Agkistrodon halys venom antitumor component-I inhibits vasculogenic mimicry in triple-negative breast cancer cells in vitro by down-regulating MMP2 / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of agkistrodon halys venom antitumor component-I (AHVAC-I) on vasculogenic mimicry (VM) formation in triple-negative breast cancer MDA-MB-231 cells and explore its possible mechanism.@*METHODS@#CCK8 assay was used to determine the optimal concentration of AHVAC-I for cell treatment based on its halfinhibitory concentration (IC50). MDA-MB-231 cells were treated with different concentrations of AHVAC-I or 5-Fu, and the changes in vasomimetic capacity of the cells were examined using Matrigel assay. The expression levels of matrix metalloproteinase-2 (MMP2) and MMP9 in the treated cells were detected using quantitative PCR and Western blotting.@*RESULTS@#Compared with the control treatment with culture medium, treatment with 5, 10 and 20 μg/mL AHVAC-I significantly reduced vasomimetic ability of MDA-MB-231 cells in a dose-dependent manner (P < 0.01). MMP2 supplementation obviously restored the vasomimetic ability of the cells inhibited by AHVAC-I.@*CONCLUSION@#AHVAC-I inhibits VM formation in triplenegative breast cancer cells in vitro by down-regulating MMP2 production.

AHVAC-I reverses tumor growth of cancer-associated fibroblasats in gastric cancer / 中国临床药理学与治疗学

AIM: To explore whether Agkistrodon Halys venom antitumor component-I (AHVAC-I) affects the migration of gastric cancer cells by human primary gastric cancer-associated fibroblast (GCAFs). METHODS: Tissue block culture and trypsin digestion were used to separate and culture human primary gastric cancer-associated fibroblasts (GCAFs); the GCAFs-CM

Agkistrodon halys venom platelet inhibitor inhibits GPVI expression and changes hemorheology in rats with acute myocardial ischemia reperfusion injury / 中国临床药理学与治疗学

AIM:To explore effect and meaning of Agkistrodon halys venom platelet inhibitor on GPVI expression and hemorheology in rats with acute myocardial ischemia reperfusion injury. METHODS: Thirty matched SD male rats were randomly divided into sham operation group (6 rats), myocardial ischemia-reperfusion injury(MIRI) model group(6 rats) and agkistrodon halys venom platelet inhibitor (AHV-PI) group(18 rats). The AHV-PI experimental group was divided into low, middle and high dose groups according to the dose of AHV-PI injected into sublingual vein (0.05, 0.1, 0.2 mg/kg), with 6 rats in each group. Electrocardiogram(ECG) changes of rats were monitored by RM6240 biological signal collection and processing system. Western blot was used to monitor the expression of platelet membrane glycoprotein VI (GPVI) in rats under the intervention of AHV-PI. Blood coagulation time (R), blood clots forming time (K), Alpha Angle (A), and maximum amplitude of blood coagulation (MA) were assayed by Thrombelastography (TEG5000).Platelet aggregation rate was measured by turbidimetry. RESULTS:Compared with MIRI model group, the expression level of GPVI AHV-PI medium dose experimental group and high dose experimental group were significantly decreased (P<0.05), and the R and K values were significantly prolonged, while the A value and MA values were significantly decreased (P<0.05). Platelet aggregation time, aggregation amplitude and aggregation rate were significantly decreased (P<0.05). CONCLUSION: AHV-PI can significantly inhibit the expression of GPVI in MIRI rats and improve the hemorheological properties related to myocardial injury in rats, thus weakening the injury process.

Study on the mechanism of AHV-PI on platelet aggregation by PI3K/Akt signal pathway / 中国生化药物杂志

Objective To investigate the mechanism of platelet inhibitor from Agkistrodon halys venom (AHV-PI) on platelet aggregation. Methods Protein kinase Akt phosphorylation levels in platelet were measured by Western blot. XS-1000I blood cell counter was used for platelet count. The plasma content of 5'-NT and platelet membrane GPIb were determined by Enzyme-Linked Immunosorbnent Assay (ELISA). The effect of AHV-PI on binding rate between the fluorescence labeled monoclonal antibody CD61 (FITC-CD61) and platelet membrae glycoprotein Ⅱb/Ⅲa (GPⅡb/Ⅲa) was observed by flow cytometry (FCM). Results AHV-PI can reduce the level of Akt-phosphorylation level and the number of platelet. AHV-PI can increase the content of 5'-NT in plasma, reduce the expression of platelet GPIb. Flow cytometry displayed AHV-PI can not affect the rate of combination between platelet GPⅡb/Ⅲa and FITC-CD61. Conclusion The mechanism of inhibition of platelet aggregation may be inhibit protein kinase Akt phosphorylation to block the signal transduction pathway of Akt. Limit cell grouth and reduce platelet number, also it may be related to its 5'-NT activity, it can degradate ADP to prevent the formation of platelet thrombus.

Acute liver injury and expression of P-selectin in serum of mice induced by agkistrodon halys pallas venom / 中华急诊医学杂志

Objective To explore the relationship between pathological changes of liver,serum ALT,expression of P-selectin in serum of mice and different dose of venom,the poisoning time induced by Agkistrodon Halys Pallas venom.Methods Using two -factor factorial design,72 male Kunming mice of clean grade were randomly ( random number) divided into 9 groups (n =8).As per the different doses of snake venom injected and different lengths of time after venom injected,the 9 groups were 0-dose (0.0 mg/kg) 3 hours after saline given group,0-dose 8 hours after saline given group,0-dose 24 hours after saline given group,low-dose (0.5 mg/kg) 3 hours after venom given group,low-dose 8 hours after venom given group,low-dose 24 hours after venom given,high-dose ( 1.0 mg/kg) 3 hours after venom given group,high-dose 8 hours after venom given group and high-dose 24 hours after venom given group.The pathological change of liver tissue was determined,and ALT and expression of P-selectin in serum were detected.Results In the venom given groups,the liver histopathologic scores,serum ALT value and expression of Pselectin in serum were significantly higher than those in control group ( P ＜ 0.05 ),and those in high-dose group were higher than those in low-dose group (P ＜ 0.05 ),and those in 8h groups were higher than those in 3h groups (P＜0.05),and those in 24 h groups were higher than those in 8 h groups (P ＜0.05).As the dose of venom increased and the length of time after venom given extended,the liver histopathologic scores,serum ALT and expression of P-selectin in serum were higher.A positive correlation between Pselectin expression and liver histopathologic scores was found after correlation analysis ( r =0.98,P ＜0.05 ).Conclusions Agkistrodon snake venom can cause acute liver injury with elevated ALT and Pselectin in mice.As higher dose of venom given and longer length of time after venom given extended,more serum ALT and P-selectin are produced.Between acute liver injury and high expression of P-selectin exists a correlation,suggesting overexpression of P-selectin is one of the mechanisms of acute liver injury induced by Agkistrodon Halys Pallas venom.

Identification and partial purification of an anticoagulant factor from the venom of the Iranian snake Agkistrodon halys

An anticoagulant factor was purified from the venom of the Iranian snake Agkistrodon halys by gel filtration on Sephadex G-50 and ion-exchange chromatography on DEAE-Sepharose. In the final stage of purification, the percentage recovery of purified anticoagulant factor was found to be 83 percent. The purified anticoagulant factor revealed a single protein band in SDS-polyacrylamide electrophoresis under reducing conditions and its molecular weight was about 22 kDa. The purified peptide did not show any effect on casein, BApNA or plasma.(AU)

Purification and partial characterization of a coagulant serine protease from the venom of the Iranian snake Agkistrodon halys

Agkistrodon halys is one of several dangerous snake species in Iran. Among the most important signs and symptoms in patients envenomated by this snake is disseminated intravascular coagulation. A thrombin-like enzyme, called AH143, was isolated from Agkistrodon halys venom by gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a DEAE-Sepharose and high performance liquid chromatography (HPLC) on a C18 column. In the final stage of purification, 0.82 mg of purified enzyme was obtained from 182.5 mg of venom. The purified enzyme showed a single protein band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions, and its molecular mass was found to be about 30 kDa. AH143 revealed clotting activity in human plasma, which was not inhibited by EDTA or heparin. This enzyme still demonstrated coagulation activity when exposed to variations in temperature and pH ranging, respectively, from 30 to 40°C and from 7.0 to 8.0. It also displayed proteolytic activities on synthetic substrate. The purified enzyme did not show any effect on casein. We concluded that the venom of the Iranian snake Agkistrodon halys contains about 0.45 percent single procoagulant protein which appears to be a thrombin-like enzyme.(AU)

Isolation and Purification of Thrombin-like Enzyme from the Venom of Agkistrodon halys Pallas / 中国药房

OBJECTIVE:To study a simple method for the isolation and purification of thrombin-like enzyme (TLE) from the venom of Agkistrodon halys Pallas. METHODS:DEAE-Sephadex A-25 vs. Sephadex G-25 chromatography in the isolation and purification of TLE from the venom of Agkistrodon halys Pallas was analyzed. RESULTS:TLE was isolated from the venom of Agkistrodon halys Pallas. SDS-PAGE electrophoresis appeared as a strap with its molecular weight at about 35.5 kDa, meeting electrophoresis purity standard. Physical-chemical character assay showed that the TLE has hemostasia activity in vitro with its specific activity at about 12.57IU?mg-1, while the arginine esterase activity was about 137.65IU?mg-1 measured by BAEE method. PMSF and EDTA were used in the inhibition experiment on this enzyme, and the enzyme was proved to be serine protease other than metalloproteinase. CONCLUSION:The methods can be used for the isolation and purification of thrombin-like enzyme (TLE) from the venom of Agkistrodon halys Pallas.

Apoptosis of human leukemia K562 cells induced by snake venom of Agkistrodon Halys Pallas in Zhejiang Province / 中华急诊医学杂志

Objective To study the apoptosis of human leukemia K562 cells induced by snake venom of Agkistrodon Halys Pallas in Zhejiang Province.Methods IC_ 50 value and cytotoxity of K562 cell were detected by MTT method.Apoptotic cells were dyed by Hoechest 33258.Sub-G1 peak and cell cycle were detected by FCM.Protein expression of Bcl-2 gene was detected by FCM and western-blot method.Results The snake venom of Agkistrodon Halys Pallas in Zhejiang Province inhibited the growth of K562 cells,which appeared dose-dependent.The snake venom induced apoptosis of K562 cells.Meanwhile,protein expression of Bcl-2 was down-regulated.Conclusion Snake venom of Agkistrodon Halys Pallas in Zhejiang could induce apoptosis of human leukemia K562 cells.The mechanism may be related to down-regulation of Bcl-2 gene expression.

Standardization of an antihemorrhagic potency test of antivenoms prepared from two different Agkistrodon halys venoms

To establish Korea National Standards for venoms and antivenoms, it is necessary to have standardized assay methods. In this study, we standardized a method to evaluate the antihemorrhagic potency of two horse-derived antivenoms using rabbit intracutaneous injection. We expressed the capability of these antivenoms to neutralize the hemorrhagic activities triggered by the venoms of Agkistrodon halys from Japan and Jiangzhe Agkistrodon halys from China as Minimum Hemorrhagic Dose (MHD). We also performed cross-neutralization tests employing the parallel line assay on different pairings of venoms and antivenoms to check the possibility of using Jiangzhe Agkistrodon halys venom as a substitute for the standard Agkistrodon halys venom in measurements of the antihemorrhagic activity, since A. halys venom is not easily available. Slope function ratio (S.R.) was 0.957 for Agkistrodon halys venom either with Agkistrodon halys antivenom or with Jiangzhe Agkistrodon halys antivenom (p>0.05). Similarly, S.R. was 0.348 for Jiangzhe Agkistrodon halys venom either with Agkistrodon halys antivenom or with Jiangzhe Agkistrodon halys antivenom (p>0.05). Thus, in this study we established antihemorrhagic potency test methods for both Agkistrodon halys and Jiangzhe Agkistrodon halys antivenoms and we could also show it is possible to use Jiangzhe Agkistrodon halys venom as a standard.

Study on quality assay of agkistrodon halys's crude venom / 中成药

Objective: To establish a method for controlling the quality of Agkistrodon halys's crude venom. Methods: The method of PAGE was carried out to identificate the Agkistrodon halys' crude venom, And the protein of crude venom was also assayed by Lowry's method. Results: The Agkistrodon halys's crude venom could be identified by the method of PAGE. The determintion of protein of crude venom showed a good linear relationship in a concentration range of 25 ?g?mL -1 ～250?g?mL -1 , the regression equation was Y=0.0147+ 1.4553X , and the correlation coefficient was 0.9992. The average recovery of the content reached 100.09, RSD=1.765(n=6). Conclusion: The method is simple, feasible, accurate and available, can be used to controlling of the quality of Agkistrodon halys's crude venom.