Wind Active Ingredient In Anti-atherosclerotic Inflammatory Response In Experimental Research

Atherosclerosis (AS) is characterized by complicate mechanisms. Since 19 century, theories concerning pathological mechanisms were raised. The most important one is inflammatory hypothesis by Ross that AS is a synthetic process caused by common actions of cytokine, vascular endothelial cell, vascular smooth muscle cell and immunity factors.Based on the theory of TCM, Fangfeng is a wind-dispersing medicine, which has the effect of activating blood circulation to dissipate blood stasis. Modern Pharmacology research discovered that Fangfeng has significant activityof anti-inflammatory, and the primary anti-inflammatory component is prim-O-glucosyl-cimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con. In this paper we investigate the effect of prim-O-glucosylcimifugin and 4'-O-β-D-gluco syl-5-O-methylvisamminol con on the inflammatory reaction in AS in order to provide the scientific evidence for wide clinical application.According to the inflammatory hypothesis of Ross, the factor like oxidizedlow density lipoprotein (ox-LDL) can cause injury or dysfunction of vascular endothelium, stimulate the release of inflammatory factor like IL-6, TNF-αin endothelial cell and macrophage,which can stimulate the proliferation of smoothmuscle cell subsequently, and result in the formation and development of AS plaque finally. We employed in vitro vascular endothelial cell injured model,adhesion model and smooth muscle cell proliferation model to investigate the anti-inflammatory effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-met-hylvisamminol con. Our research work includes three parts as follows: 1 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the survival rate and secretion of cytokines of cardiac microvas-cular endothelial cell.Referred to Nishida's method, the primary culture method of rat cardiac microvascular endothelial cell (rCMEC) in vitro was established. The rCMEC was identified by immumofluorescence method, and its growth curve was drawed by cytometry. The third generation of cells was adopted in our experiment. By determining the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the activity of EC, the concentration of two medicines was decided. By determining the effect of ox-LDL on the survival rate of EC, it's concentration to make the EC injured was decided. And then the effect ofprim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamm-inol con onthe survival rate and secretion of cytokines of EC was investigated by MTT,ELISA and radioimmunoassay respectively.The results were as follows:1．The cultured cell was identified to be rCMEC by immumofluorescence method and morphology observation, because of its positive dyeing of factorⅧrelated antigen and its appearance like paving stones.2．Based on the growth curve, the best time of experiment was limited in the period between 24h to 72h after inoculating cell into culture plates in the density of 1×10⁵mL^-1 .3．The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methy-lvisamminol con on the activity of EC was determined by MTT. It was foundthat the maximum of no toxicity concentration can not be determined because of the low toxicity of these two medicines on EC. Thus, 50μg·ml^-1 ,100μg·ml^1-1 , 200μgml^-1 were chosen as the concentration of prim-O-glucosylcimifugin and 4 '-O-β-D-glucosyl-5-O-methylvisamminol con in experiment.4．The effect of ox-LDL on the survival rate of EC was determined by MTT. The ox-LDL of 3ng·ml^-1 DA content was chosen to injure EC in experiment.5．After pretreatment with prim-O-glucosylcimifugin(50,100,200μg·ml^-1 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50,100,200μgml^-1 24 hours, the rCMEC was incubated with ox-LDL for another 24 hours, then the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the survival rate of EC injured by ox-LDL was determined by MTT. The res-ult indicated that the survival rate of EC was reduced by ox-LDL of 3ng·ml^-1 as MDA content significantly, the model group had great significant difference (P<0．01) compared with control. 200μg·ml^-1 group of prim-O-glucosyl-cimifugin, 50μg·ml^-1 , 100μg·ml^-1 , 200μg·ml^-1 groups of 4'-O-β-D-glucosyl-5-O-methylvisam-minol con can inhibit EC injury made by ox-LDL, had great significant differ-ence(P<0．01) compared with model group. 50μg·ml,100μg·ml^-1 groups of prim-O-glucosylcimifugin had the tendency to lighten ox-LDL injury, but had no significant difference compared with model group.6．After pretreatment with prim-O-glucosylcimifugin(50, 100, 200μg·ml^-1 ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50, 100, 200μg·ml) for 24 hours, the rCMEC was incubated with ox-LDL for another 24 hours, then the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the secretion of cytokines of EC was determined. The result indicated that ox-LDL can increase the secretion of IL-6 and TNF-αof EC, model group had great significant difference (P<0．01) compared with control. 100μg·ml^-1 ,200μg·ml^-1 groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the secretion of IL-6, and had significant difference (P<0．05) compared with model group. The three dose groups of prim-O-glucosylcimifugin and 200μg·ml^-1 group of 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the secretion of TNF-α, and had great significant difference (P<0．01) compared with model group. 2 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the adhesion of monocyte to rCMECReferred to Nishida's method, the primary culture method of rCMEC in vitro was established. The third generation EC was adopted to investigate the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the adhesion of monocyte to rCMEC induced by ox-LDL.The result was as follows:ox-LDL of 3ng·ml^-1 as MDA content can increase the adhesion of monocyte to EC, which has great significant difference (P<0．01) compared with control. 50μg·ml^-1 ,100μg·ml^-1 , 200μg·ml^-1 groups of prim-O-glucosylcimifugin, 200μg·ml^-1 group of 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the adhesion of monocyte to EC, which has great significant difference(P<0．01) compared with model group. 50μg·ml^-1 , 100μg·ml^-1 groups of 4'-O-β-D-glucosyl-5-O-methylvisamminol con inhibit the adhesion, which has significant difference (P<0．05) compared with model group. 3 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the proliferation of smooth muscle cell stimulated by TNF-αThe primary culture method of smooth muscle cell (SMC) was established by attachment-block. The SMC was identiflcated by immunochemistry method, and the growth curve was drawed by cytometry. The third generation of SMC was adopted in our experiment. By determining the effect of prim-O-glucosylc-imifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the activity of SMC , the concentration of the two medicine was decided. By determining the effect of TNF-αon the proliferation of SMC, it's concentration to stimulate the SMC proliferate was dicided. And then the effect of prim-O-glucosylcimif-ugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the proliferation and cell cycle of SMC was investigated by MTT and flow cytometry respectively.The result was as follows:1 The cultured cell was identificated to be SMC by immunochemistry method and morphology observation, because of its positive dyeing ofα-smooth actin and its appearance like peak valley.2．Based on the growth curve, the best time of experiment was limited in the period between 24h to 96h after inoculating cell into culture plates in the density of 1×10⁵mL^-1 .3．The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methy-lvisamminol con on the activity of SMC was determined by MTT. It was found that the maximum of no toxicity concentration can not be determined because of the low toxicity of these two medicines on SMC. Thus, 50μg·ml^-1 , 100μ·gml^-1 , 200μg·ml^-1 were chosen as the concentration of prim-O-glucosylcimi-fugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con in experiment.4．The effect of TNF-αon the proliferation of SMC was determined by MTT. The concentration of 5ng·ml^-1 was chosen to stimulate SMC in experiment. 5 The SMC was stimulated to proliferate by TNF-αof 5ng·ml^-1 , and incubated with prim-O-glucosylcimifugin (50μg·ml^-1 , 100μg·ml^-1 , 200μg·ml^-1 ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con (50μg·ml^-1 , 100μg·ml^-1 , 200μg·ml^-1 ), the effect of these two medicines was investigated. The result indicate-d that TNF-αof 5ng·ml^-1 can stimulate the proliferation of SMC, which has great significant difference (P<0．01) compared with control. The three dose groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisammin-ol con can inhibit the proliferation of SMC, which has great significantdifference(P<0．01) compared with model group.6 The SMC was stimulated to proliferate by TNF-αof 5ng·ml^-1 , and incubated with prim-O-glucosylcimifugin(50μg·ml^-1 ,100μg·ml^-1 , 200μg·ml^-1 ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50μg·ml^-1 ,100μg·ml^-1 ,200μg·ml^-1 ),the effect of these two medicines on the cell cycle was investigated. The result indicated that TNF-αof 5ng·ml^-1 can increase the proportion of G2 phase and S phase in cell cycle, which has great significant difference (P<0．01) compared with control. The three dose groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can increase the proportion of G0/G1 phase, decrease the proportion of S phase and G2/M phase, which havegreat significant difference(P<0．01) compared with model group.It can be concluded that prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the injury and secretion of inflammatorycytokines of EC, the adhesion of monocytes to EC and the proliferation of SMC stimulated by TNF-α. In conclusion, prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con has the effect of anti-inflammatory reaction in AS, and provides the scientific evidence for wide clinical application.