| Objective:The investigations were based on a systematic review of the progress of clinical research in the treatment of diabetic nephropathy (DN) by TCM and the research of pathogenesis of DN in latest years. In this study, we observed the protective Effect and Mechanism of Chaihuangyishen Granula on DN Rats and constructed recombinant plasmid of Rat Uqcrfsl, in order to find some potential clues about early diagnosis biomarkers and therapeutic targets of DN.Methods:1. Study on Protective Effect and Mechanism of Chaihuangyishen Granula on DN Rats:The1type DN rat model was induced by by right-kidney nephrectomy and peritoneal injection of low-dosage streptozotocin (40mg/kg) one week after the nephrectomy. The rats were randomly divided into three groups:model, Chaihuangyishen Granula and Monopril,12rats in each group, another12rats as the normal control. General states, weight, blood glucose,24h Urinary protein were tested in the0th,4th,8th,12th,16th and20th week. The rats were sacrificed at the end of the20th week. The serum lipid, total protein, albumin and renal function were examined by blood biochemistry. Pathological changes of glomeruli and tubular interstitium were observed by semiquantitative assessment. The expression of TGF-β1、NF-κB were detected by RT-PCR. The expression of IKappa B a and p-IKappa B a were detected by Western Blotting.2. Construction of Rat Uqcrfsl for its Expression in HEK293T cell:Rat Uqcrfs1cDNA was amplified from rat kidney total RNA by RT-PCR. The amplified DNA fragment was cloned into vector pUM-T, digested with restriction enzymes, sequenced, then sub-cloned into the eukaryotic expression vector pcDNA3.1/V5-His. After restriction enzymes digestion and sequence, the recombinant plasmid DNA was transiently transfected into HEK293T cell with calcium phosphate. The expression of rat Uqcrfsl in293T cells was detected by RT-PCR and Western Blotting.Results:1. Study on Protective Effect and Mechanism of Chaihuangyishen Granula on DN Rats: From the0th week after DN rats established, the blood glucose,24h urine protein, BUN and Scr of model group was significatively worse than those of control group; index of glomerular sclerosis and index of interstitial fibrosis in the model group were significantly higher than those in control group. At the2th week, renal pathology impairments in the model group was very serious, and Chaihuangyishen Granula group showed mild pathology impairments. Interestingly, the level of glucose in Chaihuangyishen Granula group and Monopril group showed no significant differency with the model group. Compared with the model group, the glucose in Chaihuangyishen Granula group had a degrading tendency, but without statistical differency. After the treatment, TC and TG in Chaihuangyishen Granula group and Monopril group were obviously depressed, compared with those in the model group. The expression of TGF-β1and NF-κB mRNA in renal tissues of the model group was significantly higher than those in control group, and the treated groups.. The results of Western Blotting showed that expression of KappaB and p-IKappaB protein in renal tissue of model group was significantly higher than those in control group and the treated groups..2. Construction of Rat Uqcrfsl for its Expression in HEK293T cell:Related primers was designed with the additional cutting site of EcoR V or Xho I and the PCR system was used to amplify the target Uqcrfs1sequence by the rat renal cDNA successfully. The amplified DNA fragment was cloned into vector pUM-T, digested with restriction enzymes, sequenced, then sub-cloned into the eukaryotic expression vector pcDNA3.1/V5-His. The Uqcrfsl target sequence and pcDNA3.1/V5-His vector was cut by both of EcoR V and Xho I restriction endonuclease respectively.And the digested product was linked by the T4DNA ligase. The recombinant plasmid Uqcrfs1/pcDNA3.1/V5-His was transfected into competent E.Coli and positive clones of E.Coli were screened. The recombined DNA was purified and sequenced, which indicated100%of base accuracy. After restriction enzymes digestion and sequence, the recombinant plasmid DNA was transiently transfected into HEK293T cell with calcium phosphate. The expression of rat Uqcrfsl in293T cells was detected by RT-PCR and Western Blot.Conclusion:1. Chaihuangyishen Granula could reduce24h urinary protein, lessen the impairment of kidney in DN rats., of which the mechanism may be attributed to improve the unbalance of lipid metabolism, but no relationship with blood glucose. Chaihuangyishen Granula could depress the expression of TGF-β1, affect the balance between NF-κB and Ikappa B a, which may be the potential mechanism of the protective effects.2. The eukaryotic expression vector containing Uqcrfsl was constructed and it can express Uqcrfsl in HEK293T cell. The work provided a new source for the further exploration of pathophysiological mechanism of DN and TCM treatment. |
PDF Full Text Request