A Model For ARI Screening And Effect Of Astragaloside IV On Growth Of VSMC And Its Mechanism

Diabetes mellitus (DM) is a serious disease with severe complications. Polyol pathway plays an important role during the development of degenerative diabetic complications. Aldose reductase (AR) is key rate-limiting enzyme in polyol pathway, it is the target enzyme for treatment of DM complications. Aldose reductase reduces excess d-glucose into d-sorbitol with the associated conversion of NADPH to NADP+1. Available experimental data link D-glucose metabolism to long-term diabetic complications such as cataract, neuropathy, nephropathy, and retinopathy. Aldose reductase inhibitors should therefore be able to safely prevent or arrest the development of diabetic complications. Because of the scarce of suitable drugs for treatment of DMcomplications and the now existing drug Sorbinil and Tolrestat have some side effects,it is important for looking for new aldose reductase inhibitor with more safety and better effects. The aim of this study was to develop a convenient and relable model for aldose reductase inhibitor screening. Base on this model, a number of drugs were screened and mechanisms of drugs were also studied.1 ^ In this study, we developed a model for aldose reductase inhibitor screeningfrom the traditional Chinese medicine and other chemical compounds. Changes of aldose reductase activity and ARmRNA expression in smooth muscle cells (VSMCs) cultured in various concentration of glucose for different time were determined. The effect of an AR inhinitor, Epalrestat, was also examined. Furthermore, we examined the effect of some traditional Chinese medicines on the AR activity in VSMCs. Results suggested that AR activity and ARmRNA expression in VSMCs culture in 37.5mM glucose andlO% FBS containing DMEM for 48 hours was remarkably higher than that of VSMCs cultured in 5.5mM glucose and 1% FBS containing DMEM, and aldose reductase inhibitor, Epalrestat, can almost normalize this elevation. These data suggest that VSMCs cultured in 37.5mM glucose and 10% FBS containing DMEM maybe can be used as a cell model for AR inhibitor screening from the traditional Chinese medicine and other chemicals.2, Based on this model , a number of extractive of drugs, drug -containing serumand monomers from drugs were screened. We found that Astragaloside IVand Saponins from Anemarrhena sphodeloides Bunge can inhibit AR activity in smooth muscle cells.This pharmacologic action of Astragaloside IV has not been reported before.3, During further research, we found that Astragaloside IVcan reduce the viabilityof VSMCs and enhance apoptotic rate of VSMCs significantly. Cell viability was measured by the tetrazolium salt WST-1 assay. Cell apoptosis was assayed by flow cytometry through detecting Annexin V. The results demonstrated that 2ug / ml and 20ug / ml Astragaloside IV reduced the viability of VSMCs significantly, 20ug/mlAstragaloside IV and interleukin-l]3 (lOng/ml) enhance apoptotic rate of VSMCssignificantly(p < 0.01). These indicated that Astragaloside IV can inhibit VSMCs proliferation through reducing cell viability and enhancing apoptotic rate of VSMCs.4, Nitric oxide (NO)production and intracellular Ca2+ movement were studied inthe presence or absence of Astragaloside IV using confocal laser scanning microscopy with diaminofluorescein diacetate (DAF-2, DA) and Flue-3/AM respectively. Cell aldose reductase (AR) activity was also explored. Nitric oxide production wassignificantly enhanced after the administration of Astragaloside N or interleukin-lj3.Furthermore, Astragaloside IV increased cytosolic Ca2+ significantly. The results indicated that Astragaloside IV inhibit proliferation of VSMCs associated with serum by inhibiting activity of aldose reducrase and by inducing nitric oxide release. We also found that Astragaloside IV can induce Fas protein expression and supress Bcl-2 protein expression, those indicated that the effect of Astragaloside IV on apoptosis of VSMC was through inducing Fas protein expression and supress Bcl-2 protein expression and inducing cell NO release. Astragaloside IV may exert vascular protection by inhibiting VSMCs proliferation through inhibiting AR activity and by augmenting apoptotic rate of VSMCs via cytosolic Ca2+ related NO-dependent pathway and by inducing Fas protein expression and supress Bcl-2 protein expression.? In this study, we developed a cell model for ARI screening. It supplies a new...