| Background: atherosclerosis,hypertension and other cardiovascular disease is a kind of is characterized by vascular endothelial dysfunction of chronic inflammation disease.Therefore,the study of the inflammatory response and its related mechanism of the prevention and treatment of cardiovascular disease has important clinical significance.At present,the research shows that transient receptor ppotential vanillloid type1 channel(transient receptor ppotential vanillloid type1 channel,TRPV1)has obvious inhibiting effect of inflammatory response,several studies have shown that expressed in the endothelial cells of TRPV1,type by activating endothelial nitric oxide synthase(endothelial nitric oxide sgnthase,e NOS)increase nitric oxide,(nitric oxide,NO)release,so as to achieve the purpose of inhibiting inflammation.Has a long history of traditional Chinese medicine evodia rutaecarpa treatment of cardiovascular disease,a study suggests the effective components of evodia rutaecarpa alkali(evodiamine,EVO)may be type by activating endothelial nitric oxide synthase(e NOS)increase nitric oxide(NO)release.Type,therefore,the research by activating TRPV1 receptors in endothelial nitric oxide synthase(e NOS),nitric oxide(NO)release of vascular endothelial cells mediated inflammatory reaction mechanism and the influence of the intervention mechanism of evodia rutaecarpa alkali,will lay the foundation for further clinical study.Purpose: This research in human umbilical vein endothelial cells(human umbilical vein endothelial cells and HUVEC)on the training model,through the observation by activating TRPV1 receptors type endothelial nitric oxide synthase(e NOS),nitric oxide(NO)release of lipopolysaccharide(LPS)induced endothelial cells produce cytokines and chemokines,adhesion factor and mononuclear cells and the influence of the interaction between endothelial cells,and evodia rutaecarpa alkali type by activating endothelial nitric oxide synthase(e NOS)increase the effects of nitric oxide(NO)release of the process,so as to clarify evodia rutaecarpa alkali of TRPV1 and inhibiting inflammatory reaction mechanism of molecular biology.Methods:1.The cell culture:Human umbilical vein endothelial cells(HUVECs,Lonza,Allendale,NJ,USA)were used between 3-7 passages and cultured in EGM-2 Bullet Kit medium containing growth supplements(Lonza,Allendale,NJ,USA)at 37 o C in a 5% CO2 humidified incubator.85%-95% confluent HUVECs were starved in EBM-2 containing no growth supplements for 4 h before the experiment was started.2.The experimental group:(1)normal group: in addition to adding solvent DMSO(5%),don't give any processing.(2)LPS groups: join the LPS,make its final concentration of 1(including g/ml.(3)CAP + LPS groups: add the CAP and LPS respectively make its final concentration respectively umol/L and 1(including 10 g/ml,add CAP processing after 1 h,then add the LPS.(4)CAPZ + CAP + LPS groups: add CAPZ,CAP and LPS respectively to make the final concentration of 10 umol/L respectively,1(including 10 umol/L and g/ml,join CAPZ treatment after 1 h,then add CAP fully blending processing after 1 h,the last to join the LPS.(5)L-NMMA + CAP + LPS groups: add L-NMMA,CAP and LPS respectively to make the final concentration of 1 respectively tendency for 10 umol/L and 1 L,including g/ml,join L-NMMA processing after 1 h,then add CAP fully blending processing after 1 h,the last to join the LPS.(6)EVO + LPS groups: join the EVO and LPS respectively to make the final concentration of 5 umol/L respectively and 1(including g/ml,join the EVO processing after 1 h,then add the LPS.All landowners CAPZ + EVO + LPS groups: add CAPZ,EVO and LPS respectively to make the final concentration of 10 umol/L respectively,5 umol/L and 1(including g/ml,join CAPZ treatment after 1 h,then add the EVO fully blending processing after 1 h,add LPS.Today L-NMMA + EVO + LPS groups: add L-NMMA,EVO and LPS respectively to make the final concentration of 1 respectively tendency,5 umol/L and 1 L(including g/ml,join L-NMMA processing after 1 h,then add the EVO fully blending processing after 1 h,the last to join the LPS.3.The cell activity can be determined through determined by MTT method.4.Through the use of nitrite colorimetry determination kit sample nitrite level,to reflect the NO level.5.Enzyme-linked immunosorbent assay(ELISA)to detect TRPV1 receptors to the LPS stimulation after 6 h of HUVEC cytokines TNF alpha,IL-6,and the effects of chemokines MCP-1.6.The Cell--based enzyme-linked immunosorbent method to detect TRPV1 receptors to the LPS stimulation after 6 h of HUVEC expression of Cell adhesion molecules(ICAM 1,VCAM 1).7.Detection of Calcein-AM cell adhesion experiment TRPV1 receptors to the LPS stimulation after 6 h of HUVEC and monocytes THP 1 adhesive ability.8.ELISA detection of evodia rutaecarpa alkali to the LPS stimulation after 6 h of HUVEC to express the influence of TNF alpha,IL-6.Each group experiment repeated three times.Results:1.TRPV1 receptors have NO impact on HUVEC Concentration of nitrite colorimetry the kits CAP for 3 mu mu M and 10 M have the quantity of NO on HUVEC.The results showed that activated TRPV1 receptors can increase the quantity of NO HUVEC,and are dose dependent.Nitrite colorimetry kits,check the following set of CAP,CAPZ + CAP,CAP + L-NMMA,CAP + LY294002,CAP + EGTA NO amount,the results showed that activated TRPV1 receptor is obviously improved HUVEC NO amount,the effect can be CAPZ,L-NMMA,LY294002 and EGTA block,the results suggest TRPV1 receptors by activating PI3K/Akt signaling pathway to promote vascular endothelial cells to produce NO.2.TRPV1 receptors to HUVEC by LPS stimulation induced cytokines(TNF alpha,IL-6),and the influence of chemotactic factor(MCP-1)ELISA detection of TRPV1 receptors to the LPS stimulation after 6 h HUVEC TNF alpha inflammatory mediators,IL-6 and MCP-1 the effect of protein concentration.Results show that compared with normal control group,after LPS stimulation induced HUVEC cells produce cytokines(TNF alpha and IL-6)and chemokine(MCP-1)the amount of increased obviously(P < 0.05),the use of capsaicin(CAP)after activating TRPV1 receptors significantly inhibited the effect.In addition,we further found and confirmed,CAP effect can be induced by TRPV1 receptor antagonist(CAPZ)and NOS blockers(L-NMMA),the results suggest by activating TRPV1 receptors e NOS/NO way to inhibit endothelial cells to produce inflammation medium.3.TRPV1 receptors to HUVEC by LPS stimulation induced adhesion factor ICAM 1,VCAM 1Cell--based enzyme-linked immunosorbent method to detect TRPV1 receptors to the LPS stimulation after 6 h of HUVEC expression of Cell adhesion molecules(ICAM 1,VCAM1).Results show that compared with normal control group,after LPS stimulation induced HUVEC cells adhesion factor of ICAM 1,VCAM 1 quantity increased significantly(P <0.05),the use of capsaicin(CAP)after activating TRPV1 receptors significantly inhibited the effect.In addition,we further found and confirmed,CAP effect can be induced by TRPV1 receptor antagonist(CAPZ)and NOS blockers(L-NMMA),the results suggest by activating TRPV1 receptors e NOS/NO way to inhibit endothelial cells adhesion factor.4.TRPV1 receptors of THP 1 cells induced by LPS stimulation effect with the effects of the adhesion between HUVEC.TRPV1 receptors to the LPS stimulation after 6 h of HUVEC cells with the Calcein-AM tagged THP 1 cells common incubation for 30 min,fluorescent probe results show that compared with normal control group,treated with LPS induced THP 1 cells and adhesion ability between HUVEC cells increased significantly(P < 0.05),the use of capsaicin(CAP)after activating TRPV1 receptors significantly inhibited the effect.In addition,we further found and confirmed,CAP effect can be induced by TRPV1 receptor antagonist(CAPZ)and NOS blockers(L-NMMA),the results suggest by activating TRPV1 receptors e NOS/NO way to inhibit induced by LPS THP 1 cells and adhesion between HUVEC cells.5.Evodia base on human umbilical vein endothelial cells induced by lipopolysaccharide produce cytokine TNF alpha and IL-6ELISA detection of HUVEC after LPS stimulation TNF alpha produce inflammation medium,the influence of IL-6 protein concentration.Results show that compared with normal control group,after LPS stimulation induced HUVEC cells produce cytokines TNF alpha and IL-6 volume increased significantly(P < 0.05),the use of evodia rutaecarpa after alkali activated TRPV1 receptors(Mr)significantly inhibited the effect.In addition,we further found and confirmed that the EVO effects can be induced by TRPV1 receptor antagonist(CAPZ)and NOS blockers(L-NMMA),the results suggest evodia rutaecarpa alkali by activating TRPV1/e NOS/NO way to inhibit endothelial cells produce cytokines.Conclusion:1.On HUVEC cell culture model,e NOS approach can restrain after blocking TRPV1 receptors on LPS induced by the cytokines,chemokines,and adhesion factor increases,and also affect its inducing mononuclear cell adhesion process,the results indicate that endothelial cells express by activating TRPV1 receptors e NOS ways have important anti-inflammatory effects.2.On HUVEC cell culture model,e NOS way can inhibit evodia rutaecarpa alkali after blocking of LPS induced by the increase of cytokines,the results suggest evodia rutaecarpa alkali by activating e NOS ways have important anti-inflammatory effects. |
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