Aim To investigate the effect of resveratrol ( Res) , PDTC and AG490 on adhesion molecules ex-pression induced by product of activated complement alternative pathway on human microvascular endothelial cells ( HMECs) and the possible mechanisms. Meth-ods HMECs were exposed to the product of comple-ment alternative pathway activation, then the superna-tant was removed to detect the concentration of malond-ialdehyde ( MDA ) with TBA method. ELISA method was used to detect the expression of soluble ICAM-1 , VCAM-1 ( and E-selectin) in the culture supernatant. Res, PDTC and AG490 with different concentrations were used to determine their effect on cell oxidation level and adhesion molecules expression. The phospho-rylation of NF-κB p65 was detected by Western blot, and the intervention of Res, PDTC and AG490 was as-sayed by the same way. Results The activation of complements alternative pathway resulted in the phos-phorylation of NF-κB p65 , and increased the concen-tration of MDA and up-regulated the expression of ICAM-1, VCAM-1 and E-selectin. Res reduced the concentration of MDA. Res, PDTC and AG490 inhibi-ted the phosphorylation of NF-κB p65 . Res and PDTC showed similar inhibition on expression of ICAM-1 and VCAM-1 , while exhibiting little effect on expression of E-selectin, and AG490 significantly inhibited the ex-pression of the above adhesion molecules. Conclusions Res, PDTC and AG490 could inhibit the expression of adhesion molecules induced by activated complement alternative pathway, the inhibition of NF-κB pathway activation was involved in their mechanism, and JAK2 may be a more important intervention target in regula-ting adhesion molecule expression.

A novel fibrinogenolytic protease,named atrase A,has been purified from the venom of Naja atra by sequential chromatography.Atrase A is a single chain glycoprotein with a molecular weight of 64.6 kD,an isoelectric point of pH 9.6 and a neutral sugar content of 4.16%.Atrase A specifically and slowly degraded α-chain of fibrinogen.This fibrinogenolytic activity Was inhibited by chelating agents(EDTA,EGTA and 1,10-phenanthroline)and DTY,and partially inhibited by PMSF,but not by soybean trypsin inhibitor,indicating it is a metalloproteinase.Atrase A showed edema-inducing activity and bactericidal activity against Staphylococcusa aureus.Atrase A did not show cytotoxicity on A549 and K562 cells in MTT assay,but detached adherent A549 cells from the substrate.Atrase A did not show significant inhibition of platelet aggregation induced by ADP or collagen,and did not exhibit proteolytic activities towards fibrin,azocasein and BAEE.It also did not show hemorrhage activity when injected subcutaneously into mice.

Aim To investigate the change of molecu-lar expression related to coagulation and fibrinolysis in human microvascular endothelial cells ( HMEC ) in-duced by activated complement alternative pathway and effect of pyrrolidine dithiocarbamate ( PDTC ) and res-veratrol on intervention. Methods Normal human se-rum was activated by cobra venom factor ( CVF) . After exposure of HMEC to activated complement for various times, supernatant was removed and assayed for ex-pressions of P-selectin, VWF, t-PA, PAI-1, TF, TM, and NO by using reagent kits. The expressions of the above molecules in HMEC pretreated with PDTC and resveratrol were also investigated. Results P-selectin and VWF were rapidly released by endothelial cells and the expression reached the peak at the time point of 15 min. The expressions of t-PA, PAI-1, and TF were continuously upregulated, whereas NO and TM were decreased. PDTC and resveratrol inhibited the upregulation of P-selectin, VWF, t-PA, PAI-1 and TF, and intervened the downregulation of NO. Res-veratrol further downregulated the expression of TM. Conclusion Activated complement alternative path-way can influence the expression of molecules related to coagulation and fibrinolysis in HMEC, and PDTC and resveratrol can affect this change.

Aim To investigate the effect of chlorogen-ic acid,caffeic acid,and ferulic acid on expression of molecules related with inflammatory response of HMECs induced by activated complement alternative pathway.Methods CVF was used to activate the al-ternative pathway of serum complement.After exposure of HMECs to activate complement for various times, supernatant of cell culture was removed and assayed for content of ICAM-1,IL-6,IL-8,t-PA,and PAI-1 u-sing ELISA kits.The expression of the above mole-cules induced by activated complement was measured after HMECs were pre-treated with 50,100,250 μmol ·L-1 of CGA,CA,and FA.Results After HMECs were exposed to the product of the activated comple-ment alternative pathway,the expression of ICAM-1 , IL-6,IL-8,t-PA,and PAI-1 was up-regulated.The expression of ICAM-1,IL-6,IL-8,t-PA,and PAI-1 was down-regulated by various concentrations of CGA, CA,and FA.ICAM-1 and IL-8 were inhibited most significantly in all molecules mentioned above.CA ex-hibited the best intervention effect,followed by FA. Conclusion Certain concentration of CGA,CA,and FA can inhibit the expression of ICAM-1,IL-6,IL-8, t-PA,and PAI-1 in HMECs induced by the activation of the alternative complement pathway,indicating that CGA,CA,and FA can inhibit inflammatory response of HMECs.

Aim To investigate the effect of cobra venom metalloproteinase atrase A on human endothelial cell.Methods The effects of atrase A on the growth of HMEC were measured by MTT,SRB and morphological observation methods,respectively.After exposure to atrase A,IL-8,ICAM-1,MCP-1 and E-selectin released by HMEC were detected.Caspase-8 and caspase-3/7 were detected by fluorescent luminescence method.After intravenous injected atrase A to rats,the concentrations of ICAM-1,IL-8 and TNF-? in serum were measured.Results The results showed that atrase A(400,40 mg?L-1) significantly inhibited the growth of HMEC in low cell density.The microscopic examination revealed that atrase A detached the adherent HMEC.After exposure to atrase A,IL-8,ICAM-1 and MCP-1 released by HMEC were increased.Atrase A induced HMEC to express caspase-3/7 and caspase-8.After the administration of atrase A to rats,the concentrations of ICAM-1,IL-8 and TNF-? in serum were increased.There was no difference between the low-dose group and control group in our experiments.Conclusion Cobra venom metalloproteinase atrase A inhibits the growth of HMEC,and induces HMEC releasing inflammation mediators and apoptosis.

Aim To investigate the effects of atorvas-tatin ( ATV) on activation and injury of microvascular endothelial cells induced by oxidized low density lipo-protein ( ox-LDL) . Methods Cultured human micro-vascular endothelial cells were pre-incubated with ATV for 24 h prior to exposure of endothelial cells to ox-LDL. After exposure of endothelial cells to ox-LDL, the cell viability was measured by MTT method, LDH in supernatants was determined by enzyme activity as-say kit, ICAM-1 in supernatants was assayed by using ELISA method, phosphorylation of NF-κB p65 was de-tected by western blot analysis, transcriptional activity of NF-κB signal pathway was measured by employing dual-luciferase reporter assay system. Results Hu-man microvascular endothelial cells were activated and injured by ox-LDL. Inhibition of the cell viability, re-lease of LDH, expression of ICAM-1, phosphorylation of NF-κB p65 , and up-regulated transcriptional activity of NF-κB induced by ox-LDL were attenuated by ATV. Conclusion ATV can significantly inhibit the activa-tion and injury of human microvascular endothelial cells induced by ox-LDL, and that may be related to inhibition of phosphorylation and transcriptional activity of NF-κB.

Aim To investigate the change of coagulation and fibrinolysis functions of human microvascular endothelial cells (HMEC) induced by activated complement alternative pathway and effect of pyrrolidine-dithiocarbamate (PDTC) and resveratrol (Res) on intervention.Methods Normal human serum was activated by cobra venom factor (CVF).After exposure of HMEC to activated complement for various time points, supernatant was removed and assayed for activities of hydrolysing chromogenic substrate and affecting activated partial thromboplastin time (APTT) and prothrombin time (PT).The cells exposed to activated complement were collected and washed, and then the cell suspension was assayed for activity of affecting coagulation function of normal plasma.Lastly, the coagulation and fibrinolysis functions of HMEC pretreated with PDTC and Res were also investigated after HMEC was exposed to activated complement alternative pathway.Results The hydrolysis activity of chromogenic substrate of supernatant was up-regulated significantly after HMEC exposed to activated complement alternative pathway.The supernatant induced APTT decreased significantly, and also shortened PT.The cell suspension of various time points induced APTT decreased significantly, and also shortened PT by suspension of 6 h time point.PDTC and Res failed to inhibit the up-regulation of the chromogenic hydrolysis activity, but Res showed significant intervention on decrease of APTT, and PDTC had better effect on inhibiting the decrease of PT than that of Res.Conclusion Activated complement alternative pathway can induce abnormality of coagulation and fibrinolysis functions of HMEC, and PDTC and Res can affect this change.

To reveal the underlying mechanism of doxorubicin induced hepatotoxicity, an NMR-based metabolomic approach combined with multivariate statistical analysis was used to observe its metabolic alternations of rat liver. Sixteen differential metabolites between model rats and normal rats were characterized as potential pathological biomarkers related to doxorubicin induced hepatotoxicity. Six pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis, phenylalanine metabolism, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, and tyrosine metabolism were regarded as the targeted metabolic pathways according to Metabolic Pathway Analysis (MetPA). The results suggested that the metabolic perturbations in rats with doxorubicin induced hepatotoxicity were mainly involved in amino acid metabolism, lipid pathways, purine metabolism, energy metabolism, dysfunction of biotransformation and oxidative stress. The investigation revealed the effects of doxorubicin on liver in a holistic metabolic way, which laid a foundation for further studies on its toxicity mechanism.

Aim To investigate the effect and mechanism of cobra venom metalloproteinase atrase A on inhibiting platelet aggregation.Methods Platelet aggregation induced by collagen,ADP,PAF,AA,ristocetin and thrombin,respectively,was measured turbimetrically after platelet incubated with atrase A.Western blot was used to detect the effect of atrase A on cleavage of platelet membrane glycoprotein and von Willebrand Factor.Results Atrase A significantly inhibited platelet aggregation induced by ristocetin and thrombin in a dose-and time-dependent manner.Meanwhile,atrase A just showed slight inhibitory effect on platelet aggregation induced by PAF,AA,collagen,and ADP after incubated with PRP for 5 min.After incubation time was prolonged to 30 min,significant inhibition was shown on platelet aggregation.Western blot revealed that atrase A cleaved platelet membrane glycoprotein GPIb.Conclusions Cobra venom metalloproteinase atrase A significantly inhibits platelet aggregation induced by ristocetin and thrombin due to the cleavage of platelet membrane glycoprotein Ib.And atrase A also has inhibitory effect on platelet aggregation induced by ADP,PAF,AA,and collagen.

Aim To investigate the inhibitory effect of atrase B on human platelet aggregation induced by activated complement.Methods By employing CVF to activate complement,the effect of atrase B on gel filtered platelet aggregation induced by activated complement was measured by turbidimetry and the expression of P-selectin and GPⅡb/Ⅲa on platelet membrane were detected by flow cytometry.Results Atrase B inhibited platelet aggregation and the expression of P-selectin and GPⅡb/Ⅲa on platelet membrane induced by activated complement.Conclusion Anticomplementary protein atrase B from Naja atra venom can significantly inhibit platelet activation and aggregation induced by activated complement.