| Background and objective:Snake bites(snake injuries)are listed by the World Health Organization as one of the most neglected and important tropical/subtropical diseases.In China,the number of patients with snakebite is up to 300000 every year.Chinese cobra bites are one of the most common snake bites in southern China,ranking the second in the incidence of snake bites,and the first in Guangxi.and Clinically acute.The clinical manifestations of Chinese cobra bites are very prone to local tissue edema or necrosis or even extensive muscle necrosis.In severe cases,it can cause disability or amputation.At present,due to the lack of good animal models,little is known about the pathophysiological changes in organisms caused by cobra bites.It brings a lot of difficulties to treatment.Therefore,in this study,Guangxi Bama miniature pig was used as the animal model of cobra bite,First,establish a reasonable,convenient,safe and stable animal model to solve the problem of no large animal model of cobra bite;Then,non-targeted and targeted metabolomics methods were used to screen,identify and verify the serum metabolite profile and differential metabolites of cobra bites,and combined with the serum samples of clinical patients with cobra bites to perform clinical verification of differential metabolites.In order to reveal the pathological mechanism of Chinese cobra bites from the perspective of metabolite molecules,explore the complex interaction between snake wound poisoning and body response,and provide the corresponding molecular pathology theoretical basis and potential for clinical treatment of Chinese cobra bites Target of treatment.Methods:1.Construction of animal model of Chinese cobra biteTaking Bama miniature pigs as the research object,the cobra venom was injected into the muscles of the outer legs of the piglets.By observing the biological behavior of the pigs,pathological examinations and related blood biochemical indicators,the constructed model was comprehensively judged whether it met the research requirements.And explore the optimal dose of snake venom for modeling.2.Analysis of non-targeted metabolomicsTwenty pigs were selected to be divided into a snake venom group and a control group.The snake venom group was injected with the optimal dose of snake venom for modeling,and pig serum was collected for metabolomics research experiments.UPLC-Q-TOF-MS non-targeted metabolomics method was used to analyze and identify the serum metabolites of the pigs with snake injury poisoning.Meanwhile,multivariate statistical analysis such as OPLS-DA and univariate statistical analysis were performed to screen the differential metabolites.Using bioinformatics methods,cluster analysis,correlation analysis and KEGG pathway analysis were performed on the selected differential metabolites.3.Targeted metabolomics analysisBased on non-targeted metabolomics results and combined with literature,Select key differential metabolites for targeted metabolomics analysis,First,formulate the standard curve of the differential metabolite standards to determine the quantitative results;Then perform targeted metabolomics analysis on the key serum metabolites of poisoned pigs,compare the relevant parameters of the analyte with the corresponding standard curve,and obtain quantitative results,Statistical analysis was performed using t test to verify the reliability of differential metabolites.4.Clinical validation of key metabolitesTargeted metabolomics was used to verify the key metabolites obtained from pig serum in clinical serum samples.Take patient serum and normal human serum for targeted metabolomics analysis,compare the result parameters with the corresponding standard curve,and obtain the final quantitative results of key metabolites.The t-test was used for statistical analysis of the key metabolite content of serum samples to verify the reliability and stability of the key metabolite.Result:1.Successfully constructed an animal model of Chinese cobra biting a pigletThe experimental results showed that the piglets had neurotoxin poisoning30 minutes after injection of 0.75mg/kg snake venom,and cytotoxin poisoning occurred about 3 hours later,which was consistent with clinical cobra bite poisoning symptoms;The blood indicators showed that compared with the control group,the piglets in the snake venom group had a significant increase in WBC,hs-CRP,LDH,and CK-MB within 12 hours of the snake venom injection;The histopathological staining results showed that the nucleus of muscle tissue of the piglets in the snake venom group was lysed,and the cytoplasm showed irregularly arranged muscle necrosis;myocardial cells were mostly turbid and swollen,with varying degrees of deformation,necrosis,and apoptosis;liver tissue cells had bleeding Phenomenon;nerve cells in the brain tissue swell.The results of behavioral,pathological and blood indicators showed that the pathological course of the piglets was in line with the clinical manifestations of cobra wound poisoning,and the constructed animal model of cobra bites met the requirements.2.Through the UPLC-Q-TOF-MS system to collect metabolite information in 20 serum samples,5045 chromatographic peaks were obtained in positive ion mode,and 3,871 chromatographic peaks were obtained in negative ion mode.Compared with the normal group,24 different metabolites were identified and screened in the positive ion mode,of which 3 metabolites were up-regulated and21 metabolites were down-regulated;in the negative ion mode,42 metabolites were screened and identified,of which 12 were The content of metabolites was up-regulated,and the content of 30 metabolites was down-regulated.There were8 different metabolites in both models,They were L-Arginine,L-Leucine,L-Phenylalanine,Hippuric acid,Glycocholic acid,Glycochenodeoxycholate,Chenodeoxycholate,Inosine.3.Bioinformatics analysis results of different metabolites in Chinese cobra bitesThrough matching with the KEGG database,it is found that the differential metabolites of the model group and the control group are mainly involved in 5metabolic pathways.ABC transporters,Central carbonmetabolism in cancer,Protein digestion and absorption,Aminoacyl-t RNA biosynthesis,Mineral absorption.Different metabolites involved in these pathways include,Inosine,L-Glutamine,L-Phenylalanine,D-prolin,L-Leucine.4.Targeting verification results of key serum metabolites of Chinese cobra bitten pigletsAccording to the results of non-targeted metabolomics positive and ne gative ions,4 different metabolites of inosine,hippuric acid,thymidine,an d L-glutamine were selected for absolute quantification in negative ion mo de,Select 5 different metabolites of chenodeoxycholic acid,L-leucine,L-p henylalanine,D-proline,and L-arginine for absolute quantification in positi ve ion mode.Test result display,The contents of 8 metabolites of inosine,hippuric acid,thymidine,glutamine,L-leucine,L-phenylalanine,D-proline and L-arginine were different in the two groups.The difference was stati stically significant(p<0.05),The same metabolite has the same retention time.This is the same as the results of non-targeted studies,indicating th at targeted absolute quantitative verification has been achieved.5.Clinical verification results of key metabolites from Chinese cobra bites Using targeted metabolomics to verify the above 8 metabolites in clinical serum samples,the result shows,Compared with normal human serum,The content of three metabolites of inosine,L-phenylalanine,and D-proline in the serum of the patient decreased,The quantitative results are consistent with the results of non-targeted and targeted metabolomics,and the difference is significant(p<0.05).It indicates that these three key metabolites may be closely related to the pathophysiological mechanism of cobra bite poisoning.Conclusion:1.The study took Guangxi Bama piglet as an animal model,and injected0.75 mg/kg of cobra venom into the lateral muscle of the pig’s leg.After 6 hours,a cobra bite animal model was obtained.2.The non-targeted metabolomics method comprehensively analyzes the overall metabolic profile of the blood of the cobra bitten piglet,and then uses the absolute quantitative verification of targeted metabolomics to find inosine,hippuric acid,glutamine,L-leucine,The content differences of 8 different metabolites of L-phenylalanine,D-proline,L-arginine and thymidine in the two groups were verified.3.Through further targeted verification and analysis of key metabolites in clinical serum samples from cobra bites,it was found that the levels of inosine,L-phenylalanine,and D-proline in the patients’ serum were reduced,and the difference was statistically significant.Significance(p<0.05),The results are consistent with the changes in serum metabolite content after snake wound poisoning in piglets.It fully shows that these three different metabolites play an important role in the pathophysiology of cobra bites.4.Through KEGG pathway analysis,it was found that compared with the control pigs,Cobra bites can affect five important metabolic pathways,including ABC transporters,Central carbonmetabolism in cancer,Protein digestion and absorption,Aminoacyl-t RNA biosynthesis,Mineral absorption.Different metabolites involved in these pathways include,Inosine,L-Glutamine,L-Phenylalanine,D-prolin,L-Leucine.In conclusion,this study has screened,identified and verified the metabolite spectrum and differential metabolites of cobra bites,which is expected to provide the corresponding molecular pathology theoretical basis and potential therapeutic targets for the clinical treatment of Chinese cobra bites.. |
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