| Background and Purpose:The bamboo leaf green snake(Trimeresurus stejnegeri)is one of the top ten poisonous snakes in China.Its venom mainly contains blood circulation.Guangxi is located in the south of China with a subtropical climate and a large number of snake species.According to the study of Wang Naiping et al.,among the snake wounds in Guangxi,the bamboo leaf green snake wound ranks the second(20.2%)and is the main snake species of snake wound.Bamboo leaf green snake bites are mainly found in urban garden grass,fields and remote forests,etc.Clinically,the injuries can be classified into light,heavy and critical types,which mainly cause blood dysfunction,limb swelling and even DIC-like injuries,while severe ones will cause dysfunction or failure of multiple organs and eventually lead to death.At present,due to the lack of a good animal model,it is not possible to continuously observe the physiological and pathological process of bamboo leaf snake after its bite,which makes it difficult to further study its pathological mechanism.Therefore,in this study,Bama small pig of Guangxi was used as the animal model of bamboo leaf snake bite to establish a rational,safe,continuous and stable animal model to solve the problem that there is no large animal model of bamboo leaf snake bite at present.The establishment of the porcine model of bamboo leaf snake bite can be used to continuously dynamically detect the changes of blood indexes,study the pathological mechanism of bamboo leaf snake bite,analyze the differential metabolites in serum and urine by metabonomics method,and screen the biomarkers that can be used to identify the species of snake bite and assist the diagnosis.Metabonomics is one of the hot topics in the field of medicine and pharmacy in recent years.It can directly reflect the changes of metabolites in organisms and explain the composition characteristics and change rules of metabolites under the physiological and pathological conditions of the body.It has been widely used in pharmacological toxicology,disease diagnosis,nutrition and food science and other important fields.In this study,a model of bamboo leaf bite was established,and the metabolic markers in serum and urine of bamboo leaf bite were analyzed and screened by ultra-performance liquid chromatography-quadrupole-time-of flight tandem mass spectrometry(UPLC-Q-TOF-MS),in order to provide reliable and effective clinical diagnostic markers for bamboo leaf bite.It laid a foundation for the precise treatment of bamboo leaf snake bite.Methods:1.The animal model of Bama miniature pig bitten by Trimeresurus stejnegeri was establishedThe theoretical 50% lethal dose of Trimeresurus stejnegeri venom to Ba miniature pig was calculated by equivalent dose coefficient conversion method and body surface area conversion method based on LD50 value of intramuscular injection of Trimeresurus stejnegeri venom to mice.Twelve Bama miniature pigs were randomly divided into normal group(n = 6)and model group(n = 6).The model group was injected with 0.2ml/kg intramuscular injection of Trimeresurus stejnegeri venom to Bama miniature pigs(1/3 of the theoretical lethal half dose),while the control group was injected with the same amount of normal saline.After injecting venom watch Obama small pig poisoning symptoms,before the snake venom injection,injection of snake venom section3-72 hours after collecting blood,two groups of animals testing routine blood,and clotting four,blood biochemical and electrolyte,and collection of urine in both groups,by HE staining to observe the heart,brain,lung,liver,kidney,and the injection site muscle pathological changes.2.Nontargeted metabolomics analysis of serum and urine in pigs bitten by the Trimeresurus stejnegeriUPLC-QTOF-MS/MS was used to analyze the metabolic profiles of serum and urine,including the dynamic changes of organic acids,phospholipids,amino acids,etc.at time points in the pigs of the two groups:The first step is to select the blood and urine at a certain time point to carry out the liquid-mass spectrometry test according to the basis of the change of blood index detection results.Serum samples were treated with cold acetonitrile deproteinization method,and urine samples were treated with cold methanol.Then,the metabolic profile data at the time point of the bite was obtained by liquid-mass spectrometry analysis of the two treated samples.The second step is to conduct multivariate statistical analysis on the obtained data.First through the principal component analysis(PCA)method to the overall data analysis,get together and separation of trend,then through supervised OPLS – DA method and the Change ratio(a Fold Change)method,screening compared with control group differences potential biomarkers of ions,the structure identification of the potential difference between ions.Thirdly,the precise mass number of potential differential ion mass spectrometry obtained was compared with MEDLIN database and KEGG,and the potential metabolic markers of bamboo snake bite in serum and urine were identified in combination with ion cleavage rule and standard substance verification.Results:1.Piglet model bitten by Trimeresurus stejnegeri: Piglets in the model group showed swelling at the wound site accompanied by blood blisters,redness and swelling in the affected limbs and surrounding areas,which spread rapidly to the proximal end.Piglets often kept walking due to unbearable pain,and no animals died during the experiment.Compared with the control group,the piglets in model group had higher red blood cell count,higher white blood cell count,lower platelet count,higher prothrombin time,lower fibrinogen,higher ALT,and higher D-dimer.Pathological routine H-E staining showed that compared with the control group,the pulmonary capillary permeability of piglets in the model group was increased,and there was congestion and edema.The changes of edema,hemorrhage,degeneration and necrosis were observed in the muscle tissue at the injection site.No obvious abnormalities were observed in other organs and tissues.This method can well establish the animal model of piglets bitten by bamboo snake,and can reflect the pathophysiological process of bamboo snake bite after 3-24 hours.It has good operability and repeatability,and can be used to study the pathophysiological mechanism of bamboo snake bite.2.Results of untargeted metabonomics analysis: 685 chromatographic peaks were obtained in serum UPLC-Q-TOF-MS positive ion mode,and 528 chromatographic peaks were obtained in negative ion mode.In urine,651 chromatographic peaks were obtained under the positive ion mode of UPLC-Q-TOF-MS,and 640 peaks were obtained under the negative ion mode.The reliability analysis of the data showed that PCA,PLS-DA and OPLS-DA could distinguish the serum samples of the two groups well.PCA showed better separation degree and clustering between the two groups in positive and negative ion modes.The OPLS-DA analysis could accurately separate the two groups of samples,indicating that the differences in the data of the group were mainly caused by the differences between groups.The OPLS-DA model was tested for 7 cycles of cross-validation,and the results excluded the random effect of the data in the model.Combined with the univariate analysis of multiple of variation analysis and T test and the results of R language volcano map,it is more intuitive to show that there are significant changes in metabolites between the two samples.3.Screening results of differential metabolites: Serum mass spectrometry showed that 685 compounds were identified in positive ion mode,and 367 differential metabolites were screened out.A total of 528 compounds were identified in negative ion mode and 277 differential metabolites were screened out.Nine metabolites differed in both positive and negative serum modes,including 2-acyl-1-(1-alkenyl)-Sn-glycero-3-phosphate,Lithocholyltaurine,Oleic acid,Lithocholic acid,Deoxycholic acid,Hypoxanthine,Phenylacetaldehyde,gama-linolenicacid,Tryptophan,respectively.Urine mass spectrometry showed that 651 compounds were identified under positive ion mode,and 292 differential metabolites were screened out.640 compounds were identified in negative ion mode,and 326 differential metabolites were screened out.There were 11 metabolite differences in both positive and negative serum patterns,which were 3-methoxy-4-hydroxyphenylglycol glucuronide,3,4-benzopyrene,Thiocysteine,Arginine,Indoleacetaldehyde,5-hydroxyindoleacid,D-glucurono-6,3-lactone,respectively.5-acetamidovalerate,Dihydrolipoamide,Dehydroepiandrosterone sulfate.4.Bioinformatics analysis results of differential metabolites: KEGG analysis found that the different metabolites of piglet bite by Trimeresurus stejnegeri were mainly involved in 5 metabolic pathways,including Liver disease due to cystic fibrosis,Hyperbaric oxygen exposure,Biliary cirrhosis,Tryptophanuria and Hartnup disease.The contents of the core metabolites deoxycholic acid,lysocholic acid,tryptophan and hypoxanthine in these pathways were significantly changed in the bite of green snake.There was statistical significance in urine Aromatic L-amino Acid Decarboxylase,Vitiligo,Blue Diaper Syndrome and Hyperargininemia,and the core metabolites in these pathways were dopamine,5-hydroxyindoleacetic Acid and arginine.5.Targeted metabolomic analysis results: Trimeresurus stejnegeri bites the differences in serum and urine metabolites of deoxycholic acid,tryptophan,arginine absolute quantitative validation of targeted metabolomic analysis showed that deoxycholic acid,tryptophan,arginine respectively in Trimeresurus stejnegeri bites variation of serum and urine and the targeted metabolomic analysis,and statistical differences(P < 0.05),is expected to become the Trimeresurus stejnegeri bites.Conclusion:1.This study explored for the first time the method of constructing the bite model of large animal of Trimeresurus stejnegeri.In order to provide a reliable animal model for the study of the pathophysiological mechanism and drug treatment of Trimeresurus stejnegeri bite,an animal model with clinical characteristics of Trimeresurus stejnegeri bite was established based on the Bama Miniature pig in Guangxi.2.In this study,UPLC-Q-TOF-MS non-targeted metabonomic method was used to comprehensively analyze the overall metabolic profile of blood and urine after Trimeresurus stejnegeri bite.After screening and identification,three core metabolites,deoxycholic acid(serum),tryptophan(serum)and arginine(urine),were found to be possible markers of Trimeresurus stejnegeri bite.In the absolute quantitative verification studies of targeted metabolomics,the quantitative verification results of deoxycholic acid(serum),tryptophan(serum)and arginine(urine)were consistent with those of non-targeted studies,which are expected to be markers for the bite of Apicorphias bamboides,and can further study the physiological and pathological mechanism of Apicorphias bamboides bites.3.The differences of metabolites involved in the metabolic pathways of research found that Trimeresurus stejnegeri bites can affect metabolic disorders,respiratory disorders,liver blood protein metabolism and absorption barriers serum five important metabolic pathways,such as the core of these pathways and tryptophan metabolites deoxycholic acid content in bamboo leaves a significant change occurred in the green snake bites.The dopamine and tryptophan pathways detected in urine correspond to neurotransmitter disorders and urea circulation disorders. |
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