Effect Of Three Alkaloids On Intimal Hyperplasia Of Femoral Artery In Rats

Object:To establish the dedifferentiated phenotype model of vascular smooth muscle cells (VSMCs) induced by platelet derived growth factor (PDGF-BB), on which to evaluate the intervention of VSMCS dedifferentiation of stachydrine, berberine and matrine. Employing the rat femoral artery intimal hyperplasia model by striking off endothelium evaluate the effect of stachydrine, berberine and matrine on vascular intimal hyperplasia. Through conducting the correlation between cell assessment and animal assessment evaluate the reliability of cell assessment base on characteristic of VSMCs phenotype transformation. Thereby setting up a potential compound screening model on VSMCs. Otherwise providing new ideas and experimental sustain for drug development in vascular intimal hyperplasia diseases.Methods:1. Primary cultured rat aortic VSMCs; the experimental conditions was determined through the dose-activity assay and time-activity assay on PDGF-BB induced VSMCs; the cytotoxicity assays of stachydrine, berberine and matrine; then detecting the proliferation inhibition of stachydrine, berberine and matrine on VSMCs induced by PDGF-BB.2. The effects of stachytrine, berberine and matrine on VSMCs phenotype transforming proteins undergoing PDGF-BB stimulation:the PDGF-BB induced VSMCs were treated with stachydrine, berberine and matrine, then extracted the whole cells protein for western blot to detect the SM a-actin and SM MHC expression.3. The rat femoral artery endothelium strikes off by metal wire, which results in intimal hyperplasia. Male wistar rats were randomly divided into sham Ⅰ group, sham Ⅱ group, model Ⅰ group, model Ⅱ group, rapamycin group, stachydrine high dose group, stachydrine low dose group, berberine high dose group, berberine low dose group, matrine high dose group, matrine low dose group; The second day after operation rats were injected drugs by tail vein continually 21 days. After the last drug injection the femoral artery was taken for histopathological detection with hematoxylin-eosin staining and masson staining, so as to observe the vascular tissue morphological changes and collagen fiber hyperplasia.4. The rats suffered above femoral artery intimal njury and treated with stachydrine, berberine, matrine. Blood were collected on day 3, day 6, day 10 day 15 and day 21, serum was separated for C-reactive protein (CRP), day 21 serum also for interleukin-6 (IL-6) detection.Results:1. The results of dose-activity assay and time-activity assay on PDGF-BB induced VSMCs show that the maximum proliferation effect appears after 24 hours stimulating with 20 ng/mL PDGF-BB; there is no cytotoxicity on VSMCs for stachydrine within 1-1000 μM, berberine within 1～200 μM and matrine within 1～1000 μM; there is different extent inhibition on PDGF-BB induced VSMCs proliferation with stachydrine within 1～500 μM, berberine within 1～200 μM and matrine within 5～500 μM.2. Western blot results show that, the expression of contractile phenotype characterized proteins of VSMCs, SM a-actin and SM MHC, have decreased undergoing PDGF-BBstimulation, while after treated with stachydrine and berberine, the expression of SM a-actin and SM MHC has certain increae, but not. matrine.3. The hispathological detection of femoral artery injuried show that, the vascular intimal thicken, collagen fiber and extracellular matrix increase, which have been improved by treating with stachydrine and berberine, but not matrine.4. The concentration of serum CRP and IL-6 are significantly increased in rats suffered femoral artery injuried; CRP levels has been decreased by treating with stachydrine and berberine, not matrine; while IL-6 is not affected by three drugs.Conclusion:1. The cell experiments show that, stachydrine, berberine and matrine can inhibit the PDGF-BB induced VSMCs proliferation, the strength order: stachydrine>berberine>matrine; but berberine exert the most strong action of maintaining VSMCs contractile phenotype characterized proteins.2. The animal experiments show that, stachydrine and berberine can inhibit rat femoral artery intimal hyperplasia, inhibiting intimal thickening, collagen fibers and extracellular matrix increse could be involving in regulating inflammatory reactions. Matrine has no effect on intimal hyperplasia in rats.3. We can speculate that vitro inhibition assessment of VSMCs proliferation is not a proper option. Combining with contractile phenotype dedifferentitation could be more reliable.