Effects Of Chaihuangyishen Granule On L-FABP And Its Downstream Inflammatory Factors In Diabetic Nephropathy Liver Injury Rats

Objective:Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes melli-tus, and it contributes20to40%percents of end stage renal diseases, which gives our nation a serious bur-den and a major public health problem of the whole society which should be solved. The pathogenesy of DN is very complicated, and traditional chinese medicine have evident special feature and superiority in treating DN. Recent researches indicate that inflammation molecules and inflammation pathways have emerged as being the key pathophysiological mechanism of DN and are related to the whole body mi-cro-inflammation conditions in DN. However, how it plays a role in liver injury in DN still should be ana-lyzed. This research observes the effects of Chaihuangyishen granule on L-FABP and its downstream in-flammatory factors in DN liver injury rats, and then tries to explore the protective effects of Chaihuangyi-shen granule on liver injury rats in DN. On the basis of all the results, we use cell transfection experiments to verify the action mechanism..Method:1. Effects of Chaihuangyishen granule on L-FABP and its downstream inflammatory factors in DN liver injury rats:The I type DN rat model was induced by right-kidney nephrectomy and peritoneal injection of streptozotocin (40mg/kg). Then rats were randomly divided into four groups:normal control, DN model, CHYS and monopril. General states,weigh and blood sugar were observed in the0th,4th,8th,12th,16th,20th week. Rats were sacrificed at the end of the20th week. ALT.AST, CHO,TG were detected by blood biochemistry. Pathological changes in liver tissues were observed by semiquantitative assessment. The expression of L-FABP, PPAR-a,PPAR-Y and MCP-1mRNA were detected by RT-PCR,and the expression of protein L-FABP were detected by Western blot.2. The construciton of recombinant rat L-FABP DNA and its influence on related inflammatory fac-tors:The rat L-FABP cDNA was amplified from rat kidney total RNA by RT-PCR. Amplified DNA fragment was then cloned into vector pUM-T, and digested with restriction enzymes, se-quenced, and then sub-cloned into the eukaryotic expression vector pcDNA3.1/V5-His. After re-striction enzymes digestion and sequence, the recombinant plasmid DNA was transiently trans-fected into293T cell with calcium phosphate. After72h in transfected HEK293cell culture, the total RNA and protein was extracted. The expression levels of the MCP-1, PPAR-a,PPAR-Yand TGF-β1mRNA were tested by RT-PCR. The expression of rat L-FABP in293T cells was detected by Western Blot.3. Experimental data were statistically treated with SPSS13.0.Result:1. Effects of Chaihuangyishen granule on L-FABP and its downstream inflammatory factors in DN liver injury rats:General state and weight of model group was significantly worse than that of con-trol group, and the difference was more significant followed by the progress of the experiment, and the differece was peaking in20th week. The general state of rats of every medication adminis-tration group was better than that of model group. Chaihuangyishen granule group was increased from4th week and continued until the end of the experiment. At the end of the20th week,the CHO，TG of DN group were higher than normal group,the expression of L-FABP,PPAR-a,PPAR-Y mRNA of DN group were lower than that of normal group, however, MCP-1mRNA was higher than normal group. In DN+CHYS group, the level of CHO and TG were decreased than in DN group. Expression of mRNA of L-FABPN PPAR-α、PPAR-yof liver tissue of DN+C group were higher significantly than that of control group,and the expression of MCP-1mRNA was inhabited. The effect between medicine given groups was not significantly.2. The construciton of recombinant rat L-FABP DNA and its influence on related inflammatory fac-tors:1) Rat L-FABP cDNA with the eukaryotic expression vector was successfully constructed.2) This recombinant plasmid DNA can express in HEK293T cell both in gene and protein level.3) RT-PCR showed that in transfected L-FABP group, mRNA of PPAR-a, PPAR-ywere over ex-pressed. The expression of MCP-1, p65and TGF-β1were decreased.4) Using Fugene HD trans-fected HepG2cell showed that no significant changes between transfected vector A group and transfected L-FABP group.Conclusion:1. CHYS can lessen the inflammatory impairment of liver in DN.the mechanism may be related to increase the expression of L-FABP, PPAR-a,PPAR-Y,and then decrease the expression of down-stream inflammation factors such as MCP-1. L-FABP may play an important role in liver injury in DN.2. We successfully constructed recombinant rat L-FABP DNA, then made it overexpressed in293T cell, and test the changes of downstream factors it may influenced, which provide us a powerful evidence in discussing its function. However, overexpression L-FABP in HcpG2cell did not find significantly changes in downstream.HepG2cell itself expressed the gene may be the reason.