The Protective Effects Of The Monomers And Their Combinations In Fufangdanshen On HUVECs Induced By Inflammatory Cytokines

Object:The Chinese complex formula Fufangdanshen has been found to have beneficial effects on the circulatory system including the effect of anti-atherosclerosis, while the molecular mechanism still needs further study.Atherosclerosis(AS)is a common disease in cardiovascular.It is thought to be an inflammation-related disease and many inflammatory cytokines accelerate the development of it.In this research, the protective effects of the monomers and their combinations in Fufangdanshen on vascular endothelial cells injuried by inflammatory cytokines were observed and the related mechanisms were also studied.Method:The changes of mRNA expression profile in primary cultured human umbilical vein endothelial cells(HUVECs)after tumor necrosis factor-α(TNF-α, 2ng/ml,12 h)or protocatechuic aldehyde(PCA,derived from the Chinese herb, Salvia miltiorrhiza)were observed by cDNA microarray assay.HUVECs were stimulated by lipopolysaccharide(LPS,0.5μg/ml,12 h)and the expression of intercellular cell adhesion molecule-1(ICAM-1),vascular adhesion molecule-1 (VCAM-1)and fibronectin(FN)were detected by cell surface ELISA assay to evaluate the pharmacologic action of PCA and the related mechanism.The secretion of monocyte chemoattractant protein-1(MCP-1)was also studied by ELISA assay. Heochest 33258 staining and AnnexinV/PI/FITC assay were used to detect cell apoptosis induced by LPS(15μg/ml,30 h)and the protective effect of PCA.The cell signal transduction pathways:death receptor-caspase-3 pathway,cytochondriome pathway,cell DNA damage pathway and caspase-independent pathway were observed to find the pathway by which LPS and PCA displayed their actions.The expression of ICAM-1 was also used to screening danshensu,tanshinoneⅡA,cryptotanshinone, ginsenosideRb1,ginsenosideRg1 and notoginsenosideR1 to find the monomers that can inhibit LPS-induced inflammation.Orthogonal design was used to screening the best combination of the effective formulas. Result:Compared with the control group,the genes was differentiated with the incubation of PCA(0.5 mmol/L).Among them,expression of the genes including inflammatory,adhesion molecular,and angiotasis genes were down-regulated and that of antioxidant genes were up-regulated.These results were confirmed by RT-PCR.PCA(0.25,0.5 and 1.0 mmol/L)inhibited high expression of ICAM-1,VCAM-1, FN and secretion of MCP-1 induced by LPS.The effects showed a dose-dependent manner and were related with MAPK -NF-κB pathway.The apoptotic cells stimulated by LPS displayed typical apoptotic nuclei changes. These changes were improved by PCA.The numbers of apoptotic cells were down-regulated by PCA in a dose-dependent manner.The studies on membrane receptor-caspase-3 pathway,cell mitochondrion pathway,DNA damage pathway and caspase-independent pathway suggested that the cell apoptosis induced by LPS was related with caspase-3 pathway.Cell mitochondrion pathway,DNA damage pathway and caspase-independent pathway seemed no contribution to LPS-induced apoptosis of HUVECs.Caspase-3 pathway also contributed to the protective effect of PCA on HUVEC apoptosis.The screening of the other compositions and their combinations indicated that PCA(0.25-1.0 mmol/L),tanshinoneⅡA(0.11-1.0μmol/L),ginsenoside Rb1(0.33-3.0μmol/L)and notoginsenoside R1(5.0-45.0μmol/L)inhibited high expression of ICAM-1 on HUVEC surface induced by LPS.Orthogonal experiment design was used to screen the best combination of these four formulas and PCA(1.0mmol/L)+ Tan(0.33μmol/L)+ Rb1(3.0μmol/L)+ R1(45.0μmol/L)was found.Conclusion:Collectively,these results would contribute to explain the cardiovascular protective effect,especially anti-atherosclerosis effect of Fufangdanshen and Salvia miltiorrhiza.Furthermore,this research may explain the mechanism of traditional complex formula in the view of modern life science.