible in common group, part of cells were swelling, and few lipid droplet vacuoles was visible,slightly steatosis. The structure of hepatic lobular was disordered in high-fat group,high-protein group and high-carbohydrate group, liver cells swelled extensively and presented to be ballooning degeneration, lipid droplets vacuoles in varied sizes was visible in cytoplasm. Liver tissues were moderate-severe steatosis in 12 weeks, liver tissues were severe steatosis in 16 weeks, liver cells were damaged and inflammatory cells infiltrating scatterly.6) Quantitative result of bacteria in fecal sample Compared with concurrent control group and normal group, E. coli were increased significantly in high-fat group, high-protein group and high-carbohydrate group,Lactobacilli were decreased obviously. the difference was statistically significant(P<0.05).Conclusions1. The NAFLD mode of lrats was established successfully by feeding high-fat dietafter 8 weeks. Suggesting that long-term high-fat diet may induced hepatic steatosis.2. The level of serum inflammatory cytokines IL-18, IL-1β were both increased significantly in high-fat group, high-protein group and high-carbohydrate group. The transcriptional level of IL-1β, IL-18, NLRP3, Caspase-1 m RNA were increased in high-fat group, high-protein group and high-carbohydrate group. The expression of protein of NLRP3, Caspase-1 were increased in high-fat group, high-protein group and high-carbohydrate group, and tended to be upward. It presented that IL-1β, IL-18,NLRP3, Caspase-1 participated in chronic inflammation in the course of the development of NAFLD.3. E. coli was increased significantly in high-fat group, high-protein group and high-carbohydrate group, while Lactobacilli was decreased obviously. It Suggested that intestinal dysbacteriosis may existed during the development of NAFLD. Meanwhile,intestinal dysbacteriosis may be one of the reasons for the occurrence and development of NAFLD inflammatory.4. Irrational structure of dietary can aggravate the development of NAFLD.High-fat, high-protein and high-carbohydrate diet are the risk factors of NAFLD. |
