Objective: In folk and TCM clinical medicine, Chinese herbal medicine is used to treat snakebite and has good curative effect, but its active ingredients and mechanism are still unclear. In this study, virtual screeni...Objective: In folk and TCM clinical medicine, Chinese herbal medicine is used to treat snakebite and has good curative effect, but its active ingredients and mechanism are still unclear. In this study, virtual screening and mechanism analysis of effective components from 6 Chinese herbs to inhibit phospholipase A2 of Deinagkistrodon acutus (dPLA2) venom were conducted. Methods: With advanced computing software AutoDock, Pymol and GROMACS, the molecules selected from the Chinese herbal Medicine Chemical Composition databas6e (TCMSP) were docked with the dPLA2 from the protein database (PDB). Further molecular dynamics simulation was used to evaluate the molecular binding stability. Results: Four potential dPLA2-inhibiting molecules were screened: lobelanidine, lobeline, norlobelanine and pratensein, by analyzing the spatial structure, binding energy and binding interaction of small molecular-dPLA2 complexes, as well as the RMSD and RMSF of molecular dynamics simulation. Conclusion: To our knowledge, this is the first report of lobeline has an inhibitory effect on dPLA2, and lobelanidine, as a precursor of lobeline, has a stronger inhibitory effect. According to the docking results, it is speculated that the mechanism of action of the four molecules is to form stable interactions with calcium ions and amino acid residues on the calcium ion binding ring in dPLA2. Moreover, these small molecules compete with phosphatidylcholine (the natural substrate of dPLA2) to bind dPLA2 and have a higher affinity than phosphatidylcholine, resulting in inhibition of dPLA2 activity.展开更多
In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-...In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-related variation in electrophoretic profiles was found in both species and proteolytic and fibrinogenolytic activity was higher in neonate than adult venoms. Neonate D. acutus venom had higher 5＇ nucleotidase, PLA2, hyaluronidase and gelatinolytie activity, but lower esterolytic activity, than adult venom. Neonate and adult D. acutus venoms showed identical phosphomonoesterase, LAO and fibrinolytic activities. Neonate N. atra venom had higher phosphomonoesterase and LAO activity, but lower 5＇ nucleotidase, PLA2, hyaluronidase and Ache activities than adult venom. Neonate and adult N. atra venoms showed similar gelatinolytic activity. Further, age-dependent immunoreactivity was found in both species, and cross-reactions between homologous venoms and antiserums were closely related to venom composition. We speculate that age-related variation in venom characteristics is possibly driven by evolutionary forces associated with ontogenetic shifts in dietary habits, competition and predation pressure.展开更多
Background：Snakebites are a neglected threat to global human health with a high morbidity rate.The present study explored the efficacy of antivenom with hyperbaric oxygen （HBO） intervention on snakebites,which coul...Background：Snakebites are a neglected threat to global human health with a high morbidity rate.The present study explored the efficacy of antivenom with hyperbaric oxygen （HBO） intervention on snakebites,which could provide the experimental basis for clinical adjuvant therapy.Methods：Male Sprague-Dawley rats （n =96） were randomized into four groups：the poison model was established by injecting Deinagkistrodon acutus （D.acutus） venom （0.8 LD50） via the caudal vein;the antivenom group was injected immediately with specific antivenom via the caudal vein after successful establishment of the envenomation model;and the antivenom ＋ HBO group was exposed to HBO environment for 1 h once at predetermined periods of 0 h,4 h,12 h,and 23 h after antivenin administration.Each HBO time point had six rats;the control group was left untreated.The rats in the experimental group were euthanized at the corresponding time points after HBO therapy,and brain tissue and blood were harvested immediately.Hematoxylin and eosin （H＆E） staining was used to investigate the pathological changes in the rat brain.Immunohistochemistry （IHC）,real-time polymerase chain reaction （PCR）,and Western blotting were used to detect the expression ofNestin mRNA and protein in the subventricular zone （SVZ） of the brain.The levels of coagulation function （prothrombin time,activated partial thromboplastin time [APTT],and fibrinogen） and oxidation/antioxidation index （malondialdehyde [MDA] and superoxide dismutase [SOD]） were analyzed.Data were analyzed using one-way analysis of variance.Results：The brain tissue from rats in the poison model was observed for pathological changes using H＆E staining.Tissues showed edema,decreased cell number,and disordered arrangement in the SVZ in the snake venom group.The antivenom-HBO intervention significantly alleviated these observations and was more prominent in the antivenom ＋ HBO group.The serum levels of SOD and MDA in the snake venom group were increased and the antivenom-HBO intervention further increased the SOD levels but significantly decreased the MDA levels;however,this was enhanced within 1 h after HBO administration （MDA：F=5.540,P=0.008,SOD：F=7.361,P =0.000）.Activated partial thromboplastin time （APTT） was significantly abnormal after venom administration but improved after antivenom and was even more significant in the antivenom ＋ HBO group 5 h after envenomation （F =25.430,P =0.000）.Only a few nestin-positive cells were observed in the envenomation model.The expression levels were significant in the antivenom and antivenom ＋ HBO groups within 1 and 5 h after envenomation and were more significant in the antivenom ＋ HBO group as determined by IHC,real-time PCR,and Western blotting （P 〈 0.05）.D.acutus envenomation has neurotoxic effects in the brain of rats.Conclusions：Antivenin and HBO,respectively,induced a neuroprotective effect after D.acutus envenomation by attenuating brain edema,upregulating nestin expression in SVZ,and improving coagulopathy and oxidative stress.The intervention efficacy of antivenom with HBO was maximum within 5 h after envenomation and was more efficacious than antivenom alone.展开更多
目的通过实验纯化出特异性尖吻蝮蛇蛇毒多克隆抗体并检测其生物活性。方法以尖吻蝮蛇蛇毒免疫新西兰兔,利用Econo-Pac Protein A kit及CNBr activated sepharose 4B凝胶对兔源性抗蛇毒血清抗体进行纯化,制备特异性尖吻蝮蛇蛇毒多克隆抗...目的通过实验纯化出特异性尖吻蝮蛇蛇毒多克隆抗体并检测其生物活性。方法以尖吻蝮蛇蛇毒免疫新西兰兔,利用Econo-Pac Protein A kit及CNBr activated sepharose 4B凝胶对兔源性抗蛇毒血清抗体进行纯化,制备特异性尖吻蝮蛇蛇毒多克隆抗体,经Western blot和ELISA方法对抗体进行评价,通过出血抑制实验及致死性保护实验评价抗体的生物活性。结果成功纯化出特异性抗尖吻蝮蛇蛇毒多克隆抗体,ELISA检测抗体效价>3 200,Western blot证实多克隆抗体能识别蛇毒中大多数毒素蛋白,出血抑制实验证实该抗体能有效抑制蛇毒所致的皮下出血,在致死性保护实验中,该抗体对2LD50和4LD50蛇毒分别起到100%和54%的保护作用。结论纯化获得的尖吻蝮蛇蛇毒多克隆抗体具有较好特异性和生物活性。展开更多
In this article, we report our first experience of successful artificial propagation about the five paced pit viper ( Deinagkistrodon acutus ) and breeding to its second filial generation. In May, 1994, 18 adult snake...In this article, we report our first experience of successful artificial propagation about the five paced pit viper ( Deinagkistrodon acutus ) and breeding to its second filial generation. In May, 1994, 18 adult snakes (8 males, 10 females) were captured in field, and were reared in man made environment. In July and August of the same year, 5 females laid 134 eggs in total (26 8±5 26), which were artificially incubated into 123 hatchlings. 100 hatchlings (average body weight 12 13±1 50 g)were selected to feed. Three years later, 58 snakes were alive (livability 58%). In September of 1997, April and May of 1998, some of the 58 snakes copulated. From July to August of 1998, 6 females laid 64 eggs in total, 58 of which were fertilized, and 54 were incubated into hatchlings in September of 1998. Therefore, we had successfully bred the second filial generation of the five paced pit viper in complete artificial environment.展开更多
文摘Objective: In folk and TCM clinical medicine, Chinese herbal medicine is used to treat snakebite and has good curative effect, but its active ingredients and mechanism are still unclear. In this study, virtual screening and mechanism analysis of effective components from 6 Chinese herbs to inhibit phospholipase A2 of Deinagkistrodon acutus (dPLA2) venom were conducted. Methods: With advanced computing software AutoDock, Pymol and GROMACS, the molecules selected from the Chinese herbal Medicine Chemical Composition databas6e (TCMSP) were docked with the dPLA2 from the protein database (PDB). Further molecular dynamics simulation was used to evaluate the molecular binding stability. Results: Four potential dPLA2-inhibiting molecules were screened: lobelanidine, lobeline, norlobelanine and pratensein, by analyzing the spatial structure, binding energy and binding interaction of small molecular-dPLA2 complexes, as well as the RMSD and RMSF of molecular dynamics simulation. Conclusion: To our knowledge, this is the first report of lobeline has an inhibitory effect on dPLA2, and lobelanidine, as a precursor of lobeline, has a stronger inhibitory effect. According to the docking results, it is speculated that the mechanism of action of the four molecules is to form stable interactions with calcium ions and amino acid residues on the calcium ion binding ring in dPLA2. Moreover, these small molecules compete with phosphatidylcholine (the natural substrate of dPLA2) to bind dPLA2 and have a higher affinity than phosphatidylcholine, resulting in inhibition of dPLA2 activity.
基金supported by grants from the Natural Science Foundation of China (31101635 and 31272294)Zhejiang Provincial Foundation of Natural Science (Z3090461)+1 种基金Zhejiang Provincial Department of Science and Technology (2009C13045) Hangzhou Department of Science and Technology (20080433T03)
文摘In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-related variation in electrophoretic profiles was found in both species and proteolytic and fibrinogenolytic activity was higher in neonate than adult venoms. Neonate D. acutus venom had higher 5＇ nucleotidase, PLA2, hyaluronidase and gelatinolytie activity, but lower esterolytic activity, than adult venom. Neonate and adult D. acutus venoms showed identical phosphomonoesterase, LAO and fibrinolytic activities. Neonate N. atra venom had higher phosphomonoesterase and LAO activity, but lower 5＇ nucleotidase, PLA2, hyaluronidase and Ache activities than adult venom. Neonate and adult N. atra venoms showed similar gelatinolytic activity. Further, age-dependent immunoreactivity was found in both species, and cross-reactions between homologous venoms and antiserums were closely related to venom composition. We speculate that age-related variation in venom characteristics is possibly driven by evolutionary forces associated with ontogenetic shifts in dietary habits, competition and predation pressure.
基金This study was supported by grants from the Science and Technology Foundation of Guizhou Province （No. SY [2013]3067） and National Natural Science Foundation of China （No. 81560217）.
文摘Background：Snakebites are a neglected threat to global human health with a high morbidity rate.The present study explored the efficacy of antivenom with hyperbaric oxygen （HBO） intervention on snakebites,which could provide the experimental basis for clinical adjuvant therapy.Methods：Male Sprague-Dawley rats （n =96） were randomized into four groups：the poison model was established by injecting Deinagkistrodon acutus （D.acutus） venom （0.8 LD50） via the caudal vein;the antivenom group was injected immediately with specific antivenom via the caudal vein after successful establishment of the envenomation model;and the antivenom ＋ HBO group was exposed to HBO environment for 1 h once at predetermined periods of 0 h,4 h,12 h,and 23 h after antivenin administration.Each HBO time point had six rats;the control group was left untreated.The rats in the experimental group were euthanized at the corresponding time points after HBO therapy,and brain tissue and blood were harvested immediately.Hematoxylin and eosin （H＆E） staining was used to investigate the pathological changes in the rat brain.Immunohistochemistry （IHC）,real-time polymerase chain reaction （PCR）,and Western blotting were used to detect the expression ofNestin mRNA and protein in the subventricular zone （SVZ） of the brain.The levels of coagulation function （prothrombin time,activated partial thromboplastin time [APTT],and fibrinogen） and oxidation/antioxidation index （malondialdehyde [MDA] and superoxide dismutase [SOD]） were analyzed.Data were analyzed using one-way analysis of variance.Results：The brain tissue from rats in the poison model was observed for pathological changes using H＆E staining.Tissues showed edema,decreased cell number,and disordered arrangement in the SVZ in the snake venom group.The antivenom-HBO intervention significantly alleviated these observations and was more prominent in the antivenom ＋ HBO group.The serum levels of SOD and MDA in the snake venom group were increased and the antivenom-HBO intervention further increased the SOD levels but significantly decreased the MDA levels;however,this was enhanced within 1 h after HBO administration （MDA：F=5.540,P=0.008,SOD：F=7.361,P =0.000）.Activated partial thromboplastin time （APTT） was significantly abnormal after venom administration but improved after antivenom and was even more significant in the antivenom ＋ HBO group 5 h after envenomation （F =25.430,P =0.000）.Only a few nestin-positive cells were observed in the envenomation model.The expression levels were significant in the antivenom and antivenom ＋ HBO groups within 1 and 5 h after envenomation and were more significant in the antivenom ＋ HBO group as determined by IHC,real-time PCR,and Western blotting （P 〈 0.05）.D.acutus envenomation has neurotoxic effects in the brain of rats.Conclusions：Antivenin and HBO,respectively,induced a neuroprotective effect after D.acutus envenomation by attenuating brain edema,upregulating nestin expression in SVZ,and improving coagulopathy and oxidative stress.The intervention efficacy of antivenom with HBO was maximum within 5 h after envenomation and was more efficacious than antivenom alone.
文摘目的通过实验纯化出特异性尖吻蝮蛇蛇毒多克隆抗体并检测其生物活性。方法以尖吻蝮蛇蛇毒免疫新西兰兔,利用Econo-Pac Protein A kit及CNBr activated sepharose 4B凝胶对兔源性抗蛇毒血清抗体进行纯化,制备特异性尖吻蝮蛇蛇毒多克隆抗体,经Western blot和ELISA方法对抗体进行评价,通过出血抑制实验及致死性保护实验评价抗体的生物活性。结果成功纯化出特异性抗尖吻蝮蛇蛇毒多克隆抗体,ELISA检测抗体效价>3 200,Western blot证实多克隆抗体能识别蛇毒中大多数毒素蛋白,出血抑制实验证实该抗体能有效抑制蛇毒所致的皮下出血,在致死性保护实验中,该抗体对2LD50和4LD50蛇毒分别起到100%和54%的保护作用。结论纯化获得的尖吻蝮蛇蛇毒多克隆抗体具有较好特异性和生物活性。
文摘In this article, we report our first experience of successful artificial propagation about the five paced pit viper ( Deinagkistrodon acutus ) and breeding to its second filial generation. In May, 1994, 18 adult snakes (8 males, 10 females) were captured in field, and were reared in man made environment. In July and August of the same year, 5 females laid 134 eggs in total (26 8±5 26), which were artificially incubated into 123 hatchlings. 100 hatchlings (average body weight 12 13±1 50 g)were selected to feed. Three years later, 58 snakes were alive (livability 58%). In September of 1997, April and May of 1998, some of the 58 snakes copulated. From July to August of 1998, 6 females laid 64 eggs in total, 58 of which were fertilized, and 54 were incubated into hatchlings in September of 1998. Therefore, we had successfully bred the second filial generation of the five paced pit viper in complete artificial environment.
