| Background: as we all know,inflammation is a major and common pathophysiological feature in many stages of atherosclerosis,ischemic heart disease and sepsis.Therefore,the study of the pathological mechanism of inflammatory reaction mediated by endothelial cells can provide important significance for improving the clinical treatment of cardiovascular diseases.Traditional Chinese medicine Tripterygium wilfordii has been applied in ancient Chinese medicine for nearly a hundred years.It has the effects of dispelling wind and removing dampness,activating blood circulation and dredging collaterals,relieving swelling and alleviating pain,killing insects and detoxifying.Tripterygium glycosides,as the main form of Tripterygium wilfordii,has been effective in the treatment of rheumatoid arthritis,various primary and secondary kidney diseases,such as autoimmune and chronic inflammatory diseases.It is suggested that triptolide,as the most important active component of Tripterygium wilfordii,regulates the inflammation mediated by endothelial cells.Therefore,we studied the effect of triptolide on lipopolysaccharide(LPS)-induced inflammation of vascular endothelial cells and the related mechanisms,providing a solid pharmacological basis for the clinical application of Tripterygium Wilfordii and its extract,tripterygium glycosides.Objective: this experiment in human umbilical vein endothelial cells(human umbilical vein endothelial cell,HUVECs)training model,to observe the effect of triptolide on HUVECs LPS induced inflammatory cytokines,chemokines,adhesion factor release,HUVECs and monocyte adhesion and the nuclear transcription factor kappa NF-B p65 DNA binding force and so on,to clarify the effects of triptolide on vascular endothelial cell inflammation effects and the related mechanism.Method:1.cell intervention: at 37 C,5%CO2 and under suitable humidity,HUVECs(Lonza,USA)was placed in a EGM-2 medium containing growth factor(Lonza,USA),and was routinely cultured.The cells were transferred to the 3-7 generation,and the condition was stable.Before the experiment,the HUVECs fusion degree of85%-95% was placed in the EGM-2 medium without growth factor to cultivate 4H.2.experimental grouping1)normal control group: in addition to vehicle,do not do any treatment.2)group LPS: adding LPS,the final concentration is 1 u g/ml.3)LPS+ triptolide 25 nM group: at 37 OC,1H was incubated with 4H without growth factor medium and 1H at the final concentration of 25 nM,then added LPS(14h g/ml)to incubate.4)LPS+ triptolide 50 nM group: at 37 OC,1H was incubated with 4H without growth factor medium and 1H at the final concentration of 25 nM,then added LPS(14h g/ml)to incubate.5)LPS+ triptolide 100 nM group: at 37 OC,1H was incubated with 4H without growth factor medium and 1H at the final concentration of 50 nM,then added LPS(14h g/ml)to incubate.3.MTT assay was used to detect the activity of triptolide on HUVECs incubated with 6h,12 h and 24 h under LPS stimulation4.,enzyme linked immunosorbent assay(ELISA)was used to detect the effect of triptolide on the release of inflammatory cytokines(TNF-,IL-6)and chemokines(MCP-1)in HUVECs stimulated by LPS.The effect of triptolide on HUVECs expression of adhesion factor(ICAM-1,VCAM-1)under LPS stimulation by 5.cell enzyme-linked immunosorbent assay(Cell-based ELISA)The effect of triptolide on the adhesion of HUVECs to mononuclear cell THP-1under LPS stimulation by 6.CFSE fluorescence labeled probe cell adhesion assay7.effect of triptolide on DNA binding force of nuclear factor NF-kappa B p65 under LPS stimulation based on ELISA methodThe experimental data of each group were repeated three times to reduce the error Result:1.when using MTT to detect cell viability,LPS 1 HUVECs g/ml HUVECs 6h was selected.Cell viability did not change(data).Therefore,LPS 1 g/ml was used for follow-up test.2.enzyme linked immunosorbent assay(ELISA)was used to detect the effect of triptolide on the release of inflammatory cytokines(TNF-,IL-6)and chemokines(MCP-1)in HUVECs stimulated by LPS.Compared with the control group,the amount of TNF-,IL-6 and MCP-1released by HUVECs increased significantly under the stimulation of LPS(P<0.05),which could be inhibited by Triptolide(P<0.05)and dose dependently.3.The effect of triptolide on HUVECs expression of adhesion factor(ICAM-1,VCAM-1)under LPS stimulation by cell enzyme-linked immunosorbent assay(Cellbased ELISA)Compared with the control group,the expression of HUVECs(ICAM-1 and VCAM-1)increased significantly(P<0.05)under the stimulation of LPS(P<0.05),which could be inhibited by Triptolide(P<0.05)in a dose-dependent manner.4.The effect of triptolide on the adhesion of HUVECs to mononuclear cell THP-1 under LPS stimulation by CFSE fluorescence labeled probe cell adhesion assayCompared with the control group,the adhesion ability of HUVECs to monocyte THP-1 increased significantly under the stimulation of LPS,(P<0.05),which could be inhibited by Triptolide(P<0.05)in a dose-dependent manner.5.effect of triptolide on DNA binding force of NF-kappa B p65 stimulated by LPS based on ELISA methodCompared with the control group,the DNA binding force of HUVECs nuclear transcription factor NF-kappa B p65 increased significantly under the stimulation of LPS(P<0.05),which could be inhibited by Triptolide(P<0.05)in a dose-dependent manner.Conclusion:1.through the HUVECs cell culture model,we found and confirmed that triptolide significantly inhibited the inflammatory cytokines induced by LPS(TNFand IL-6),chemokine(MCP-1)and adhesion molecules(ICAM-1,VCAM-1)release and expression,at the same time,the adhesion process between the inhibition of HUVECs and mononuclear cells and,in a dose-dependent manner,all experimental results suggest that the inflammatory reaction of triptolide can inhibit vascular endothelial cell mediated.2.through the HUVECs cell culture model,we further found and confirmed that triptolide alleviated and alleviated the inflammatory response mediated by vascular endothelial cells by inhibiting the nuclear factor NF-kappa B signaling pathway. |
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