Screening and identification of differential metabolites in serum and urine of bamaxiang pigs bitten by trimeresurus stejnegeri based on UPLC-Q-TOF/MS metabolomics technology.

Trimeresurus stejnegeri is one of the top ten venomous snakes in China, and its bite causes acute and severe diseases. Elucidating the metabolic changes of the body caused by Trimeresurus stejnegeri bite will be beneficial to the diagnosis and treatment of snakebite. Thus, an animal pig model of Trimeresurus stejnegeri bite was established, and then the metabolites of serum and urine were subsequently screened and identified in both ESI+ and ESI- modes identified by ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) methods. There are 9 differential metabolites in serum, including Oleic acid, Lithocholic acid, Deoxycholic acid, Hypoxanthine, etc. There are 11 differential metabolites in urine, including Dopamine, Thiocysteine, Arginine, Indoleacetaldehyde, etc. Serum enrichment pathway analysis showed that 5 metabolic pathways, including Tryptophanuria, Liver disease due to cystic fibrosis, Hartnup disease, Hyperbaric oxygen exposure and Biliary cirrhosis, the core metabolites in these pathways, including deoxycholic acid, lithocholic acid, tryptophan and hypoxanthine, changed significantly. Urine enrichment pathway analysis showed that 4 metabolic pathways, including Aromatic L-Amino Acid Decarboxylase, Vitiligo, Blue Diaper Syndrome and Hyperargininemia, the core metabolites in these pathways including dopamine, 5-hydroxyindole acetic acid and arginine. Taken together, the current study has successfully established an animal model of Trimeresurus stejnegeri bite, and identified the metabolic markers and metabolic pathways of Trimeresurus stejnegeri bite. These metabolites and pathways may have potential application value and provide a therapeutic basis for the treatment of Trimeresurus stejnegeri bite.

Uncommon defibrinogenation and coagulopathy caused by Trimeresurus stejnegeri stejnegeri envenomation in a patient with swelling above the ankle.

In Taiwan, Trimeresurus stejnegeri stejnegeri (Stejneger's Bamboo pitviper) is responsible for more than half of all venomous snakebites annually. This species often causes local envenoming characterized by tissue swelling and pain, occasional local ecchymosis, bullae and blister formation, and lymphangitis and lymphadenitis. The pathophysiology and treatment of potentially life-threatening coagulopathy and defibrinogenation induced by T. s. stejnegeri systemic envenoming have not been specifically addressed. Here, we describe the case of a man who was bitten by T. s. stejnegeri on his right first toe, which later developed into swelling above the ankle. It was found that there was severe hypofibrinogenemia, prolonged prothrombin time, and reduced activities of factors V and XI, plasminogen, and α2-antiplasmin. Even though a favorable outcome was achieved after repeatedly administering specific antivenom, fresh frozen plasma, and cryoprecipitate, probably low effectiveness of antivenom against the coagulopathy and prodigious amounts of replacement products were observed. To control coagulopathy early and avoid the needless replacement of coagulation factor, which are associated with inherent adverse reactions, more frequent serial blood assessment (e.g., every 6 h) and higher initial antivenom doses may be helpful. Knowledge of the specific coagulation factor deficiencies may improve our understanding of the relationship between hemotoxins and the resulting envenoming syndromes in this snakebite.

Acute myocardial infarction with no chest pain following a Trimeresurus stejnegeri snakebite: a case report.

A 74-year-old female was bitten by a Trimeresurus stejnegeri, which is an unusual but dangerous type of snakebite. After the snakebite, the patient developed oedema, pain and numbness in the injured limb, and acute myocardial infarction, but no chest pain. The patient received base treatment, including anti-venom serum, statins and wound cleaning. After treatment, the pain in the injured limb disappeared and the swelling decreased. The patient underwent a coronary angiogram the next day, and severe stenosis of the anterior descending branch of the left coronary artery was found. She was given coronary stent implantation. After surgery, she was treated with anticoagulants, and antiplatelet medication and was discharged from the hospital on the sixth day after the condition improved. This case report of myocardial infarction-related snake envenomation aims to increase the awareness that snakebites may cause AMI and therefore, multidisciplinary management particularly from emergency physicians and cardiologists may be necessary.

A guangxi bama minipig model of trimeresurus stejnegeri snakebite: Modeling and evaluation / 中国组织工程研究

BACKGROUND: At present, there are not many research methods on the pathological mechanism of Trimeresurus stejnegeri snakebite, and there are few methods for constructing animal models. OBJECTIVE: To establish and evaluate the Guangxi Bama minipig model for research on the pathological mechanism, diagnosis and treatment of Trimeresurus stejnegeri snakebite. METHODS: Based on the 50% lethal dose of intramuscularly injected Trimeresurus stejnegeri venom to mice, the theoretical 50% lethal venom dose for Bama minipigs was calculated by the equivalent dose coefficient conversion and reduction algorithm, and the body surface area conversion algorithm. Twelve Bama minipigs were randomly divided into a normal group (n=6) and a model group (n=6). The model group was injected intramuscularly with 0.2 mL/kg snake venom 1/3 of the theoretical 50% lethal dose (0.643 mg/kg). The control group was injected with the same amount of normal saline. After snake venom injection, the poisoning symptoms of Bama minipigs were observed. Two groups of animal blood samples were collected before, 6 hours and 24 hours after snake venom injection. Blood routine test, four coagulation items, blood biochemistry and electrolyte were detected. Histopathological changes of the heart, brain, lung, liver, and kidney as well as the injection site were observed by hematoxylin-eosin staining. The study protocol was approved by the Laboratory Animal Ethics Committee (approval No. 201909013). RESULTS AND CONCLUSION: There was swelling at the wound of the piglet accompanied with blood blisters after snake venom injection. The affected limbs and the surrounding area were swollen and spread rapidly to the proximal end. The piglets walked all the time because of the pain, and no animal died during the experiment. Compared with the control group pig, the model group had higher red blood cell count, white blood cell count, alanine aminotransferase, D-dimer count, longer prothrombin time, and lower fibrinogen and platelet count. Hematoxylin-eosin staining showed that, compared with the control group, capillary permeability of piglet lung tissues in the model group was increased with hyperemia and edema. Edema, bleeding, degeneration and necrosis were seen in the muscle tissue on the injection site. No obvious abnormalities in other organs and tissues were observed. To conclude, this method can be used to establish a pig model of Trimeresurus stejnegeri snakebite that can reflect the pathophysiological process of Trimeresurus stejnegeri snakebite. It has operability and repeatability that can be used to study the pathophysiological mechanism of Trimeresurus stejnegeri snakebite.

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan.

BACKGROUND: The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. METHODS: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. RESULTS: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. CONCLUSION: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.

Envenomation by Trimeresurus stejnegeri stejnegeri: clinical manifestations, treatment and associated factors for wound necrosis.

BACKGROUND: Trimeresurus stejnegeri stejnegeri bite induces tissue swelling, pain, thrombocytopenia, rhabdomyolysis, and acute renal failure. However, the incidence of coagulopathy, factors associated with wound necrosis, and the appropriate management of this condition have not been well characterized yet. MATERIALS: This study included patients bitten by T. s. stejnegeri that were admitted to the study hospitals from 2001 to 2016. Patient characteristics, laboratory data, and management approaches were compared in victims with and without wound necrosis. RESULTS: A total of 185 patients were evaluated: three patients (1.6%) were asymptomatic; whereas tissue swelling and pain, local ecchymosis, wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and renal impairment were present in 182, 53, 13, 15, 10, 1, and 3 patients, respectively. One patient died from coagulopathy and hemorrhagic shock. Antivenom was administered to all envenomed patients at a median time of 1.8 h after the bite. The median total dose of antivenom was five vials. Chi-square analysis showed that bitten fingers, using cold packs during first aid, presence of bullae or blisters, lymphangitis or lymphadenitis, local numbness and suspected infection to be significantly associated with wound necrosis. After adjustment using a multivariate logistic regression model, only cold packs as first aid, bulla or blister formation, and wound infection remained significant. CONCLUSIONS: The main effects of T. s. stejnegeri envenomation are tissue swelling, pain, and local ecchymosis. We do not recommend the use of cold packs during first aid to reduce wound pain, as this may be a risk factor for wound necrosis. In addition, patients with bulla or blister formation should be carefully examined for subsequent wound necrosis. Antiplatelet use may worsen systemic bleeding. No severe rhabdomyolysis or renal failure was observed in this large case series, we therefore considered that they were not prominent effects of T. s. stejnegeri bite.

Envenomation by Trimeresurus stejnegeri stejnegeri: clinical manifestations, treatment and associated factors for wound necrosis

Trimeresurus stejnegeri stejnegeri bite induces tissue swelling, pain, thrombocytopenia, rhabdomyolysis, and acute renal failure. However, the incidence of coagulopathy, factors associated with wound necrosis, and the appropriate management of this condition have not been well characterized yet. Materials: This study included patients bitten by T. s. stejnegeri that were admitted to the study hospitals from 2001 to 2016. Patient characteristics, laboratory data, and management approaches were compared in victims with and without wound necrosis. Results: A total of 185 patients were evaluated: three patients (1.6%) were asymptomatic; whereas tissue swelling and pain, local ecchymosis, wound necrosis, coagulopathy, thrombocytopenia, rhabdomyolysis, and renal impairment were present in 182, 53, 13, 15, 10, 1, and 3 patients, respectively. One patient died from coagulopathy and hemorrhagic shock. Antivenom was administered to all envenomed patients at a median time of 1.8 h after the bite. The median total dose of antivenom was five vials. Chi-square analysis showed that bitten fingers, using cold packs during first aid, presence of bullae or blisters, lymphangitis or lymphadenitis, local numbness and suspected infection to be significantly associated with wound necrosis. After adjustment using a multivariate logistic regression model, only cold packs as first aid, bulla or blister formation, and wound infection remained significant. Conclusions: The main effects of T. s. stejnegeri envenomation are tissue swelling, pain, and local ecchymosis. We do not recommend the use of cold packs during first aid to reduce wound pain, as this may be a risk factor for wound necrosis. In addition, patients with bulla or blister formation should be carefully examined for subsequent wound necrosis. Antiplatelet use may worsen systemic bleeding. No severe rhabdomyolysis or renal failure was observed in this large case series, we therefore considered that they were not prominent effects of T. s. stejnegeri bite.(AU)

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

The influence of purging fire and removing toxin on chemokines and adhesion factors related to inflammation induced by trimeresurus stejnegeri bites / 中国中西医结合急救杂志

Objective To investigate the influence of the purging fire and removing toxin method on chemokines and adhesion factors related to vascular endothelialitis injury induced by toxin of trimeresurus stejnegeri bite.Methods ① Animal experiment:50 healthy New Zealand white rabbits were chosen.According to random numbers generated by statistical software,they were divided into normal control group,model group,low,middle and high dose Sheshang capsule groups,10 in each group.Trimeresurus stejnegeri bite model was replicated by injecting 0.75 mL/kg snake venom into subcutaneous tissues of rabbits' right hind legs.And the same volume of normal saline was injected into the rabbit in the normal control group.After the model was established for 6 hours,the rabbits in low,middle and high dose Sheshang capsule groups received 174,348 and 522 mg· kg-1 · d-1 of Sheshang capsule solution respectively (the content of capsules was dissolved in normal saline to make liquid with 17.4,34.8 and 52.2 g/L Sheshang solution respectively,so the volume of gavage of each group was 10 mL· kg-1 · d-1);in the model and normal control groups,the same amount of normal saline was given by gavage,once daily for consecutive one week.24 hours after the last gavage,the blood of the rabbits was collected through an auricular border vein and the serum was separated by centrifuge ready for use.Meanwhile,the whole abdominal aorta segment of the rabbit was harvested and kept them in liquid nitrogen ready for use.② Cell experiment:human umbilical vascular endothelial cell (HUVEC) was cultured with MEM for 24 hours.The solution was replaced and according to the random number generated by statistical software,the cells were divided into blank control group,model group and low,middle,high dose Sheshang capsule medicinal serum groups,10 wells in each group.Trimeresurus stejnegeri toxin cell model was reproduced by addition of 5 mg/L snake venom into the cell culture medium.After 6-hour culture,the cells of model group and blank control group received 10％ normal rabbit serum,and the cells of low,middle and high dose Sheshang medicinal serum capsule groups received serum containing 5％,10％ and 15％ drug,respectively.After culture for 72 hours,the cells were collected and the total RNA was extracted.The real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to detect the levels of mRNA of interleukin-8 (IL-8),monocyte chemoattractant protein-1 (MCP-1),intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial cell adhesion molecule-1 (VCAM-1) in the vascular endothelial cells of rabbit aorta abdominalis and human umbilical vein,and the content of serum E-select element (CD62E) was measured by enzyme linked immunosorbent assay (ELISA).Results In model group,the expression levels of mRNA in IL-8,MCP-1,ICAM-1,VCAM-1 and the content of CD62E were all increased significantly in the endothelial cells of rabbit aorta abdominalis and HUVEC compared with those in control group [when the mRNA expression levels of IL-8,MCP-1,ICAM-1 and VCAM-1 in normal and blank control group were all being 1,the mRNA expression levels (2-△ △Ct) of the above mentioned inflammatory factors and adhesion molecule in animal model group were 3.96 ± 0.39,3.07 ± 0.27,3.71 ± 0.26,3.94 ± 0.26,and the mRNA expression levels (2-△ △Ct) of the above mentioned inflammatory factors and adhesion molecule in HUVEC model group were 3.53±0.70,2.24±0.48,3.13±0.44,2.80±0.13,respectively,all P ＜ 0.01].The content of CD62E in serum was increased significantly in model group compared with that in normal control group (μg/L:1.31 ± 0.22 vs.0.82 ± 0.13,P ＜ 0.01),the mRNA expression levels of IL-8,MCP-1,ICAM-1 and VCAM-1 were decreased significantly in low,middle,high dose Sheshang capsule groups compared with those in model group in endothelial cells of aorta abdominalis of rabbits and HUVEC [abdominal aorta:IL-8 mRNA (2-△ △Ct) were 1.13 ± 0.19,1.26 ± 0.16,1.27 ± 0.17 vs.3.96 ± 0.39,MCP-1 mRNA (2-△ △ Ct) were 1.79 ± 0.24,2.22 ± 0.38,1.76±0.19 vs.3.07±0.27,ICAM-1 mRNA (2 △△Ct) were 2.05±0.11,1.68±0.09,2.37±0.48 vs.3.71±0.26,VCAM-1 mRNA (2-△△Ct) were 1.59±0.08,1.40±0.11,1.84±0.11 vs.3.94±0.26;HUVEC:IL-8 mRNA (2-△△Ct) were 2.33±0.59,2.82±0.82,2.51±0.77 vs.3.53±0.70,MCP-1 mRNA (2-△△Ct) were 1.59±0.35,1.48±0.36,1.54±0.29 vs.2.24±0.48,ICAM-1 mRNA (2-△△Ct) were 1.46±0.38,1.77±0.65,1.73±0.50 vs.3.13±0.44,VCAM-1 mRNA (2-△△Ct) were 2.49±0.24,2.18±0.19,2.45±0.24 vs.2.80±0.13,all P ＜ 0.05].The contents of CD62E were decreased significantly in middle,high dose Sheshang capsule groups compared with the content in model group (μg/L:1.01 ±0.14,1.04±0.13 vs.1.31 ±0.22,all P ＜ 0.01),but there were no statistical significant differences among the three drug group (all P ＞ 0.05).Conclusion The therapy of purging fire and removing toxin can treat vascular endothelial injury by inhibiting the inflammatory response induced by Trimeresurus stejnegeri bites.

A study on effects of Sheshang capsule on blood coagulation function of patients bitten by Trimeresurus stejnegeri snake / 中国中西医结合急救杂志

Objective To investigate the mechanism of traditional Chinese medicine (TCM) Sheshang capsule for treatment of blood coagulation dysfunction in patients bitten by Trimeresurus stejnegeri snake. Methods A prospective study was conducted. Seventy Trimeresurus stejnegeri snake envenoming patients whose manifestations conformed to the diagnostic criteria of the fire toxin syndrome in TCM were assigned into therapy group and control group by random number table (each, 35 cases). The basic treatments (including wound disinfection, intramuscular injection of 1 500 U tetanus antitoxin, conventional dose of antibiotics, 10 mg dexamethasone, 40 mg omeprazole) and 10 Jidesheng Sheyao tablets three times a day were applied in the control group. In the therapy group, the basic treatments the same as those of the control group were given, and in the mean time 5 Sheshang capsules (the drug was prepared in our hospital including ingredients:rhubarb, coptidis rhizoma, pleione bulbocodioides, elecampane inula root, bayberry bark, borneol and so on) were administered three times a day. The therapeutic course in the two groups was 1 week. The levels of platelet α-granule membrane protein (CD62p), thromboxane B2 (TXB2), platelet factor 3 (PF3) and von Willebrand factor (vWF) in serum were measured by enzyme linked immunosorbent assay (ELISA) before and after treatment. Results Before treatment, there were no significant differences in CD62p, TXB2, PF3 and vWF between therapy group and control group [CD62p (μg/L):3.81±1.64 vs. 3.52±1.43, TXB2 (μg/L):13.04±1.67 vs. 13.31±1.14, PF3 (μg/L): 2.84±1.08 vs. 2.88±1.23, vWF (μg/L):12.36±2.42 vs. 11.89±2.08, all P>0.05]. After treatment, the levels of CD62p, TXB2 and PF3 were increased, while vWF decreased compared with those before treatment in both groups, the level changes in therapy group being more remarkable [CD62p (μg/L): 6.73±1.77 vs. 5.81±1.62, TXB2 (μg/L):18.65±1.77 vs. 17.90±1.68, PF3 (μg/L):5.61±1.48 vs. 4.77±1.24, vWF (μg/L):3.87±1.01 vs. 4.58±1.09, P < 0.05 or P < 0.01]. Conclusion The Sheshang capsule is capable of treating patients with blood coagulative disorder after Trimeresurus stejnegeri snake bite, and its mechanism is possibly related to the improvement of platelet activation function and amelioration of the damage of vascular endothelial cells.