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

Abstract

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.

INTRODUCTION

The World Health Organization (WHO) has indicated snakebites as a neglected tropical disease. In more than 160 countries around the world, about 200 kinds of snakes can cause serious harm to humans, killing > 100,000 people and maiming > 400,000 people every year (Gutiérrez et al., 2017). Southeast Asia and southern China are subtropical regions, and the climate environment is suitable for snakes to breed. Therefore, snakebite cases remain high, and many people are bitten by snakes every year (Gutiérrez et al., 2013; Isbister and Silva, 2018; Liu et al., 2020). Epidemiological survey shows that Trimeresurus stejnegeri bite is one of the main snakebites in Southeast Asia (Bolon et al., 2019). In recent years, with the development of artificial snake breeding and the snake-eating/catering industry, the number of clinical snake injuries is also increasing (Kasturiratne et al., 2008).

Trimeresurus stejnegeri belongs to Viperfidae. Studies showed that its venom components are complex, and it is a mixed venom based on blood circulating toxins, including hemorrhagic toxins, cytotoxins, procoagulants and other enzymes. Trimeresurus stejnegeri bite can cause systemic intoxication and local wound changes. The mainly manifested symptoms include wound pain, bleeding, congestion, tissue edema and necrosis, and some other symptoms (Chan et al., 1993a, 1993b; Chan and Hung, 2010; Cockram et al., 1990; Yang et al., 2007); the main reason is that hemorrhagic toxins, proteolytic enzymes, phospholipases and snake venom metalloproteinases have proteolytic enzyme activity, which can destroy capillaries, increase vascular permeability, and cause bleeding, swelling, tissue necrosis, visceral bleeding and some other symptoms. Lectins, procoagulant and other coagulation toxins can cause abnormal coagulation function, longer prothrombin time, and lower fibrinogen (Chiang et al., 2020; Hutton et al., 1990; Isbister, 2010). At present, the clinical identification of Trimeresurus stejnegeri bite is mainly based on patient description, laboratory blood examination, bite site tooth printshape and wound condition, making it difficult to quickly diagnose the snake species. Once diagnosed, the mainstay treatment is antivenom and combination with other treatment, but the type and specificity of antivenom are limited, and the treatment cost of antivenom is relatively high (Huang et al., 2021; Sharma et al., 2017; Sunitha et al., 2015). In the process of clinical treatment, different hospitals and doctors have different intervention methods for snakebite, which increases the difficulty of treatment. In addition, although polyvalent antivenoms are useful in cases where the snake species has not been identified, they may bring more risk to patients (Pandey et al., 2019). Therefore, biomarker studies of Trimeresurus stejnegeri bite are required to provide guidance for the diagnosis and treatment of Trimeresurus stejnegeri bite.

Metabolomics is a method of quantitatively analyzing all metabolites in organisms and searching for the relative relationship between metabolites and physiology. It is an integral part of systems biology. Metabolomics can reveal the overall metabolic spectrum of individuals, as many life activities in cells occur at the level of metabolites. Metabolites are an important part of cell function and susceptible to environmental, genetic and other factors, metabolomics have been established as new tools for exploring the potential biomakers (Rinschen et al., 2019; Schrimpe-Rutledge et al., 2016). In metabolomics studies, UPLC-Q-TOF/MS is a new metabolomics detection technology. Compared with other metabolomics tools, it is an effective analysis tool with the advantages of fast speed, high resolution and high accuracy (Zhong et al., 2021). In this study, we established a Trimeresurus stejnegeri bite model using the Guangxi Bama miniature pig, and UPLC-Q-TOF/MS technology was used to screen and identify the differential metabolites in serum and urine, and to explore the bio-markers of the Trimeresurus stejnegeri bite, elucidate the relationship between snakebite and changes in body metabolism, and help the diagnosis and treatment of snakebite.

MATERIALS AND METHODS

Venom

Trimeresurus stejnegeri venom was purchased from the Snake Venom Research Institute of Guangxi Medical University. The venom was pooled, lyophilized, and stored at −20°C until used. Venom solutions were prepared immediately before use, using 0.9% NaCl as solvent.

Experimental Animals and Design

Fifty KM mice (Kunming mice) (18–22 g, half of which were female) were purchased from the Experimental Animal Center of Guangxi Medical University. After 7 days of acclimation to laboratory conditions, the mice were randomized into 5 groups (n = 10 per group). To calculate the LD50 value of Trimeresurus stejnegeri venom in mice according to the references, mice were intramuscularly injected with 17.47 mg/kg, 14.85 mg/kg, 12.62 mg/kg, 10.73 mg/kg, 9.12 mg/kg of Trimeresurus stejnegeri venom, and the death of mice was then observed and recorded continuously for 7 days, and the results were analyzed with the Bliss method (LD50 CALC 2.0 version). Next, based on the LD50 dose of Trimeresurus stejnegeri venom injected into mice intramuscularly, the theoretical 50% lethal dose of Trimeresurus stejnegeri venom to Bama miniature pig was obtained by an equivalent dose coefficient conversion algorithm and body surface area method.

Twenty-two healthy Guangxi Bama miniature pigs, weighing approximately 10 kg, were purchased from Experimental Animal Center of Guangxi University (Guangxi, China, approval no. SCXK [Gui] 2018-0003). The animal experiments were reviewed and approved by the University’s Animal Ethics Committee. The pigs were raised in the Experimental Animal Center of Guangxi Medical University. After 1 week of acclimation, twelve Bama minipigs were randomly divided into a control group (n = 6) and a snake venom group (n = 6). According to previous research, the snake venom group was intramuscularly injected with a dose of 0.643 mg/kg (1/3 of the theoretical lethal dose) into the left legs of the pigs, and the control group was treated with an equal volume of physiological saline. After injection of snake venom, the local wound changes of the pigs were observed.

Detection of hematological indexes

Two tubes of blood were collected from the anterior vena cava plexus of pigs before, 6 hr after, 12 hr after, and 24 hr after the injection of snake venom, and about 2.5 mL of blood was collected from each tube. The blood was placed in sodium citrate anticoagulant tubes and EDTA anticoagulant tubes respectively. The blood in the EDTA anticoagulant tube was inverted 3–5 times to ensure even mixing, and then the blood routine was detected by a BC-6800PLUS automatic blood routine analyzer. The blood in the sodium citrate coagulation test tube was inverted 3–5 times for mixing, and then it was subsequently centrifuged to extract the plasma. After that step, the alanine aminotransferase (ALT), prothrombin time (PT), fibrinogen (FIB), D-Dimer (D-Dimer), potassium ion (K+) and platelet (PLT) were detected by the CS-5100 automatic coagulation analyzer.

Hematoxylin and eosin (HE) staining

Twenty-four hours after blood collection, the heart, liver, lung, duodenum and the wound area of muscle tissue of three pigs in the snake venom group and control group were respectively removed, and were irrigated 3 times with saline. The tissues were fixed with 4% paraformaldehyde solution, and then subjected to paraffin embedding, conventional section, xylene immersion, dehydration and HE staining. The pathomorphological changes were observed under an optical microscope.

Preparation and handling of samples

The remaining 10 pigs were randomly divided into a control group and a snake venom group, with 5 pigs in each group. Six hours after injection, approximately 5 mL blood samples from the vena cava were collected in vacuum containers. The blood was naturally coagulated at room temperature for 1 hr, centrifuged at 3000 r/min for 15 min at 4°C to separate the serum, and then stored at −80°C. One-hundred µL serum was added to 400 µL of cold precooled acetonitrile solution and vortexed for 1 min, and then each sample stood at −20°C to precipitate protein for 1 hr. The samples were then centrifuged at 14000 g for 20 min at 4°C, the supernatants were obtained and freeze-dried using a vacuum freeze dryer. The residues were redissolved in 120 µL acetonitrile solution, were filtered with a disposable vacuum filter membrane, and take the filtered solution into the liquid phase injection vial for loading.

The urine of the pigs was collected 6 hr after injection of Trimeresurus stejnegeri venom, centrifuged at 3000 r/min for 10 min at 4°C. The supernatant was centrifuged at 12000 r/min for 15 min at 4°C. Then 0.5 mL of samples were added to 0.5 mL of methanol and vortexed for 1 min, and each sample stood at 4°C for 3 hr. After centrifuging at 12000 r/min at 4°C for 10 min, the samples were filtered through a 0.22 mm membrane for LC-MC analysis.

UPLC-Q/TOF-MS conditions

The samples were analyzed by UPLC-QTOF-MS using ultra-high performance liquid chromatography (ACQUITY UPLC I-class, Waters, Milford, MA, USA), and the separation was carried out on an UPLC column (C18, 1.7 μm, 2.1 × 50 mm, Waters). The column temperature was maintained at 25°C. The flow rate was 0.35 mL/min. Mobile phase A: high purity water (containing 25 mmol ammonium acetate + 25 mmol ammonia), Mobile phase B: 100% acetonitrile. The gradient elution procedure is shown in Table 1. Continuous analysis of samples was carried out in random order to avoid the influence caused by the fluctuation of instrument detection signal. In order to monitor and evaluate the stability of the instrument system and improve the reliability of experimental data, QC samples were inserted before sampling and in sample sequencing. During the whole analysis process, the samples were placed in an automatic sampler at 4°C.

Table 1. UPLC-MS gradient elution table.

Time (min)	Mobile phase A (%)	Mobile phase B (%)	Flow rate (mL/min)
0	5	95	0.35
1	5	95	0.35
14	35	65	0.35
16	60	40	0.35
18	60	40	0.35
18.1	5	95	0.35
23	5	95	0.35

All samples were tested in ESI+ and ESI- modes respectively. The samples were separated by ultra-high performance liquid chromatography and analyzed by mass spectrometry (Xevo G2-S Q TOF, Waters). ESI conditions after chromatographic separation: ion atomization gas: 60 psi, ion auxiliary gas: 60 psi, curtain gas: 30 psi, temperature: 600°C, ion voltage: ± 5500 eV (both positive and negative modes), mass spectrum scanning range: 60–1000 Da, product scanning range: 25–1000 Da, mass spectrum scanning time: 0.20 sec/spectrum, product ion accumulation scanning time: 0.05 sec/spectrum, the secondary mass spectrum was acquired in a high-sensitivity mode, with the declustering voltage DP: ± 60 eV (both positive and negative modes), the collision energy (CE): 35 ± 15 eV, the isotope exclusion 4 Da, and the ion detection cycle: 6.

Data processing and analysis

The original data were converted into MzXML format by ProgenesisQI. Firstly, the peak area was identified and aligned, the peak baseline and peak area were processed and corrected, and the retention time was corrected. Finally, the data matrix containing ion fragment information was output to compare the differences in serum and urine metabolic profiles between the snake venom group and control group. Accurate mass matching (< 25 ppm) and secondary spectrum matching were used to identify and analyze the metabolite structure. The data were processed by SIMCA14.1, for performing multi-dimensional statistical analysis, including principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA). The R programming language was used to draw the volcano plot for one-dimensional statistical analysis. Then, the OPLS-DA model was used to maximize the differences among different groups within the model, and VIP values of each metabolite in this model were calculated, then, combined with t-test, the criteria for screening for differential metabolites were the projection (VIP) value > 1 and a t-test P value < 0.05. The differential metabolites were analyzed by hierarchical cluster analysis and correlation analysis, and the rationality and correlation of differential metabolites were evaluated. In addition, the differentially abundant metabolites were imported into the KEGG database (http://www.genome.jp/kegg/) for enrichment analysis of metabolic pathways.

Statistical analysis

The data were presented as the mean ± standard deviation. SPSS software (version 20.0, IBM Corp.), GraphPad Prism software (version 5.0, GraphPad Software, Inc.) and Origin8 (OriginLab, Corp.) were used to analyze all data. P < 0.05 was considered to indicate a statistically significant difference.

RESULTS

Exploration of snake venom dose in model construction

The LD50 value of Trimeresurus stejnegeri venom in KM mice was calculated by Bliss (LD50CALC2.0). The result was 10.074 mg/kg (the 95% confidence interval was 7.5785–11.383 mg/kg), which is shown in Table 2. According to the equivalent dose coefficient method and body surface area, the theoretical median lethal dose (LD50) of model pigs bitten by Trimeresurus stejnegeri was 1.929 mg/kg. Thus, we used 1/3 of the LD50 (0.643 mg/kg) for modeling based on the preliminary experimental results.

Table 2. Death of mice within 7 days of intramuscular injection of Trimeresurus stejnegeri venom.

Dose/ (mg/kg)	Logarithmic measurement/X	Number of animals	Number of deaths	Death percentage/%	Experimental probability unit /Y	Regression probability unit /Y
17.47	1.242	10	10	1	——	6.873
14.85	1.172	10	9	90	6.282	6.320
12.62	1.101	10	7	70	5.524	5.767
10.73	1.031	10	6	60	5.253	5.215
9.12	0.960	10	4	40	4.747	4.661

Regression equation y(Probit) = 2.8591 + 7.834Log(D).

Changes in bite site

Compared with the control group (Fig. 1A), the local wound changes results showed that within after the injection of Trimeresurus stejnegeri venom, the pigs in the snake venom group clearly exhibited poisoning symptoms, such as swelling that began to appear at the wound 3 hr after Trimeresurus stejnegeri venom injection, with the swollen area continuing to expand (Fig. 1B). Subsequently, the swelling site became larger, forming bruises accompanied with bleeding (Fig. 1C, D, E). After 48 hr, the edema was relieved, accompanied by congestion, ecchymosis and other symptoms (Fig. 1F, G). These results indicated that the pigs develop the symptoms of snakebite after injection with Trimeresurus stejnegeri venom, and the local wound changes of pigs was consistent with that of Trimeresurus stejnegeri bite in clinical practice.

Fig. 1

Wound changes in pigs before (A) and 3 hr (B), 6 hr (C), 12 hr (D), 24 hr (E), 48 hr (F), and 72 hr (G) after injection of snake venom

Hematological examination results

The hematological examination was applied to analyze the hematological changes in each group, as shown in Table 3. Compared with the control group, the red blood cells, white blood cells, alanine aminotransferase and D-dimer of piglets and the prothrombin time were increased, while the platelet, K+ concentration and fibrinogen were decreased 6 hr after injection, indicating that the symptoms of metabolic disorder combined with infection, inflammation, liver function injury and bleeding may occur after Trimeresurus stejnegeri bite, which was consistent with the hematological characteristics of Trimeresurus stejnegeri bite. After 12 hr, the red blood cells and white blood cells were decreased, which may be related to the self-healing of the body. The difference was statistically significant (P < 0.05).

Table 3. Blood indexes of pigs before and after injection of Trimeresurus stejnegeri venom.

Blood indexes	Before venom injection						6 hr after injection of venom						12 hr after injection of venom						24 hr after injection of venom
	Control group			Snake venom group			Control group			Snake venom group			Control group			Snake venom group			Control group			Snake venom group
RBC (10^9/L)	6.00	±	0.38	6.13	±	0.17	6.06	±	0.22	8.18	±	0.53**	5.97	±	0.51	7.04	±	0.66*	6.08	±	0.32	6.42	±	0.74
WBC (10^12/L)	35.54	±	2.77	33.75	±	2.15	35.59	±	0.85	45.86	±	4.35*	29.47	±	6.60	44.09	±	7.38*	32.28	±	5.41	34.01	±	11.07
ALT (U/L)	50.47	±	2.54	47.20	±	5.12	48.90	±	1.37	57.93	±	26.33	51.65	±	2.71	154.83	±	62.48*	49.71	±	2.96	103.87	±	6.68*
PT (S)	12.09	±	0.59	11.60	±	1.88	11.87	±	1.10	14.17	±	0.21*	11.64	±	1.51	15.75	±	1.99*	11.58	±	1.61	17.23	±	0.63**
FIB (g/L)	1.56	±	0.32	1.60	±	0.39	2.39	±	0.14	0.89	±	0.58**	2.30	±	0.29	0.81	±	0.96**	3.18	±	0.27	2.53	±	0.23*
D-Dimer	0.27	±	0.36	0.33	±	0.29	0.23	±	0.04	0.96	±	0.24*	0.46	±	0.30	3.00	±	1.69**	0.40	±	0.24	0.80	±	0.13*
K+ (mmol/L)	4.54	±	0.49	4.80	±	0.50	6.15	±	0.34	4.46	±	0.38*	5.73	±	0.99	5.32	±	0.44	5.80	±	0.50	5.35	±	0.74
PLT	627.71	±	52.62	666.83	±	46.23	681.00	±	53.23	491.67	±	63.85**	675.71	±	61	447.00	±	48.03**	684.28	±	49.96	398.33	±	61.40**

*P < 0.05.

**P < 0.01 vs control group.

HE staining results

Compared with the control group, HE staining (Fig. 2) showed that the muscle tissue at the injection site of the snake venom group, serous exudate could be seen diffusing and infiltrating the tissues, with local inflammatory edema, bleeding, degeneration and necrosis. In addition, the capillary permeability of the lung tissue was increased, and there was hyperemia and edema in the capillaries of the alveolar. A large number of red blood cells were found in the alveolar cavity. Moreover, the results of liver tissue staining showed that the cells had hemorrhage and necrosis, and the nucleus was partially dissolved. Duodenal mucosal edema, hyperemia, erosion.

Fig. 2

Pathological changes of muscle, heart, liver, lung, duodenum.

UPLC-Q-TOF/MS Analysis

To ensure the stability and repeatability of mass spectrometry and samples, the mass spectrometry quality control analysis was performed on the serum and urine samples. According to the test results, the metabolites in both blood and urine were analyzed at 6 hr after injection of snake venom. Due to the metabolites relatively complex, the UHPLC-Q-TOF-MS technique was used in this study to comprehensively analyze the differential metabolites, with an electrospray ionization source in the negative mode (ESI-) and positive mode (ESI+). The total ionizing flow diagrams of serum and urine in snake venom group and control group are shown in Fig. 3 and Fig. 4.

Fig. 3

(A) Total ion chromatogram of serum metabolites in the control group and snake venom group in ESI+ mode, (B) Total ion chromatogram of serum metabolites in the control group and snake venom group in ESI- mode.

Fig. 4

(A)Total ion chromatogram of urine metabolites in the control group and snake venom group in ESI+ mode, (B) Total ion chromatogram of urine metabolites in the control group and snake venom group in ESI- mode.

Multivariate statistical analysis of metabolites

The serum and urine PCA score diagrams of the snake venom group and the control group were obtained by Pareto-scaling, as shown in Fig. 5A and B. The results showed that the parameter R2X of PCA score in the serum ESI+ was 0.609, while that in ESI- was 0.516, indicating that the PCA model could effectively separate the two groups. The parameter R2X of PCA score in urine ESI+ was 0.414, while that in ESI- was ESI-, indicating that the overall group classification and inter-group clustering were poor, and it was necessary to continue to study the difference between groups with supervision and discriminant analysis method. Then, OPLS-DA models were established (Fig. 5C and D). The OPLS-DA model could effectively separate the two groups, which had good prediction ability and reliability. In order to verify whether the established model was over-fitting and repeatable, the OPLS-DA model was used to verify response permutation testing (PRT). A seven-fold cross-validation permutation test was applied, with a total of 200 operations to exclude random effects in the constructed model. The R2 and Q2 intercept values of serum in ESI+ were 0.416 and −0.597, and those in ESI- were 0.957 and −1.15 (Fig. 6A). The R2 and Q2 intercept values of urine in ESI+ were 0.557 and −0.578, and those in ESI- were 0.358 and 0.392 (Fig. 6B). These results indicated that the constructed models in this study were valid and repeatable. In addition, volcano maps were used to analyze the differences between the control group and the snake venom group. As shown in Fig. 7, the red dots in the map are the differential metabolites with FC > 1.5 and P value < 0.05, indicating a significant difference in metabolite changes between the two groups under ESI+ and ESI- modes.

Fig. 5

(A) PCA score plots of serum metabolic profiling of the experimental group and control group, (B) PCA score plots of urine metabolic profiling of the experimental group and control group, (C) OPLS-DA score plots of serum metabolic profiling of the experimental group and control group, (D) OPLS-DA score plots of urine metabolic profiling of the experimental group and control group.

Fig. 6

(A) permutation plots of the serum OPLS-DA models, (B) permutation plots of the urine OPLS-DA models.

Fig. 7

Volcano plot of serum metabolic profiling of the Snake venom group and control group in ESI+ (A) and ESI- (B) mode. Volcano plot of urine metabolic profiling of the snake venom group and control group in ESI+ (C) and ESI- (D) mode.

Screening and identification of differential metabolites

With VIP > 1.0 and P values < 0.05 serving as the screening criteria in the OPLS-DA models, the serum analysis results are shown in Table 4. A total of 367 differential metabolites were identified in the ESI+ mode, and 277 differential metabolites were identified in the ESI-. There were 9 differential metabolites in both ESI+ and ESI- serum modes, which are 2-acyl-1-(1-enyl)-sn-glycero-3-phospholecithin (2-Acyl-1-(1 -alkenyl)-sn-glycero-3-phosphate), Oleic acid, Lithocholyltaurine, Lithocholic acid, Deoxycholic acid, Hypoxanthine, Phenylacetaldehyde, Tryptophan and Gamma-Linolenic acid. The urine analysis results are shown in Table 5. A total of 292 differential metabolites were identified in the ESI+ mode, and 326 differential metabolites were identified in the ESI-. There were 11 differential metabolites in both ESI+ and ESI- urine modes, namely 3-Methoxy-4-hydroxyphenylglycol glucuronide, Thiocysteine, Thiocysteine, Dopamine, 3,4-Benzopyrene, Arginine, Indoleacetaldehyde, D-Glucuronic-6,3- Lactone, 5-Hydroxyindoleacetic acid, Dihydrolipoamide, 5-Acetamidovalerate and Dehydroepiandrosterone sulfate.

Table 4. Distinct metabolites identified in serum samples.

Metabolites	VIP	P	m/z	RT(s)	Trend	Ion mode
2-Acyl-1-(1-alkenyl)-sn-glycero-3-phosphate	1.01	0.002	725.47	7.9	↑	ESI+
Lithocholyltaurine	1.48	0.000	502.32	6.37	↑	ESI+
Oleic acid	1.93	0.000	321.21	8.03	↑	ESI+
Lithocholic acid	1.15	0.001	435.26	7.5	↑	ESI+
Deoxycholic acid	1.97	0.001	357.28	5.55	↑	ESI+
Hypoxanthine	4.38	0.000	159.02	0.93	↑	ESI+
Phenylacetaldehyde	1.04	0.003	121.07	5.63	↑	ESI+
Gamma-Linolenic acid	1.11	0.000	301.21	5.48	↑	ESI+
tryptophan	1.02	0.001	205.10	8.56	↓	ESI+

Table 5. Distinct metabolites identified in urine samples.

Metabolites	VIP	P	m/z	RT(s)	Trend	Ion mode
3-Methoxy-4-hydroxyphenylglycol glucuronide	5.4	0.001	378.06	4.65	↓	ESI+
3,4-Benzopyrene	1.88	0.000	275.08	2.33	↑	ESI+
Dopamine	1.05	0.000	108.09	6.03	↓	ESI+
Thiocysteine	1.25	0.000	110.01	8.3	↑	ESI+
Arginine	1.78	0.000	175.12	7.41	↓	ESI+
Indoleacetaldehyde	1.38	0.000	160.08	5.88	↓	ESI+
5-Hydroxyindoleacetic acid	1.12	0.004	156.04	0.7	↑	ESI+
D-Glucurono-6,3-lactone	1.25	0.000	199.02	1.71	↓	ESI+
5-Acetamidovalerate	1.19	0.005	114.09	4.99	↓	ESI+
Dihydrolipoamide	1.73	0.000	190.07	2.31	↑	ESI+
Dehydroepiandrosterone sulfate	1.09	0.001	427.13	2.26	↑	ESI+

Metabolic pathway analysis

Fisher’s exact test was used to analyze the enrichment of the KEGG pathways of differential metabolites between the two groups. The results showed that the five serum pathways of Liver disease due to cystic fibrosis, Hyperbaric oxygen exposure, Biliary cirrhosis, Tryptophanuria and Hartnup disease are closely related (Fig. 8 and Table 6). The contents of Deoxycholic acid, Lithocholic acid, Tryptophan and Hypoxanthine, the core metabolites of these pathways, changed significantly during the bite process of Trimeresurus stejnegeri. The four urine pathways of Aromatic L-Amino Acid Decarboxylase, Vitiligo, Blue Diaper Syndrome and Hyperargininemia in urine are closely related (Fig. 9 and Table 7), dopamine, 5-hydroxyindole acetic acid and arginine undergoes are the core metabolites of these pathways.

Fig. 8

Enrichment analysis of serum differential metabolite pathways.

Table 6. Enrichment analysis of serum differential metabolite pathways.

Metabolite Set	total	expected	hits	Raw p	Holm p	FDR
Liver Disease Due to Cystic Fibrosis	6	0.097	2	0.00332	1	1
Hyperbaric Oxygen Exposure	9	0.145	2	0.00779	1	1
Biliary Cirrhosis	10	0.162	2	0.00965	1	1
Tryptophanuria	1	0.0162	1	0.0162	1	1
Hartnup Disease	2	0.0323	1	0.0321	1	1
Extrahepatic Biliary Atresia	3	0.0485	1	0.0578	1	1
Lesch-Nyhan Syndrome	5	0.0808	1	0.0785	1	1
Spastic Ataxia	5	0.0808	1	0.0785	1	1
Stroke	5	0.0808	1	0.0785	1	1
Metabolites Affected by Exercise	5	0.0808	1	0.0785	1	1
Critical Illness (Major Trauma, Severe Septic Shock, or Cardiogenic Shock)	6	0.097	1	0.0936	1	1
Acute Seizures	14	0.226	1	0.206	1	1
Early Markers of Myocardial Injury	14	0.226	1	0.206	1	1
Diabetes Mellitus (Mody), Non-Insulin-Dependent	19	0.307	1	0.271	1	1
Different Seizure Disorders	24	0.388	1	0.33	1	1
Schizophrenia	26	0.42	1	0.353	1	1

Fig. 9

Enrichment analysis of urine differential metabolite pathways.

Table 7. Enrichment analysis of urine differential metabolite pathways.

Metabolite Set	total	expected	hits	Raw p	Holm p	FDR
Aromatic L-Amino Acid Decarboxylase Deficiency	5	0.106	2	0.00392	1	1
Vitiligo (Non-Segmental)	9	0.19	2	0.0135	1	1
Vitiligo	1	0.0211	1	0.0211	1	1
Blue Diaper Syndrome	3	0.0633	1	0.0211	1	1
Hyperargininemia	3	0.0633	1	0.0211	1	1
Tyrosine Hydroxylase Deficiency	3	0.0633	1	0.0621	1	1
Cystinuria	4	0.0845	1	0.082	1	1
Hyperdibasic Aminoaciduria I	4	0.0845	1	0.082	1	1
Beta-Thalassemia	4	0.0845	1	0.082	1	1
Hyperornithinemia With Gyrate Atrophy (Hoga)	4	0.0845	1	0.082	1	1
Dopamine Beta-Hydroxylase Deficiency	5	0.106	1	0.102	1	1
Lysinuric Protein Intolerance (Lpi)	5	0.106	1	0.102	1	1
Spastic Ataxia	5	0.106	1	0.102	1	1
Rheumatoid Arthritis	7	0.148	1	0.14	1	1
Monoamine Oxidase-A Deficiency (Mao-A)	7	0.148	1	0.14	1	1
Tyrosinemia I	8	0.169	1	0.158	1	1
Maple Syrup Urine Disease	9	0.19	1	0.176	1	1
Propionic Acidemia	20	0.422	1	0.353	1	1
Alzheimer’s Disease	23	0.486	1	0.394	1	1
Schizophrenia	32	0.676	1	0.506	1	1

Comparative analysis of metabolic results

For the credibility of the screened metabolic markers, we compared metabolomic results with other snakes (Table 8). It can be seen that the candidate markers of Trimeresurus stejnegeri were Deoxycholic acid, Lithocholic acid, Tryptophan, Hypoxanthine, Dopamine, Arginine, 5-hydroxyindoleacetic, and the candidate markers of Bungarus multicinctus were L-Glutamine, L-Citrulline, D-Proline, L-Leucine, L-Tryptophan, Alpha-D-Glucose and Glycine (Huang et al., 2021). At present, there is no metabolomic study of other snakes, so only the Bungarus multicinctus has been compared.

Table 8. Comparison of metabolomics results.

Snake species	Metabolic markers
Trimeresurus stejnegeri	Deoxycholic acid, Lithocholic acid, Tryptophan, Hypoxanthine, Dopamine, Arginine, 5-hydroxyindoleacetic
Bungarus multicinctus	L-Glutamine, L-Citrulline, D-Proline, L-Leucine, L-Tryptophan, Alpha-D-Glucose and Gly-cine

DISCUSSIONS

Snakebite is one of the most neglected global problems identified by the World Health Organization, with about 400,000 cases of snakebite reported worldwide every year, and at least 95,000 deaths (Chippaux, 1998; Gutiérrez et al., 2013; Kasturiratne et al., 2008). The Trimeresurus stejnegeri bite ranks in the forefront of the viper bite cases in China, especially in the south. Since the whole body of Trimeresurus stejnegeri is green, people often mistakenly enter or approach the hiding place of the Trimeresurus stejnegeri or touch the its body by mistake, resulting in bites. Studies have shown that the venom components of Trimeresurus stejnegeri is complex, mainly toxins circulating in the blood, and snakebite can lead to coagulation disorders, serious complications such as cerebral hemorrhage, and even death (Chiang et al., 2020; Hutton et al., 1990). Current studies primarily used rats, mice or rabbits to establish the animal model of viper bite (Rivel et al., 2016; Vieira et al., 2016). However, the body structure, lifespan and gene expression of these animals are quite different from those of humans, and thus there are limitations to simulation of the occurrence and development of human disease (Dos Anjos-Garcia and Coimbra, 2019). The Guangxi Bama miniature pig was considered to be an ideal experimental animal model due to its stable genetics, small size, easy experimental operation, low feeding cost, strong adaptability to the environment, and similarities to human skin structure, physiology and anatomy (Bendixen et al., 2010; Bian et al., 2015; Liedtke et al., 1975; Sullivan et al., 2001). In a recent study, we established a Guangxi Bama miniature pig model of Bungarus multicinctus bite, and systematically analyzed the metabolite changes of Bungarus multicinctus bite, proving the reliability of the pig model (Huang et al., 2021). This experiment refers to the previous scheme of the laboratory. Trimeresurus stejnegeri venom was intramuscularly injected into the legs of pigs at the injection dose of 0.6 mg/kg, and after 3 hr, the piglets had symptoms such as swelling, bleeding and pain at the injection site. The results of comprehensive evaluation of local wound changes, blood indexes, tissue and other pathological characteristics of poisoned pigs were consistent with those of clinical patients bitten by Trimeresurus stejnegeri, providing reliable models for subsequent metabolomic analysis.

In this study, we established the Trimeresurus stejnegeri bite model, and we further employed the metabolomics methods based on UPLC-Q-TOF-MS to screen the differential metabolites and metabolic pathway changes during the snakebite process, and explore the metabolic markers and pathophysiological mechanisms of the Trimeresurus stejnegeri bite. Five metabolic pathways were found in serum that are related to the Trimeresurus stejnegeri bite, namely Tryptophanuria, Liver disease due to cystic fibrosis, Hartnup disease, Hyperbaric oxygen exposure and Biliary cirrhosis, and the core metabolites of these pathways, deoxycholic acid, lithocholic acid, tryptophan, and hypoxanthine, were significantly changed. Four pathways of Aromatic L-Amino Acid Decarboxylase, Vitiligo, Blue Diaper Syndrome and Hyperargininemia in urine are associated with the Trimeresurus stejnegeri bite, and the core metabolites are dopamine, 5-hydroxyindoleacetic acid and arginine.

Compared with the control group, there were significant differences in Deoxycholic acid, Lithocholic acid, Dopamine and other lipid compounds after Trimeresurus stejnegeri bite, suggesting that it might play a role in the pathophysiology of the Trimeresurus stejnegeri bite by regulating lipid metabolism. Moreover, several studies have reported that lipid metabolism is also associated with intestinal disease, cancer and other digestive diseases (Bian et al., 2021; Schoeler and Caesar, 2019). Lipid metabolism is a complex physiological process, and it also play an important role in many life processes, such as nutrient adjustment, hormone regulation, cell development and differentiation (Li et al., 2017; Ma et al., 2014). The content of Tryptophan and Arginine was significantly changed in the snake venom group compared with control group, indicating that protein digestion and absorption pathways may play an important role in after Trimeresurus stejnegeri bite metabolism through amino acid metabolism. As the basic unit of protein and polypeptide synthesis, amino acids are essential biologically active substances for all living cells and organisms, and their abnormal levels are associated with many diseases (Marazzi et al., 2008; Wu, 2009). In addition, Hypoxanthine as a purine metabolite, it was also down-regulated, indicating that nucleotide metabolism pathway may play a role in the pathophysiological process of Trimeresurus stejnegeri bite. These results indicate that lipid metabolism, amino acid metabolism, and nucleotide metabolism change significantly after Trimeresurus stejnegeri bite, which affects the metabolism function of the body. We will reveal the pathological mechanism of Trimeresurus stejnegeri bite from the perspective of metabolite molecules, elucidate the interaction between snakebite and the immune response of body, to provide theoretical basis and therapeutic targets for prevention and early diagnosis of Trimeresurus stejnegeri bite.

Deoxycholic acid (DCA) is produced by primary bile acids under the action of intestinal flora, and is a naturally occurring secondary bile acid (Xu et al., 2021a), hydroxyl-substituted 5β-cholic acid at positions 3 and 12, respectively, which is human serum metabolite. DCA acts as a detergent to dissolve fat and promote intestinal absorption. Excessive DCA-induced gut dysbiosis and intestinal inflammation may result in the metabolic disorder of bile acids in the liver and small intestine, which may be related to the disruption of intestinal flora balance and the promotion of bile acid synthesis in liver through the FXR-FGF15 signaling pathway (Ding et al., 2015; Xu et al., 2021b). Studies have shown that DCA is considered to be associated with the development of inflammatory bowel disease (IBD). At present, more and more evidence has shown that the level of intestinal bile acid, especially the increase of DCA, plays an important role in the process of inducing the pathogenesis of IBD (Xu et al., 2021b; Zhao et al., 2018). After Trimeresurus stejnegeri bite, the toxin will attack the intestine, stimulate the production of DCA, and lead to the occurrence of intestinal inflammation, eventually causing diarrhea and reduced food intake in pigs.

Lithocholic acid (LCA) is a monohydroxy bile acid, one of the major bile acid components, formed by bacterial biotransformation (7a-dehydroxylation) of the primary bile acid chenodeoxycholic acid (CDCA) in the terminal small intestine (Abrahamsson et al., 2005). It is the most toxic monomeric bile acid in the liver. Normally, the body mainly degrades LCA in the liver to maintain low concentration of LCA and avoid damage to the body (Xu et al., 2020). Studies have found that LCA is cytotoxic, especially in the liver, and supraphysiological levels of LCA are important risk factors for digestive diseases such as gastric cancer, colon cancer and liver damage (Alkhedaide, 2018; Ding et al., 2015; Zheng et al., 2021), cause oxidative stress and DNA damage, and induce apoptosis in hepatocytes and colonic epithelial cells (Barrasa et al., 2013; Ward et al., 2017). After Trimeresurus stejnegeri bite, the increase of LCA of blood may be due to the toxin-induced the liver damage in pigs, resulting in the inability to degrade lithocholic acid, another possibility is that the intestinal tract was stimulated by toxin to produce a large amount of LCA, which leads to the accumulation of bile acids in the liver.

Tryptophan is an essential amino acid that can only be obtained from external food and cannot be synthesized by the human body. Tryptophan is essential for the production of protein, enzymes and muscle tissue in the human body. It is also essential for the production of niacin, synthesis of the neurotransmitters serotonin and melatonin. Tryptophan metabolites and related neurotransmitters play an important role in physiological functions, which are involved in regulating the function of the nervous system and inflammatory response. Tryptophan plays an important regulatory role in the metabolism of body, growth, and development, and has been implicated in the pathology of depression, Alzheimer’s disease, and other diseases (Shi et al., 2017; Wang et al., 2019). Low levels of tryptophan can lead to anxiety, emotional depression, and various mental system diseases, and the mucosa of the gastrointestinal tract will also be damaged, resulting in gastric ulcers, gastritis and other gastrointestinal tract diseases (Moehn et al., 2012; Yap et al., 2020). The results of this study found that the tryptophan content of the snake venom group was significantly lower than that of the control group in serum. The reason was that after the Trimeresurus stejnegeri bite, the toxin attacks the intestines and hearts of pigs. Pigs need to increase the consumption of tryptophan in the body to protect the intestinal and nerve functions in case of poisoning, resulting in a decrease in the content of tryptophan in the blood.

Hypoxanthine is the metabolic intermediate of purine base in nucleic acid metabolism. In nucleotide synthesis, hypoxanthine synthesizes inosinic acid under the action of transferase. Hypoxanthine plays a direct role in vascular endothelial cell dysfunction in humans, is cytotoxic, and can cause cell death and production of ROS (Kim et al., 2017). In purine metabolism, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase, and the process also involves ROS generation. ROS-induced endothelial cell damage and dysfunction are related to the pathogenesis of vascular diseases (Robillard et al., 2008). Studies have shown that metabolic disorder of the liver could lead to the deficiency of xanthine oxidase, hypoxanthine cannot be converted into xanthine, xanthine cannot be converted into uric acid, and the accumulation of hypoxanthine induces inflammatory response, tissue hypoxia and oxidative stress (Furuhashi, 2020; Ohtsubo et al., 2009). The hypoxanthine content of the snake venom group was significantly higher than that of the control in serum. The reason was that after the Trimeresurus stejnegeri bite, hypoxanthine is severely affected, and the liver metabolism disorder of poisoned pigs, hypoxanthine cannot be further metabolized, and hypoxanthine accumulates in the blood.

Dopamine is a neurotransmitter, which synthesis directly from tyrosine, and since L-phenylalanine can be converted to tyrosine by phenylalanine hydroxylase, dopamine can also be synthesized indirectly from phenylalanine, which works by binding to G protein-coupled receptors (Fernstrom and Fernstrom, 2007; Klein et al., 2019; Nagatsu et al., 1964). Dopamine is the most abundant catecholamine neurotransmitter in the brain; as a neurotransmitter, it regulates various physiological functions of the central nervous system. Dysregulation of the dopamine system is associated with Parkinson’s disease, schizophrenia, Tourette syndrome, attention deficit hyperactivity syndrome and pituitary tumors (Cools et al., 2019; Juárez Olguín et al., 2016). Trimeresurus stejnegeri venom contains neurotoxins. The piglets of the snake venom group showed symptoms of neurotoxicity, and the dopamine level decreased in the body. It may be that the emergency response of the body regulates dopamine to repair the poisoned nervous system.

Arginine is a conditionally essential amino acid that has been identified as an important player in many biological processes, including the normal function of the cardiovascular and immune systems, and is also used in the synthesis of creatine to meet muscle metabolic needs and urea synthesis to maintain systemic nitrogen balance (Albaugh et al., 2017).Arginine can catalyze the cycle of ornithine and promote the formation of urea, so that the ammonia in the human body becomes non-toxic urea and is excreted through urine. Arginine in the body mainly comes from dietary intake and synthesis. Trauma, injury, sepsis, intestinal and renal failure and other conditions will affect the synthesis and bioavailability of arginine. Arginine supplementation has been reported to reduce inflammation at the wound site and improve wound healing after blood loss (Loehe et al., 2007). The arginine content in urine of the snake venom group was significantly lower than that of the control; the reason may be that the hemorrhagic toxin in the Trimeresurus stejnegeri venom caused local bleeding, and the body mobilized the function of arginine to try to repair the bleeding wound, so the consumption was excessive and the content was significantly reduced.

5-hydroxyindoleacetic acid is the final product of the tryptophan-5-serotonin (5-HT) metabolic pathway, and is converted from 5-HT under the action of monoamine oxidase (MAO). It is an important neurotransmitter compound that has an important effect on neurological diseases (Lin et al., 2013). 5-hydroxyindoleacetic acid as a 5-HT final product has been implicated in the pathophysiology of Parkinson’s disease, especially non-motor symptoms such as depression, fatigue, sleep disturbance, sensory and autonomic dysfunction (Tong et al., 2015). Studies had shown that 5-hydroxyindoleacetic acid plays an important role in the development of diseases such as hepatic encephalopathy, acute appendicitis and neuroendocrine tumors (Adaway et al., 2016; Jangjoo et al., 2012; José and J., 1979). The 5-hydroxyindoleacetic acid content in urine of the snake venom group was significantly lower than that of the control. Trimeresurus stejnegeri venom contains neurotoxins, and the poisoning is accompanied by damage to peripheral nerves. Symptoms of neurotoxicity appeared in piglets of the snake venom group, and the consumption of 5-hydroxyindoleacetic acid in the body increases, which may be the body’s emergency response regulating 5-hydroxyindoleacetic acid, and attempting to repair the poisoned nervous system.

In addition, we compared metabolomics results with other snakes. The metabolic markers of Trimeresurus stejnegeri bite were Deoxycholic acid, Lithocholic acid, Tryptophan, Hypoxanthine, Dopamine, Arginine and 5-hydroxyindoleacetic, while the metabolic markers of Bungarus multicinctus bite were L-Glutamine, L-Citrulline, D-Proline, L-Leucine, L-Tryptophan, Alpha-D-Glucose and Glycine. It can be seen that the metabolic markers were screened by us are representative compared with other snakes, and these seven metabolites may be potential markers for the treatment of Trimeresurus stejnegeri bite.

In addition, we enriched the metabolic pathways of the above differential metabolites by KEGG, and 7 metabolites were involved in the changes of 9 metabolic pathways in total. It can be seen that the physiological mechanism response of the body involves multiple metabolic pathways after the Trimeresurus stejnegeri bite, and in particular lithocholic acid, tryptophan and arginine were significantly changed, which means that they may become the key metabolites in the diagnosis of Trimeresurus stejnegeri bite.

In conclusion, the present study used Bama Xiang pig bitten by the Trimeresurus stejnegeri as the research object, and UPLC-Q-TOF-MS technology combined with multivariate statistical methods was used to screen and identify potential biomarkers in blood and urine after Trimeresurus stejnegeri bite for the first time. Then, the function of the core metabolites was analyzed, the physiological and pathological mechanisms of the Trimeresurus stejnegeri bite were further discussed, and the complex relationship between the snakebite and the body’s reaction was solved, providing a theoretical basis for the prevention and early diagnosis of Trimeresurus stejnegeri bite.

This study is only preliminary experimental results, and it is necessary to expand the verification of metabolites to obtain more complete data. Then, absolute quantitative validation of candidate biomarkers is used for a large number of samples by targeted metabolomics.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (grant no.81860344) and the Guangxi Natural Science Foundation (grant no. 2021GXNSFAA075025).

Conflict of interest

The authors declare that there is no conflict of interest.

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