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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.
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On basis of the idiosyncratic lipopolysaccharide(LPS)-mediated hepatotoxicity model, liver injury induced by Zhuangguguanjie wan(ZGW)was evaluated, and the mechanism was explored. Idiosyncratic hepatotoxicity model was established in rats by injecting LPS at a dosage of 2.8 mg·kg-1. Rats were randomly divided into the normal control group, LPS group, ZGW group and LPS+ZGW group. Alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities were analyzed in serum; pathological changes(HE staining)and the content of cytokines of liver were tested; and immune cell subpopulation ration were determined in blood and liver. Compared with the control group, the ZGW group and LPS group had no significant changes in ALT, AST and liver pathology(P> 0.05); while the ZGW+LPS group exhibited an elevation in ALT and AST(P< 0.05). Disorder of liver lobular arrangement and irregular island-like or massive necrosis of liver cells were observed in the group. Several cytokines in the liver were increased in LPS group and ZGW+LPS group(P< 0.05 or P< 0.01), and the level in ZGW+LPS group was higher than that of LPS group. Compared with the control group, the ratio of CD3+ T cell/lymphocyte of blood in LPS group was significantly decreased(P< 0.01); while the percentage of CD3+ T cells in the liver were significantly increased(P< 0.05). The contents of immune cells of blood had no significant changes between LPS group and ZGW+LPS group(P> 0.05). CD3+ T cell in the liver of ZGW+LPS group was significantly increased over the LPS group(P< 0.05). Aggregation or activity of CD3+ T cell was increased by ZGW combined with LPS. These results suggest that ZGW could promote T lymphocyte recruitment to liver under the immune activation state leading to inflammatory response, which may contribute to idiosyncratic liver injury.
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>
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<p><b>OBJECTIVE</b>To investigate the changes of serum levels of chitinase-3-like-1 protein (YKL-40) and high-sensitivity C-reactive protein (hs-CRP) in patients with non-ST segment elevation acute coronary syndrome (ACS), to explore its correlation with its severity, and to observe the effects of Guizhi Fuling Decoction (GFD) on levels of blood lipids, YKL-40, and hs-CRP.</p><p><b>METHODS</b>Recruited were 72 patients with unstable angina (UA) or non-ST segment elevation myocardial infarction (NSTEMI) at Department of Integrative Medicine, First Affiliated Hospital of Xinxiang Medical College from August 2010 to June 2011. They were randomly assigned to the treatment group (36 cases) and the control group (36 cases). All patients were treated by routine treatment, but patients in the treatment group took GFD additionally. The course of treatment was four weeks. According to the severity degree, all patients were graded to four ranks: low-risk group of UA, medium-risk group of UA, high-risk group of UA, and NSTEMI. The levels of YKL-40 and hs-CRP, and the correlation of severity degree were analyzed. Before and after treatment levels of triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) were measured. Before treatment, at two weeks, and after treatment the serum levels of YKL-40 and hs-CRP were detected. The relationship of YKL-40, hs-CRP and the severity of the disease were analyzed.</p><p><b>RESULTS</b>Levels of YKL-40 and hs-CRP were positively correlated with the severity of the disease respectively (r = 0.729, P < 0.05; r = 0.655, P < 0.05). The positive correlation also existed between YKL-40 and hs-CRP (r = 0.848, P < 0.05). There was no statistical difference in the levels of blood lipids, YKL-40, or hs-CRP between the two groups before treatment (P > 0.05). Compared with before treatment, the levels of YKL-40 and hs-CRP significantly decreased in both groups after two weeks of treatment (P < 0.05). The levels of TG, TC, LDL-C, YKL-40, and hs-CRP significantly decreased, while the HDL-C level increased in both groups after treatment (P < 0.05). The level of HDL-C in the treatment group was higher, while levels of YKL-40 and hs-CRP were lower after treatment, when compared with the control group (all P < 0.05).</p><p><b>CONCLUSION</b>On the basis of anti-inflammation and adjusting blood lipids by Western medicine, GFD could further reduce the serum levels of YKL-40 and hs-CRP of ACS patients, elevate the HDL-C level, and play anti-atherosclerosis effects.</p>