ABSTRACT
AIM:To explore the role of nuclear factor-κB(NF-κB)and activator protein-1(AP-1)signaling pathway in the inhibitory effects of Agkistrodon acutivirus protein C activator(PCA)on lipopolysaccharide(LPS)-induced tissue factor(TF)expression in human umbilical vein endothelial cells(HUVECs).METHODS: The viability of the HUVECs was measured by MTT assay.The protein distribution of tumor necrosis factor-associated factor 6(TRAF6)in the cells was detected by immunohistochemical staining.The protein expression of NF-κB p65,TF,c-Fos and c-Jun was deter-mined by Western blot.The mRNA expression of TF in the HUVECs was detected by qPCR.The content of TF in the me-dium of each group was measured by ELISA.RESULTS:Compared with the control group,the viability of the HUVECs in LPS group decreased significantly(P<0.01), obvious yellow dye particles appeared in the cytoplasm, cytoplasmic stai-ning deepened,and the average absorbance of TRAF6 was increased(P<0.01).The protein expression of NF-κB p65, c-Jun and c-Fos were significantly increased(P<0.01).The expression of TF at mRNA and protein levels were signifi-cantly increased(P<0.01).Compared with the LPS group,the cell viability in PCA +LPS group was slightly increased (P<0.05),the cell morphology was normal,cytoplasmic yellow dye particles were not obvious, and the average absor-bance of TRAF6 was significantly lower than that in LPS group(P<0.01).The protein expression of NF-κB, c-Jun and c-Fos was significantly decreased(P<0.01),and the expression of TF at mRNA and protein levels were decreased(P<0.01).CONCLUSION:PCA significantly reduces the damage of HUVECs induced by LPS.The mechanism may be a-chieved by reducing the activation of TRAF 6,NF-κB and AP-1 nuclear transcription factors,thereby reducing the release of tissue factor.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of Agkistrodon acutus venom protein C activator(PCA) on ultrastructure of human umbilical vein endothelial cells(HUVEC), and the levels of tissue factor(TF), vascular von Willebrand factor (vWF) and endothelin-1 secreted by HUVEC and to clarify the anti-thrombotic mechanism of PCA.</p><p><b>METHODS</b>The experiments were divided into control group(DMEM), LPS group (LPS 0.1 µg/ml), PCA group(PCA 1 µg/ml) and PCA+LPS group (1 µg/ml PCA+ 0.1 µg/ml LPS). The morphology of endoplasmic reticulum, mitochondria and the number of autophagosome in HUVEC were observed by transmission electron microscopy. The TF, vWF and ET-1 were measured in the medium of each group by ELISA; RT-PCR was used to detect mRNA expression level of vWF and ET-1 in cells; and the protein expression level of TF in cells was detected by Western blot.</p><p><b>RESULTS</b>Compared with the control group, the ultrastructural changes of HUVEC in the LPS group included the cell membrane getting rough, swelling of mitochondria and endoplasmic reticulum, and autophagosome increase, however, the ultrastructure differences between PCA and control group were not significant. Compared with the ultrastructure of HUVECs in LPS group, the swelling of mitochondria and endoplasmic reticulum disappeared in the LPS+PCA group, and the number of autophagosome decreased obviously. Compared with the control group, the content of ET-1, vWF and TF in cell culture supernatant, and the protein expression level of vWF, ET-1 gene and TF protein were significantly increased in LPS group (P<0.05); the expression levels of the 3 factors in the cell culture supernatant and cells in PCA group were not significantly different from the control group (P>0.05). The expression levels of TF, vWF and ET-1 in LPS group were significantly lower than those in LPS+PCA group (P<0.05).</p><p><b>CONCLUSION</b>PCA(1 µg/ml) can reduce the ultrastructural changes of HUVEC induced by LPS, and inhibit the increase of TF, vWF and ET-1 secretion from HUVEC induced by LPS.</p>