Mechanism Of PCA From Agkistrodon Acutus Affecting TF Secretion In HUVEC

Objective: To explore the role of NF-?B and AP-1 signaling pathway in the inhibition effects PCA on LPS-induced tissue factor(TF)expression in human umbilical vein endothelial cells.Methods: HUVECs were conventionally cultured.The experiment was divided into blank control group,PCA group,LPS group and PCA+LPS group.The blank control group was added with DMEM complete medium,The concentrations of PCA solution 1.25?g/ml were added to the PCA group,0.1?g/ml LPS solution to the LPS group,A mixture of 1.25 ?g/ml PCA and 0.1 ?g/ml LPS was added to the PCA+LPS group.After 12 h culture,the morphology of endothelial cells was observed directly under inverted fluorescence microscope,Hoechst33258 was used to detect the effect of PCA on HUVEC,MTT assay was used to detect the HUVECs viability,the protein expression level of TRAF6 was detected by immunohistochemistry,and cellular localization of NF-?B was detected with immunofluorescence staining.The NF-? B p65,FOS,JUN and TF was detected by Western blot,the TF mRNA expression in HUVEC was performed by Q-PCR,ELISA was used to determine the content of TF in the cell culture supernatant.Results:1.Effect of PCA on Cellular Structure and Growth ProliferationDirect observation of cell morphology inverted microscope found that the control group was found HUVEC oval,or spindle changeable,clear boundary,plump cytoplasm,cobblestone-like uniform adherent growth.Compared with the control group,the cells in LPS group were significantly spindle-shaped,irregular in shape and smaller in volume,but the border was still clear.The morphological changes in PCA group were not obvious.The normal group of HUVEC nuclei showed a uniform blue under the microscope with a clear boundary and uniform color.LPS group showed changes in nuclear morphology,absent,was flaky staining.HUVEC nuclei in PCA group and PCA + LPS group were still blue under microscope,with clear boundary and clear color.The results of MTT indicated that the viability of LPS group was significantly lower than that of control group(P <0.01),and the viability of PCA + LPS group was slightly higher than that of LPS group(P <0.05).2.PCA(1.25?g / ml)can reduce the intracellular expression of TRAF6 induced by LPSImmunohistochemical detection showed that the control group of HUVEC cells were spindle-shaped,cytoplasmic yellow particles were not obvious,clear boundary.The cytoplasm of LPS group showed obvious yellow staining particles,the staining of cytoplasm deepened and the mean optical density of TRAF6 increased,Compared with the control group,Visible expression of TRAF6 protein increased significantly(P <0.01).The LPS+PCA experimental group was compared with the LPS group.LPS + PCA experimental group showed normal cell morphology and no significant cytoplasmic yellow staining particles,and the average optical density of TRAF6 was significantly lower than that of LPS group(P <0.01).3.PCA(1.25?g / ml)can attenuate LPS-induced nuclear NF-? B activation-nuclear transportThe results of immunofluorescence staining showed that the intensity of red fluorescence in normal HUVEC nuclei was very weak in the control group.Compared with the control group,the red fluorescence in the LPS group nucleus was strong in most cells,and the expression of NF-? B(red fluorescence intensity)was significantly increased(P <0.01).Thus,NF-?B was activated and transported to the nucleus.The red fluorescence intensity of PCA group was not significantly increased compared with control group(P> 0.05).The red fluorescence intensity of LPS + PCA group was weak.Compared with LPS group,NF-?B nuclear transport was significantly inhibited(P <0.01).4.PCA(1.25 ?g/ml)Decreases Protein Expression of NF-?B p65,JUN and FOS in LPS-Induced HUVECThe results of Western Blot showed that the optical density ratios of NF-?B p65,JUN and FOS in LPS group were significantly increased(P <0.01)compared with those in control group,while there was no significant difference in PCA group.PCA + LPS group three proteins was significantly lower than the ratio of the LPS group(P <0.01).5.PCA(1.25?g / ml)reduced the expression level of TF in intracellular TF gene,protein and cell supernatant induced by LPSThe results of qPCR showed that compared with the control group,the mRNA expression of TF in LPS group was significantly increased(P <0.01).There was no significant difference between PCA group and control group(P> 0.05).Compared with LPS group,the mRNA expression of TF in LPS + PCA group was significantly decreased,which was less than that in LPS group(P <0.01).Compared with the control group,the TF ratio of TF protein in LPS group increased significantly(P <0.01),while the ratio of PCA group did not change significantly.PCA + LPS group protein ratio is compared with LPS group was significantly(P <0.01).ELISA method to detect the amount of TF in cell culture supernatant results,Compared with the control group,the expression of TF in LPS group increased significantly(P <0.01),while there was no significant change in PCA group.PCA + LPS group was significantly lower than LPS group(P <0.01).Conclusion:1.PCA has no significant effect on the structure and growth of HUVEC,But it can reduce the change of spindle HUVEC induced by LPS and the cell proliferation activity.2.PCA can antagonize the damage of endotoxin to HUVEC through the NF-kB and AP-1 pathways and inhibit the TF gene expression through this antagonism to reduce the secretion of TF,PCA may be one of the mechanisms to control thrombotic diseases.