Metabolic profiles in plasma of patients with herpes labialis based on GC–MS

This study aims to compare the plasma metabolic profiles of patients with herpes labialis with healthy controls and identify the biomarkers of herpes labialis.

the primary symptoms of herpes labialis are mild and self-limited, 3,4 herpes labialis may frequently relapse and lead to unexplained pain, 5,6 neurodegeneration, 7 cognitive dysfunction, 8 and atherosclerosis. 9However, since herpes labialis or infection with HSV have an unclear biological basis, their relationship with these specific diseases is still unclear.
In traditional Chinese medicine (TCM), herpes labialis is called "Kou-Chuang", which can be categorized as "Shang-Huo" based on the clinical manifestation of obvious pain, redness, and swelling in the area of the ulceration.The term "Shang-Huo" indicates an exuberant endogenous fire in the human body.It is characterized by "redness, swelling, fever, and pain" in the skin and mucosa of the head and face. 10According to modern medicine, there are many factors contributing to "Shang-Huo" including microbiome disorders and immunological abnormalities. 11ss spectrometry (MS) is a method for analyzing the diverse biological properties of small molecules that result from metabolic pathways. 12The most common separation methods for MS are gas chromatography (GC) and liquid chromatography (LC).4][15] Here, we use GC-MS to compare the plasma metabolic profiles of patients with herpes labialis with healthy controls and identify biomarkers associated with herpes labialis.

| Patients
We collected samples from 20 healthy controls and 18 patients with herpes labialis at the First Affiliated Hospital of Zhejiang Chinese Medicine University.Informed consent was obtained from all participants after a detailed explanation of the procedure was given.
This study was approved by the institutional review board (IRB) of Zhejiang Chinese Medicine University (IRB number: 2014zjtcm-001).Herpes labialis is clinically characterized by erythema and blisters around the lips, nostrils, and cheeks.Diagnostic criteria of herpes labialis followed the textbook compiled by Chen, 16 including major aphthae and minor aphthae with recurrent onset.The basic information of all participants is shown in Table 1.The age (29 ± 8 for patients with herpes labialis vs. 26 ± 3 for healthy controls, p = 0.199, unpaired t-test) and gender (female: 72.2% for the patient with herpes labialis vs. 70.0%for healthy control, p = 1.0,Fisher's exact test) were comparable between groups.The inclusion and exclusion criteria were based on recent studies. 17

| Blood sample collection
Samples were prepared as in our previous study. 18Briefly, venous blood was collected from the fasting patients in the morning and stored in EDTA coated tubes.The samples were centrifuged at 1000g for 15 min.The plasma was stored at −80°C until further analysis.

| GC-MS analysis conditions
Agilent 7890/5975C GC-MS (Agilent Technologies.)was used to run the samples.Briefly, 1 μL of sample was injected into a quartz capillary column of dimensions (DB-5 MS, 30 m × 0.25 mm × 0.25 μm, J&W Scientific.).Helium (99.9996%) was used as the carrier gas at the flow rate of 1.2 mL/min.The column oven temperature was initially set at 70°C for 3 min, and then increased to 300°C across 5 min at a rate of 5°C/min.The split ratio was set to 5:1, the inlet temperature was 300°C, the transmission line temperature was 280°C, and the temperature of ion source (EI) was 230°C.The scanning range was m/z 33 ~ 600, and the solvent delay time was 4.8 min.

| Data analysis
The raw GC-MS data was first transformed into CDF format files by the MSD ChemStation.Then, peak integration and alignment were performed using XCMS toolbox (the parameters: snthresh = 10, TA B L E 1 The basic information of participants.

| Comparing metabolic profiles between patients with herpes labialis and controls
The representative total ion current chromatogram is shown in (Figure 1A,B).Multivariate analysis was used to process the data.
As seen in the PCA score diagram, controls and patients had separate trends based on PC1 and PC2 (Figure 1C).Further analysis using PLS-DA showed a clear separation between the herpes labialis and control samples (Figure 1D).

| Differential plasma metabolites in the plasma of patients with herpes labialis
In the total ion current profiles, 1368 valid peaks were identified.
Based on variable importance in the projection (VIP) value (threshold >1)and Student's t-test (p < 0.05), 296 different metabolites with significant differences between the patient and the control group were screened.The possible structures of the metabolites were matched to the substances in the NIST library according to their mass spectra (matching degree >700), and the reliability of the results was verified by standard products.Finally, 12 metabolites were structurally identified with matching degree >700 and the heatmap of normalized intensity of these differential metabolites was shown in Figure 2.Among them, sedoheptulose and ethylamine were upregulated, while lactic acid, pyroglutamic acid, alanine, ethanedioic acid, cyclohexaneacetic acid, pyruvic acid, d-mannose, phosphoric acid, l-amphetamine, and citric acid were downregulated in patients with herpes labialis (Table 2).(TCA cycle) pathway, pyruvate metabolism pathway, glycolysis/gluconeogenesis pathway, glyoxylate, and dicarboxylate metabolism pathway.Of note, as shown in Figure 3B, citrate cycle (TCA cycle) pathway, pyruvate metabolism pathway, and glycolysis/gluconeogenesis pathway were connected in the network analysis, indicating that herpes labialis may affect the amino acid and energy metabolism.The details were shown in Table 3.

| DISCUSS ION
Herpes labialis results in blisters or erosions on the lips and skin around the mouth and nose, which is also one of the common symptoms regarding "Shang Huo" in TCM.Here, we focused on the metabolic changes in plasma patients with herpes labialis by GC-MS.We found (1) PLS-DA model based plasma metabolic profiles of patients with herpes labialis were significant discriminated from control; (2) totally 12 metabolites of difference were screened, of which two were upregulated and 10 were downregulated; (3) KEEG pathway analysis based on these metabolites indicated that the alanine, aspartate, and glutamate metabolism pathway may be downregulated and citrate cycle pathway and pyruvate metabolism pathway may be upregulated in patients with herpes labialis.Thus, our findings may contribute to elucidating the metabolic basis of herpes labialis and provide a new perspective for further research on "Shang Huo" state in TCM.
GC-MS is a highly sensitive and accurate assay that can be used to detect changes in the plasma metabolites. 20In our study, we identified 12 metabolites that were differentially expressed between the patients with herpes labialis and healthy controls.It has been reported that inflammatory factors, which produced during the development of herpes labialis, may stimulate the requirement of energy. 21Here we found that several intermediate metabolites in the tricarboxylic acicycle (TCA) cycle, such as pyruvate and citric acid, are significantly reduced due to excessive consumption to produce energy in the plasma of patients with herpes labialis.Pyruvate can be decomposed into acetyl CoA and enter the tricarboxylic acid cycle to produce energy, and it can also aminosylate alanine in the glycolysis pathway. 22,23Here we also found that the levels of alanine and lactic acid in pyruvate metabolism were downregulated in patients with herpes labialis.Alanine, as glucogenic amino acids, could be turned into glucose to provide energy. 24Taken together, as the levels of intermediate metabolites in the TCA cycle, pyruvate metabolism, and glycolysis/gluconeogenesis, which are directly or indirectly related to energy production, were all downregulated, our study suggested that the glucometabolism is increased in patients with herpes labialis.This phenomenon may be due to the growing demand for energy in patients with herpes labialis.
Fatty acids are important nutrients and metabolites in the body. 25We found acid ethylamine was upregulated.However, none of the other fatty acid substances was changed, and the ketone bodies changes were not detected.These results indicate that the upregulated fatty acid may not to be used for producing energy in patients with herpes labialis, though the glucometabolism is increased as discussed above.These results indicates a potential increase in fat mobilization in patients with herpes labialis.Besides, we found that seroheptanose was upregulated, and D-mannose and cyclohexanoacetic acid were downregulated in patients with herpes labialis.Patients with herpes labialis may metabolize energy through the conversion of ketoalcohols and aldoalcohols in the pentose phosphate pathway and provide raw materials for the biosynthesis of fatty acids and cholesterol.Thus, our results also provide metabolic evidence to support the previous finding that infection of HSV may contribute to atherosclerosis, 26,27 but may be not related to obesity. 28V is the main cause of herpes labialis and there is no proven method of eradicating HSV completely to date.Metabolism is a crucial frontier of host-virus interaction as viruses rely on their host cells to provide nutrients and energy for propagation.29,30 In these studies, a total of 12 metabolites were significantly changed in plasma of herpes labialis patients, and most of them were amino acids and tricarboxylic acid (TCA) cycle intermediates.KEEG indicates that glycolysis and amino acid metabolism are the most significant pathways of change in plasma of herpes labialis patients.
Consistent with our findings in herpes labialis patients, Abrantes et al. found that HSV-1 increased glucose uptake, lactate efflux, and ATP content in HSV-1 infected cells. 31Our results indicate that in herpes labialis patients, HSV-1 infection promoted host TCA cycle and amino acid metabolism, suggesting that the virus has evolved to reprogram the hub of cellular metabolism to create a favorable environment for its replication.Besides, citric acid acts as a bridge between carbohydrate and fatty acid metabolism, which promotes the proliferation and differentiation of immune cells such as B cells. 32Pyroglutamic acid was produced by the Inclusion criteria of the patients were as follows: (a) meeting the clinical diagnosis of herpes labialis; (b) presence of red skin lesions with clusters of papulovesicles, and painful, itchy blisters and pus-filled scabs that peel off due to skin erosion; (c) accompanying fever in patients with low resistance and repetitive infections, and (d) presence of lesions in and around the lips, nostrils or cheeks, and other parts.In addition, health adults without any above symptom above were included for comparison.Exclusion criteria of all participants were as follows: (a) patients with psychosis, cancer, autoimmune diseases, and any severe diseases were excluded; (b) pregnant and breastfeeding women were excluded; (c) patient who used any medicine within one mouth were also excluded.
bw= 10).The obtained peak area was normalized to the total peak area and entered into the Simca-P software for further analysis.Principal components analysis (PCA) was conducted to observe the metabolic differentiation between healthy controls and patients with herpes labialis and measure the intragroup variance.The partial least squares-discriminant analysis (PLS-DA) was used after the metabolic data were filtered (orthogonal signal correction) to classify intragroup plasma samples.According to the VIP and Student's ttest, the significantly different metabolites were screened and were structurally identified with the deconvolution and NIST 2011 standard spectrum search strategy.Some metabolites were verified with standards.Pathway analysis were conducted by MetaboAnalyst (https://www.metaboanal yst.ca).19 3 | RE SULTS

Five
related KEEG pathways were found with p < 0.05 based on all these different metabolites in patients with herpes labialis, including alanine, aspartate and glutamate metabolism pathway, citrate cycle F I G U R E 1 PCA and PLS-DA plot.(A) The total ion chromatogram of the plasma metabolites of patients with herpes labialis.(B) The total ion chromatogram of the plasma metabolites of healthy controls.(C) PCA, principal components analysis.(D) PLS-DA, partial least squaresdiscriminant analysis.| 3155LUO et al.

F I G U R E 3
Enriched metabolic pathways of the different metabolites.(A) Dot Plot view of the enriched metabolite sets.(B) Network View of the enriched metabolite sets.TA B L E 3 Details of Enriched metabolic pathways of the different metabolites.
Heatmap of different metabolites.
TA B L E 2 Summary of the different metabolites.