Establishment Of The Eukaryoticcell-model Containing The Human Aldose Reductase Gene And Its Application In Screening AR Inhibitors

Diabetes can develop to Diabetic chronic complications (DCC). Effective, targeted and safe diabetes medicines remain the urgent need for preventing the development of the DCC. Although much progress has been made in understanding the pathogenesis of DCC, its fundamental pathophysiological causes remain to be proved. AR is the first and rate-limiting enzyme of a two-step biochemical pathway called the "Polyol Pathway" (PP) , which plays an important role in DCC. Animal experiments and studies in vitro showed ARIs could improve the abnormal PP and prevent or delay DCC, such as neuropathy, retinopathy and nephropathy. However, most clinical trials have been more or less disappointing, in part because of side effects and low specificity. Therefore, it becomes a hot spot and new frontier to find and screen ARIs especially in Chinese herbal medicine with high efficiency and low adverse reaction.The AR gene and the chimeric gene AR::GFP were recombinated and transfected into the yeast INVScl strain and HEK293 cells by DNA recombination methods, respectively. The aims of this study were to establish a sensitive and efficient aldose reductase gene-based model, to investigate the possible contribution of AR in PP and to screen the potential effective AR inhibitors from Chinese herbal medicines. This study included three parts:(1) Construction of a yeast model of AR expression: The recombinant plasmids (pYEX-BX-AR, pYEX-BX-AR::GFP) were transformed into the yeast INVSC1 cells, named XAR, XAG strains, respectively. The interested proteins expressed by the induction of CUSO₄ (timing Ohr). Northern blot, Western blot and UV-spectrophotometry were performed to detect the expressions of AR, AR::GFP and their enzyme activities. The result showed AR expression in XAR strain abundantly under the high glucose concentration. The expression level of the AR mRNA reached the maximum at 3hr, and the protein at 9hr after the induction of CuSO₄. The AR activity was the highest at 9hr (about 140U/g), as well. Although AR::GFP mRNA and protein signals were detectable in XAG stain with high level, there was no significant AR activity in this fusion protein.(2) Construction of a HEK293 cell-model of AR expression: the fragments of AR and AR::GFP were inserted, respectively into the eukaryotic expression vector, pcDNA3.1/myc-HisA. The recombinant plasmids pHAR and pHAG were transfected into HEK293 cells by liposome-mediated gene transfer method. The expression levels of AR and AR::GFP were assayed by UV-spectrophotometry and Western blot. The results were similar to those of the yeast model, including the abundant mRNA of AR and AR::GFP in the HEK293 cells, the significant AR activity in AR protein product, and none AR activity in the fusion protein of AR::GFP. Finally, the purified AR protein was obtained through 6×His label by Ni-NTA Magnetic Agarose Beads.(3) Chinese herbal medicine ARI screening: The two models were verified by Sorbinil and Zopolrestat as the ARI controls. The outcome revealed the HEK293 cell-model was more effective in screening ARI than the yeast model. Several Chinese herbals (baicalin, potentilla anserine polysaccharide JM, and polydatin) were tested by the established ARI screening model (HAR). Enzyme kinetics experiments revealed the similar inhibition effects of the Chinese herbal medicines on AR activity to that of Sorbinil.In conclusion, we constructed two models based on AR gene in yeast and HEK293 cells successfully. Although they have the similar characteristics on the whole, the HEK293 model is more suitable in screening ARI than the yeast model. Meanwhile several selected Chinese herbal medicines showed the potential AR inhibition. Hopefully the preliminary established ARI screening model is efficient, economic and AR target-specific. It is expected to play a possible role in ARI screening.