The Protective Effect And Immunological Mechanism Of 1,25（OH）₂D₃ On Non-alcoholic Fatty Liver Hepatitis Induced With A High-fat And High-fructose Diet In Mice

Part I Experimental study on establishing mouse model of nonalcoholicsteatohepatitis by different diet structuresObjective: To establish the mouse model of nonalcoholic steatohepatitis induced by high fructose diet,high fat diet,and compound high fructose diet and explore the best diet structure.Methods:A total of 72 male C57 BL /6J mice were randomly divided into the high fructose t（HFr group）,high fat group（HF group）, high-fructose and high-fat group（HFHFr group）, and standard control group. Differences of basic data, liver function, lipid metabolism, TNF-α, IL-6, HOMA-IR, 25（OH）D, and pathological changes of groups were compared after the model was established for 4, 8, and 12 weeks.Result: Compared with the control group, the body weight, wet liver weight and Lee’s index of other groups were significantly higher（P<0.01）.The increase of body weight of HFHFr group was more significant than that of the HF group and HFr group after 12 weeks（P<0.05）. HE staining indicated that except the control group, mice of other three groups had different degrees of macrosteatosis and significant intralobular inflammatory cell infiltration was only observed in mice of the HFHFr group. Results of serum test showed that after 12 weeks, TCHO and LDL levels of the HFHFr group and high fat group significantly increased and increases of ALT, AST, TNF-α, and IL-6 of the HFHFr group were more significant than those of the HF group and HFr group（P<0.05）.Compared with the control group, HOMA-IR and 25（OH） D levels of other groups were showed an increasing and decreasing tendency respectively.There are significant difference between HFHFr group and HFr group、HF group（P<0.05）.Conclusion: High fat and fructose diet for 12 weeks successfully establish the mouse model for studying the nonalcoholic steatohepatitis.Part II The intervention effect of the different doses of 1,25（OH）₂D₃on nonalcoholic steatohepatitis（NASH） induced by high-fat andhigh-fructose diet（HFHF diet）Objective: The aim of this research was to evaluate the effect of the different doses of 1,25（OH）₂D₃ on nonalcoholic steatohepatitis（NASH） induced by high-fat and high-fructose diet.Methods: 90 male C57 BL /6J mice were randomized into five experimental groups（n = 18 each）: A group（control group, normal diet+0.9%Na Cl 0.2ml by gavage, qod）,B group（model group, HFHF diet + 0.9%Na Cl 0.2ml by gavage, qod）, C group（low dose group, HFHF diet +1,25（OH）₂D₃, 4μg/kg, by gavage, qod）, D group（moderate dose group,HFHF diet + 1,25（OH）₂D₃, 20μg/kg, by gavage, qod）, E group（high dose group,HFHF diet + 1,25（OH）₂D₃, 40μg/kg, by gavage, qod）. 6 mouse of each group were euthanized at 4weeks、8weeks、12 weeks.（1） Body weight and wet liver weight were weighed. Lee’s index also were counted.（2） Hematoxylin and eosin staining was used to assess the degree of hepatic steatosis, lobular inflammation, hepatocyte ballooning, for counting non-alcoholic fatty liver disease（NAFLD） activity score（NAS）.（3） Liver function, blood lipid 、TG、TC in liver tissue 、blood glucose and serum insulin were determined. Homeostasis model assessment of insulin resistance（HOMA-IR） was counted.（4） Enzyme-linked immuno sorbent assay（ELISA） was used to determine the serum levels of TNF-α、IFN-γ、IL-2、IL-4、IL-6 、IL-17.Result:（1） The body、liver weight and Lee’s index increased in a time-dependent manner in all groups. Compared with the B group, the body、liver weight and Lee’s index of intervention groups were decreased at all points, but a statistically significant reduction was induced by D group（P< 0.05）.（2） The B group developed hepatocyte steatosis at the 4th weeks, fatty liver at the 8th weeks, steaohepatitis at the 12 th weeks. Compared with the B group, the degrees of hepatic steatosis changes of in liver tissue showed reduced, and no NASH was observed in intervention groups.（3） Compared with the A group, the levels of serum ALT、AST、TG 、TC of other four groups were all increased from the 4th weeks, and the level of B group were higher than all intervention groups. At the 12 th weeks, the levels of serum ALT、AST、TG 、TC of D group were lower than C、E group（P<0.05）. The concentrations of TG、TC in liver tissue of intervention group were significantly decreased at all points（ P<0.05）.（4） In comparison with the B group, HOMA-IR levels of intervention groups showed an decreasing tendency（P<0.05） after the 8th weeks. At the 12 th weeks,the HOMA-IR levels between the D group and B、E group had significantly differences（P<0.01, P<0.05）.（5） Compared with B group, the levels of serum TNF-α、IL-2、IFN-γ、IL-17、IL-6 of intervention groups were significantly decreased（P<0.05）, but there was no obvious influence on the levels of serum IL-4（P>0.05）.Conclusion: Different doses of 1,25（OH）₂D₃ can statistically decrease the levels of serum of ALT、AST、TG、TC、insulin resistance、inflammatory cytokine levels, which can improve liver function and protect the liver cells. The effect of the D group is the best.Part III The immune mechanism of 1,25（OH）₂D₃ on nonalcoholicsteatohepatitis（NASH） induced by high-fat and high-fructosediet（HFHF diet）Objective: The aim was to evaluate the immune mechanism of 1,25（OH）₂D₃ on nonalcoholic steatohepatitis（NASH） induced with high-fat and high-fructose diet.Methods: 60 male C57BL/6J mice were randomized into five experimental groups（n =12 each） mentioned in Part II. All mouse were euthanized at the 12 th weeks.（1） Th l、Th 2、and Th17 cells in mice peripheral blood were analyzed by flow cytometry.（2） The levels of TNF-α、IFN-γ、IL-2、IL-4、IL-6 、IL-17 in liver tissues were measured by Realtime-PCR.（3） Kupffer cells were isolated from perfused livers and identified by flow cytometry. Lymphocyte proliferation were measured by MTT assay after cultured with Kupffer cells.（4） Kupffer cells were isolated from livers to evaluate expressions of TLR4, P-p38 MAPK, and p38 MAPK by Western blotting.Result:（1） At the end of 12 weeks, the percentages of Thl cells（6.13±0.72、5.44±0.98、6.36±1.29 respectively） in three intervention groups was lower than B group（8.45±1.51, P< 0.05）.Meanwhile, the percentages of Thl7 cells were significantly lower than B group（P< 0.05）.（2） The levels of TNF-α、IFN-γ、IL-2、IL-6 and IL-17 in B group were significantly increased when compared with A group（P<0.01）, and significantly decreased in intervention groups when compared with B group（P< 0.05）. Compared with A group, the levels of TNF-α、IFN-γ and IL-2 in intervention groups were significantly increased（P< 0.05）, but the levels of IL-6 and IL-17 were not significantly changed.（3） Lymphocyte proliferation in intervention groups were significantly decreased when compared with B group（P< 0.05） and not significantly changed at 1:10、1:20 when C、D group compared with A group.（4） Expressions of TLR4, P-p38 MAPK and p38 MAPK in Kupffer cells were decreased in all intervention groups, and the D group had the lowest expression levels of TLR4,P-p38 MAPK and p38 MAPK.Conclusion: 1,25（OH）₂D₃ might plays a protective role against NASH by reducing the levels of Th1 and Th17, inhibiting the associated cytokine expression in liver tissue, suppressing Kupffer cells activity and inactivation of p38 MAPK signaling pathways.