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.

Aim To provide practical microassays for screening acetylcholinesterase (AChE) inhibitors in drug discovery.Methods The optimal conditions of assaying the activity of AChEs from electric eel,rat brain homogenate and cobra venom were determined in 96-well plates under physiological pH value and temperature by orthogonal matrix method.The concentrations of AChE,substrate and DTNB,and reaction time were optimized.After the effects of sample solvent (DMSO) used in the assay and stopping reagent on enzyme activity were assessed,the assay conditions were finally selected,and 492 kinds of extracts from Guizhou ethno-drugs were screened.Results Practical microassays for screening AChE inhibitors were successfully constituted by using AChEs mentioned above.The data analysis of screening results revealed that electric eel AChE possessed a high sentivity to inhibitors,and cobra venom AChE shared high similarity with rat brain homogenate in positive results.Conclusion Microassays constituted in this work possessed advantages of being easy,rapid,reliable,cost saving and flexible.AChE from electric eel was especially suitable for screening AChE inhibitors from extracts,and AChE from cobra venom was more suitable to be used in screening AChE inhibitors from large numbers of compounds.

Aim To provide practical microassay for screening butyrylcholinesterase(BChE) inhibitors in drug discovery.Methods The enzyme reaction was optimized in 96-well plates under physiological pH value and temperature by orthogonal matrix method.After the assay conditions were finally selected,3 inhibitors and 115 extracts from Guizhou ethno-drugs were tested.Results A practical microassay for screening BChE inhibitors was successfully constituted by using rat serum as the source of BChE.Conclusion The microassay constituted in this work possess advantages of being practical,rapid,reliable and economical.

Aim To provide practical microassay for screening ?-glucosidase inhibitors in drug discovery.Methods The optimal conditions of assaying the activity of ?-glucosidase were determined in 96-well plates under physiological pH value and temperature by orthogonal matrix method.Reaction time and the concentrations of ?-glucosidase and substrate were optimized.After the effects of sample solvent(DMSO) used in the assay and stopping reagent on enzyme activity were assessed,the assay conditions were finally selected.Then 492 kinds of extracts from Guizhou ethno-drugs were screened.Results Practical and sensitive microassay for screening ?-glucosidase inhibitors was successfully constituted.And in 492 kinds of extracts,145 kinds of samples effectively inhibited the enzyme activity.Conclusion The microassay constituted in this work possesses advantages of being rapid,sensitive,reliable,cost saving,easy and flexible.

OBJECTIVE To investigate the effect of Protobothrops mucrosquamatus venom (PMV) and its fractions on functions of the circulatory system in vitro in order to better understand its toxicity mechanism. METHODS PMV was isolated to three fractions FⅠ, FⅡ and FⅢ with a different molecular mass range by Sephadex G-75 gel filtration chromatography. Platelet rich plasma was adjusted to 3×1011 L-1 by platelet poor plasma. Platelet suspension was incubated with PMV and its fractions 0.03 g.L-1 for 5 min, respectively, and platelet aggregation was determined on an LBY-NJ4 aggregometer. PMV and its fractions 0.05 g.L-1 were preincubated with plasminogen 0.1 U.L-1 for 10 min before chromogenic substrate cleavage activity was measured by endpoint and enzyme kinetics determination. PMV and its fractions 1.0 g.L-1 were incubated with rat plasma for 5 or 30 min, and thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FlB) content were assayed. The microvascular endothelial cells were exposed to PMV and its fractions 10, 50 and 250 mg.L-1 , respectively, for 24 h, while the morphological change was observed using an inverted phase contrast microscope, and the cell viability was determined by MTT method. PMV and its fractions were incubated with guinea pig red blood cell suspension in the presence or absence of lecithin for different time, and hemolysis was measured. RESULTS Compared with normal control, platelet aggregation rate was significantly increased by PMV and FⅠ (>71 ku)〔(12.4±4.1)%,(61.0±5.8)% and (56.9±5.9)%〕(P<0.01). PMV and FⅡ (18-37 ku) significantly hydrolyzed chromogenic substrate S-2251(P<0.01). PMV and FⅠ caused plasma coagulation. Compared with normal control, FⅡ and FⅢ (<10 ku) remarkably prolonged TT, APTT and PT( P<0.01). Morphological observation revealed that PMV, FⅠ and FⅡdetached the adherent cells. Compared with normal control group, PMV, F Ⅰ and F Ⅱ inhibited cell viability, and the survival rate of the cells decreased to (56.8±3.6)%,(71.6±3.8)% and(58.2±5.5)%, respectively. PMV and FⅡ slowly caused slight hemolysis in absence of lecithin. PMV and FⅡ caused significant hemolysis in the presence of lecithin, and the hemolytic rate increased to (81.0±4.0)% and (81.0±1.0)%( P <0.01) in 0.5 min, respectively, compared with (17.7±1.0)% of the control group. CONCLUSION PMV possesses different activities that affect the functions of the circulatory system in vitro, and the fractions play different roles in toxicity mechanisms.

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 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 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 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 study the hemolyzation of a highly active anti complementary protein (cobra venom factor, CVF) isolated from the venom of Naja kaouthia distributed in the south of Yunnan Province, China. METHODS Guinea pig red blood cell and serum were employed to evaluate the hemolyzation of this anticomplement factor, and the effects of temperature, pH, various bivalent mental ions and EDTA on its hemolyzation were investigated. RESULTS This anticomplement factor possessed hemolytic activity of 1 391 kU?g -1 protein. It showed high stability to heat, alkalinity and acidity. Ca 2+ , Mg 2+ , Mn 2+ promoted the hemolyzation of this anticomplement factor at 1, 2 and 5 mmol?L -1 . Zn 2+ , Co 2+ also promoted the hemolyzation of this anticomplement factor at 1 and 2 mmol?L -1 , but inhibited the hemolyzation at 5 mmol?L -1 . Cu 2+ strongly inhibited the hemolyzation at 1, 2 and 5 mmol?L -1 . EDTA also strongly inhibited the hemolyzation of this anticomplement factor. CONCL- USION The anticomplement factor showed hemolytic activity. This ability could be influenced by bivalent metal ions and EDTA, but rather stable when temperature or pH value changed.