Study On Effect And Mechanism Of Extracts Of Rehmanniae On Protection Of Vascular Endothelial Cells By Tumor Necrosis Factor

Objectives:Observe the protection of extracts of different parts Rehmanniae for the damageon vascular endothelial cells. And then, explore that the effection of extracts ofRehmanniae for cure the AS and mechanism. While laying the foundation for furtherdefine Rehmanniae vascular protective effects play an active ingredient.Methods:1. Choose the human umbilical vein endothelial cells in vitro for researchobjective,use the tumor necrosis factor inductes HUVEC to copy vascularendothelial cell damage model.2. To choose the concentration-response and limitation and dose effective,agingof extracts of Rehmanniae by MTT.Then,detected the influence of the HUVEC cellvitality.Flow cytometry was detected cell cycle to check the influence of theHUVEC cell vitality on effective parts of Rehmanniae3. The tumor necrosis factor inducted HUVEC to replication vascular endothelialcell damage model in the basis of. dose effective,aging.Nitrate reductase enzymaticwas detected the content of NO which was induced by TNF-α on effective parts ofRehmanniae.Enzyme-linked immunosorbent experiments detecting ET-1.Detection kitnitric oxide synthase (iNOS) which was induced by TNF-α on effective parts ofRehmanniae.4. Enzyme-linked immunosorbent experiments detecting VCAM-1,MCP-1,ET-1which was induced by TNF-α on effective parts of Rehmanniae. PDTC after blockingadhesion molecules,chemokines protection levels to explore the possible mechanismof vascular injury.5. Western-blot was utilized to investigate the expression of nucleus NF-κBp65,IκBα,cytoplasmNF-κBp65protein.Results:1MTT results showed:Rehmannia extract effective parts are:the water part and95%ethanol50%methanol part.The best dose of waterl parts,95%ethanol extractsites,50%methanol site is150μg·mL-1,75μg·mL-1,60μg·mL-1.Best time is36h(P<0.01,P<0.05).The results showed that the proliferation of HUVEC cells:cellviability compared to all parts of the control group improved significantly(P<0.01,P<0.05).The flow cytometry was shown that,compared with the modelgroup,the synthesis of DNA was significant enhanced of effective parts ofRehmanniae (P<0.01,P<0.05).2. For TNF-α-induced HUVEC cell proliferation results showed that:comparedwith the control group,model group decreased cell viability;Compared with themodel group,the effective parts of different concentrations significantly increasedcell viability(P<0.01,P<0.05).Detection of NO,iNOS and ET-1results showthat:compared with the control group,the concent of NO was reduced and the contentof ET-1,iNOS was increased of model group;Compared with the model group,theconcent of NO was increased and the content of ET-1,iNOS were reduced ofeffective parts of Rehmanniae at best dose (P<0.01,P<0.05).3. ELISA detection ofVCAM-1,MCP-1showed that:compared with the controlgroup,the protein expression of VCAM-1、MCP-1were increased of model group(P<0.01);Compared with the model group,the protein expression of VCAM-1,MCP-1were reduced of effective parts of Rehmanniae(P<0.01,P<0.05).Blockers showedthat:compared with the control group,the protein expression of VCAM-1,MCP-1were increased of model group(P<0.01);Compared with the model group,the proteinexpression of VCAM-1,MCP-1were reduced of effective parts ofRehmanniae(P<0.01,P<0.05).Suggested effective parts of Rehmanniae haveanti-inflammatory,protective role of vascular endothelial.4. NF-κB signaling pathway results show that:compared with the normalgroup,the protein expression of IκBα,cytoplasm NF-κBp65was reduced and nucleusNF-κBp65was increased of model group(P<0.01);Compared with the modelgroup,the the protein expression of IκBα,cytoplasm NF-κBp65wasincreased,nucleus NF-κBp65was reduced of effective parts of Rehmanniae.595%ethanol parts and water parts of rehmannia under optimum conditions ofdose and time optimal protective effect on HUVEC comparative study,the detection of cell proliferation,iNOS,NO/ET-1,VCAM-1,MCP-1results of the study showthat:compared with the control group,cell proliferation model group significantlyreduced,NO lower,iNOS,ET-1increased,VCAM-1,MCP-1increases;Compared withthe model group,the two part can make a significant proliferation increased,NOincreased,iNOS,ET-1reduced,VCAM-1,MCP-1reduction,and95%ethanol part isbetter than water part.6.50%methanol part is main component ingredients from95%ethanol parts themain component of the type of ring dilute ether and benzene terpene glycosidesConclusions:1. Effective parts of Rehmanniae could inhibit the activation of NF-κBsignaling pathway in TNF-α-induced HUVEC,which could be one of its possiblemechanisms for treatment of AS,leading to down-regulation of adhesivemolecular,chemokines endothelin protein expression、 inhibition of vascularendothelial damage,protecting vascular function.2. Rehmanniae play vascular protective effects of the active ingredients aremainly located in parts of50%methanol, the main component of the type of ringdilute ether and benzene terpene glycosides.