The Role Of TIM-1 Signaling Pathway In The Pathogenesis Of IgA Nephropathy Mouse

Objective:Through the establishment of mouse IgA nephropathy model study TIM-1 signaling pathway in mouse IgAN, and its effect on IgAN renal pathological damage in mice, as well as IgAN mice Th1, Th2, TLR4 signaling pathway. Explore the role of TIM-1 signaling pathway in the pathogenesis of IgAN.Method:Select SPF level BALB / c male mice 36 to establish an experimental mouse model of IgA nephropathy. Divided into normal control group, the control group + anti-TIM1 group, IgAN model group, IgAN + anti-TIM1 group. The control group + anti-TIM1 group and IgAN + anti-TIM1 group were given anti-TIM1 weekly intraperitoneal injection of 100 ug for 7 weeks. During the experiment, observe the following indicators: 1.the general situation of each group of mice; 2.light microscopy and immunofluorescence pathology identified modeling success; 3. using Westernblot measured the mice kidneys TIM-1, TLR4, MyD88, NF-?B, IFN-?, IL-4 expression; 4.semi-quantitative RT-PCR and immunohistochemistry to detect the mouse kidney tissue TIM1, TLR4, MyD88, NF-?B, IFN-?? IL-4expression.5. Pearson correlation analysis TIM-1 expression of IL-4, IFN-?, IFN-? / IL-4 ratio and renal pathology index in IgAN model group and IgAN + anti-TIM1 group.Result:1. In this study the control group of mouse body weight increment is slightly larger than the remaining treated mouse, especially in the six weeks, mouse treated hair gloss deteriorates, and varying degrees of mental malaise, eating less, but four groups of mouse were not died during the experiment.2. The four groups of renal pathological changes in mouse and renal damage Rating: renal tissue under light microscope IgAN model group showed mesangial cells, severe hyperplasia, renal interstitial fibrosis, and is usually accompanied by inflammatory cell infiltration, and IgAN + anti-TIM1 renal tissue diffuse light microscope glomerular mesangial cell proliferation.Katafuchi rated Ig AN model group(6.56 ± 1.13), IgAN + anti-TIM1 group(3.22 ± 0.97) was significantly higher than the control group(0.33 ± 0.50), with statistical significance(P <0.01); IgAN model group Katafuchi score significantly higher than IgAN + anti-TIM1 group, with statistical significance(P <0.01).In IgAN model group kidney tissue IgA immunofluorescence at high magnification was seen +++ to ++++ granular or lumpy green phosphor deposited along the mesangial area,but IgAN + anti-TIM1 group was seen precipitate + to ++. Using Kruskal-Wallis H-test analysis obtained Ig AN model group, IgAN + anti-TIM1 group classification with respect to the control group was statistically significant(P <0.01); IgAN + anti-TIM1 group classification with respect to IgAN model group was statistically significant(P <0.01)..3. Immunohistochemistry results: IgAN model group and IgAN + anti-TIM-1 group TIM1, TLR4, MyD88, NF-?bp65 expression of glomerular and tubular increased significantly compared with normal control group, the difference was statistically significance(P <0.01); IgAN + anti-TIM-1 group the expression of TIM1, TLR4, MyD88, NF-?bp65 than IgAN model group was significantly lower, the difference was statistically significant(P <0.01).4. Western Blot test results: IgAN model group and IgAN + anti-TIM-1 group TIM1, TLR4, MyD88, NF-?bp65, IL-4, IFN-? expression levels higher than normal control group, and the difference statistically significant(P <0.05). Between the normal control + anti-TIM-1 group and the normal control group TIM1, TLR4, MyD88, NF-?bp65, IL-4, IFN-?differences expression level in protein was not statistically significant(P> 0.05). IgAN + anti-TIM-1 group TIM1, TLR4, MyD88, NF-?bp65, IL-4, IFN-? protein expression is lower than IgAN model group, and there was statistically significant(P <0.05).5. The results of real-time quantitative detection method: IgAN model group and IgAN + anti-TIM-1 group TIM1, TLR4, MyD88, NF-?bp65,IL-4 mRNA expression levels were higher than normal control group, the difference was statistically significant(P <0.01). Between the normal control + anti-TIM-1 group and the normal control group TIM1, TLR4, MyD88, NF-?bp65, IL-4,IFN-?mRNA differential expression level was not statistically significant(P> 0.05). IgAN + anti-TIM-1 group TIM1, TLR4, MyD88, NF-?bp65, IL-4 mRNA expression lower than IgAN model group, and there was statistically significant(P <0.01). IgAN + anti-TIM-1 expression in IFN-?mRNA group than in IgAN model group, but the difference was not statistically significant(P> 0.05).6. Correlation analysis: IgAN group and IgAN + anti-TIM1 group TIM-1mRNA and IL-4, IFN-? and IFN-? / IL-4 correlation, TIM-1 and IL-4, IFN- ? are positive correlation, but not statistically significant(P> 0.05); TIM-1 and IFN-? / IL-4 was a negative correlation, correlation was not statistically significant(P> 0.05). IgAN group and IgAN + correlation analysis of anti-TIM1 group TIM-1mRNA Katafuchi with renal pathology scores and kidney IgA immunofluorescence classification, TIM-1 and Katafuchi rated positively correlated correlation was statistically significant(P <0.05); TIM-1 and IgA immunofluorescence grade was positively correlated, and the correlation was not statistically significant(P> 0.05)Conclusion:1.IgA nephropathy mouse model was successfully constructed in this study.2. After discovery TIM-1 upregulation, and positively correlated with the degree of pathological damage was, but given anti-TIM-1 blocking TIM-1 signaling pathway in the kidney tissues IgAN model group, TIM-1 downregulation, and histopathology still positive, suggesting that mice IgAN in TIM-1 signaling pathway may be involved in the pathogenesis and progression of the disease.3. TIM-1 and IL-4, the expression of IFN-? expression in renal tissue of IgAN model group have raised, and TIM-1 and IL-4, IFN-? were positively correlated with IFN-? / IL- 4 negative correlation in the model group administered TIM-1 antibody treatment, TIM 1-IL-4 expression are reduced, and the TIM-1 remains positive correlation between IL-4, and IFN-? / IL-4 was negative correlation, suggesting that in IgAN pathogenesis of Th2 dominant response is regulated TIM-1 signaling pathway.4. In the mouse model of IgA nephropathy in renal tissues TLR4 and MyD88 are upregulated,the expression of a downstream factor NF-?B is also upregulated, suggesting that TLR4 signaling pathway plays a role in the pathogenesis of IgA nephropathy. After the TIM1 antibody treatment of IgAN group, TLR4, MyD88, NF-?B expression were decreased, suggesting that blocking the TIM1 signaling pathway through downregulation of TLR4, etc., and thus play a role in delaying disease IgA nephropathy.