Serum lipidomics‐based study of electroacupuncture for skin wound repair in rats

Abstract Lipid metabolism plays an important role in the repair of skin wounds. Studies have shown that acupuncture is very effective in skin wound repair. However, there is little knowledge about the mechanism of electroacupuncture. Thirty‐six SD rats were divided into three groups: sham‐operated group, model group and electroacupuncture group, with six rats in each group. After the intervention, orbital venous blood was collected for lipid metabolomics analysis, wound perfusion was detected and finally the effect of electroacupuncture on skin wound repair was comprehensively evaluated by combining wound healing rate and histology. Lipid metabolomics analysis revealed 11 differential metabolites in the model versus sham‐operated group. There were 115 differential metabolites in the model versus electro‐acupuncture group. 117 differential metabolites in the electro‐acupuncture versus sham‐operated group. There were two differential metabolites common to all three groups. Mainly cholesteryl esters and sphingolipids were elevated after electroacupuncture and triglycerides were largely decreased after electroacupuncture. The electroacupuncture group recovered faster than the model group in terms of blood perfusion and wound healing (p < 0.05). Electroacupuncture may promote rat skin wound repair by improving lipid metabolism and improving local perfusion.


| INTRODUC TI ON
The skin is the largest organ of the body and is the first line of defence against external invasion and has properties that protect against microbial, mechanical, chemical, osmotic and thermal damage. 1,2Skin trauma is very common in surgery and is characterized by high morbidity and complications which adversely affect the quality of life of patients and cause high global health system costs. 3,4As patients' expectations of the speed and quality of wound healing increase, there is a need to seek additional treatment strategies that can promote rapid and high-quality healing of skin wounds.
Acupuncture is an important part of Chinese traditional medicine, and plays an important role in clinical treatment.Acupuncture can promote wound healing through anti-inflammatory effects improving microcirculation, and increasing re-epithelialization and angiogenesis. 5,6Electrical stimulation is widely used as a physical stimulus in the medical field and is gaining more and more attention in the area of skin wound repair. 7,8Electrical stimulation accelerates wound healing by promoting cell proliferation and migration and regulating the expression of growth factors and is a green therapy that mobilizes endogenous currents in the skin and corrects the internal environment of the wounds. 9ectroacupuncture, on the other hand, embodies the combination of traditional Chinese medicine and modern medicine, combining acupuncture with electrical stimulation to bring out its greater benefits.Electroacupuncture for wound repair is safe, non-invasive, effective and easy to use.However, the role and mechanism of electroacupuncture for cutaneous wound healing are less reported and need further study.
Skin wound healing involves several processes, including extracellular matrix remodelling, synthesis of pro-inflammatory mediators and angiogenesis.The new blood vessels formed by repair can promote tissue blood perfusion and is a key factor in determining the quality of skin wound healing.Therefore, blood perfusion is essential in skin wound healing. 10,11Human skin homeostasis requires the regulation of lipid metabolic balance and lipids secreted by sebocytes together with lipids derived from keratin-forming cells form an important component of the skin barrier. 12Chromatography-mass spectrometry (LC-MS) combines the capabilities of both HPLC and MS, significantly improving the analytical platform and providing a sensitive and specific tool for the identification and quantification of lipids. 13,14However, there are fewer reports on the mechanism of whether electroac upuncture can promote skin wound healing by regulating serum lipid metabolism.In order to better understand the mechanism of skin wound healing by electroacupuncture, we established a rat sk in model of total skin defect.This study focused on the changes in serum lipid metabolites, related lipid pathways and wound blood perfusion during electroacupuncture intervention in rats with total skin defects to promote skin wound repair.

| Experimental animals
Eighteen male SD rats (weight 160 ± 20 g) were grouped in the Animal Experimentation Centre of Zhejiang University of Traditional Chinese Medicine for 1 week after acclimatization.SD rats were purchased and fed, and other animal procedures followed the animal re-

| Model preparation
Combined with the group's previous modelling basis, a full-layer skin defect model was prepared.After anaesthesia, the modelling area (2 cm on the left and right side of the spine) was fixed at five points, trimmed and dehaired, rinsed with saline, disinfected with iodophor and deiodinated with ethanol.A 1*1 cm square model of the full skin defect was created using surgical scissors.Post-operatively, the wound was naturally haemostatic and the wound was kept dry to prevent wound infection.

| Grouping and processing
Eighteen SD rats were randomly divided into three groups of six rats each according to the table of random number methods, as follows: sham-operated group, model group and electroacupuncture group.(1) Sham-operated group: only hair clipping was done in the modelling area, and no wound model was made.(2) Model group: full skin defect model was prepared, and only iodophor was given daily for routine disinfection to prevent wound infection.(3)   Electroacupuncture group: Based on the operation of the model group, electroacupuncture treatment was started on the same day.
The central point of the wound and the normal skin at the edge of the wound (the edge of the midpoint of the four sides of the square wound) were selected and given electroacupuncture stimulation.
The negative electrode was located at the centre and the positive electrode was located at the edge of the midpoint of the four sides of the square wound.Continuous pulses with a frequency of 2 Hz and an output current of 0.3 mA was used to stimulate each point successively for 5 min at each point, once a day for a total time of 20 min to prevent wound infection.

| LC-MS model preparation
Orbital venous blood was taken from each group after 7 days.Take 200 μL of serum into a 1.5 mL centrifuge tube, add 800 μL of isopropanol and vortex for 1 min.After 30 min of ultrasonication in an ice bath, the samples were vortexed for 1 min and placed in a refrigerator at −20°C overnight.The next day, the centrifuge was pre-cooled to 4°C and centrifuged at 13000 rpm for 15 min.600 μL of Serum supernatant was taken, blown to dryness with nitrogen and 150 μL of isopropanol was re-dissolved and centrifuged at 13000 rpm for 15 min.100 μL of supernatant was taken into the injection vial for sample injection.The supernatant of the homogenate was centrifuged in equal amounts, blown dry with nitrogen and re-dissolved to prepare QC samples.The liquid conditions were set and the samples were analysed by UHPLC-QTOF/ MS.

| Mass spectrometry
To provide high-resolution detection, an X500B Q-TOF mass spectrometer (AB Sciex) equipped with an electrospray ionization source (Turbo Ionspray) was applied.MS detection was implemented both in negative and positive ion mode with the mass rang at m/z 150-1050.The parameters of the mass spectrometer were summarized as follows: gas1 and gas2, 45 psi; curtain gas, 35 psi.Heat block temperature, 550°C; ion spray voltage, − 4.5 kV in negative mode and 5.5 kV in positive; declustering potential, 50 V; collision energy, ±35 V; and the collision energy spread (CES) was ±15 V. To monitor the reproducibility and stability of the acquisition system, QC samples were prepared by pooling small aliquots of each sample.The QC specimens were analysed every six samples throughout the whole analysis procedure.

| Data processing
The raw profiles were extracted by SCIEX OS Analytics and transformed into a data matrix, mainly including information on the mass-to-charge ratio (m/z) and retention time (Rt) and peak area (intensity).All data were normalized by the total peak area and the Excel sheet was generated for subsequent metabolome analysis.To reduce signal interference from chance errors, variables with RSD ≥40% in QC were excluded in excel first.
The Excel files were imported into SIMCA 14.1 (Umetrics) software for multivariate mathematical and statistical analysis.Principal component analysis (PCA) was used to observe the overall distribution of the samples.In addition, the consistency of the samples within the group was analysed by PCA-Class analysis.Generally, when a sample falls outside the '2-std.dev.' line under a principal component, the sample was considered abnormal data and the sample data was considered to be excluded before the subsequent analysis.
The OPLS-DA replacement test statistically analyzes the validity of the OPLS-DA model when Q2 intersects with the y-axis at a negative value, indicating that the model is valid, which in turn screens for differential metabolites.Based on this model, the differential variables were screened according to the variable projection importance index (VIP value), and the variables with VIP >1 were generally considered to be meaningful variables causing the differences.The variables with a high impact on OPLS-DA model building were further screened by the partial correlation coefficient (pcorr).Finally, the screened variables were tested for significance (Mann-Whitney Test), and a p < 0.05 was considered a significant difference variable.Potential markers were identified by HMDB (http://www.hmdb.ca/)and LIPID MAPS (https://www.lipidmaps.org/).All differential metabolites involved in the three groups were summarized by Venn diagrams.Heat maps of the differential metabolites were produced to visualize the high and low levels of the response of the compounds after the intervention, and corresponding bar charts were produced for further analysis.Based on the results of the significantly different metabolites screened and identified, the compound name results for each group were imported into Metabo Analyst 5.0 (http://w w w.Metab oAnal yst.ca/) for metabolic pathway analysis.

| Laser doppler perfusion imaging test
The groups were examined at 1d, 4d and 7d using laser Doppler perfusion imaging with a distance of 10 cm between the probe and the test object and an imaging range of 1.0 cm × 1.0 cm. and the PIMSoft software was applied to the record, analyse, process and store the body surface flow maps.The changes in perfusion in each group were compared.

| Post-operative wound observation
The model and electroacupuncture groups were photographed in 1d, 4d and 7d using a digital camera, and trauma healing was analysed using image-pro Plus 6.0 image analysis software.Percentage of trauma healing = [(original trauma area -trauma area at the time of observation) ÷ original trauma area] × 100%.

| Histological testing
Skin tissues of each group were taken from the moulded area after 7 days, fixed with 4% PFA solution for more than 24 h, dehydrated, and paraffin-embedded to make 4 um sections.H&E and Masson staining were performed, and after completing the steps, the sections were dehydrated and sealed, observed under the microscope and photographed for comparison.

| Statistical analysis
All of the experimental results were expressed as the mean ± SD (standard deviation).All statistical analyses were performed using SPSS 21.0 software.The significance of differences between groups was determined by 2-tailed unpaired Student's t-test or one-way anova with Dunnett's post hoc test when samples were not distributed normally.A value of p < 0.05 was considered to be statistically significant.within the group was analysed by PCA-Class analysis, and all data could be retained as seen from the results in Figure 1 (C-F) and Figure 1 (G-J).

| Post-modelling affects 11 lipid metabolites in serum
Figure 2 (A-F) shows the OPLS-DA plots for model versus shamoperated, which shows that the two groups are clearly differentiated and the model is valid.Figure 2 (G-H

| Different metabolite profiles in different groups
PLS-DA analysis was performed for all groups to observe the trend of the overall metabolic group of the three groups.The results are shown in Figure 6 (A-D), model, electro-acupuncture and sham-operated were distinguishable between all three groups, the model group was     4), mainly included glycerol esters, phospholipids and sphingolipids, and the trend of response changes after electroacupuncture was the same, which likewise indicated that the electroacupuncture had a much more pronounced effect on the serum lipid group than the model group.

| Metabolomics pathway analysis of SD rat venous serum after electro-acupuncture
The results of pathway enrichment analysis from different lipid classification levels showed that electroacupuncture mainly affected the lipids associated with four pathways: cholesteryl esters, sphingolipids, glycerides and phospholipids (the redder colour, the more significant the expression of the pathway; the longer the bar or the larger the point, the more lipids matched in the pathway).And the metabolic pathways were further refined and analysed from the intermediate and lowermost lipid categories, see Figure 7 (A-F).

| Post-operative wound perfusion volume and area changes
On post-operative day 1, the haemoperfusion volume in the model and electroacupuncture groups was comparable but significantly greater than that in the sham-operated group, and the difference was statistically significant (p < 0.05).On post-operative days 4 and 7, the perfusion volume of the model group and electroacupuncture group gradually decreased, but the perfusion volume of the model group was greater than that of the other two groups, and the difference was statistically significant (p < 0.05) and the results are shown in Figure 8 (A,B).On post-operative days 4 and 7, the wound healing rate of the electroacupuncture group was faster than that of the model group, and the difference was statistically significant (p < 0.05), and the results are shown in Figure 8C.| 3137 DU et al.

| Histological and immunohistochemical results
On the 7th post-operative day, epithelial cells and fibroblasts were hyperplastic in the electro-acupuncture group, collagen fibres were densely arranged, muscle fibres were more abundant and a thinner new epidermal layer was visible.And the skin repair status of the model group was weaker than that of the electroacupuncture group.See Figure 8A.

| DISCUSS ION
The stratum corneum (SC) of the skin plays a key role in the formation and maintenance of the skin barrier, and lipids are an important component of the SC, forming a well-structured lipid matrix. 15,16fferentiated keratin-forming cells in the epidermis synthesize cholesterol, ceramide (CER), and free fatty acids (FFA), which form the major lipid classes present in the SC. 17 Therefore, the metabolic processes of these lipids may play a key role in the skin wound repair process.
In this study, changes in lipid metabolism of serum were found in both the electroacupuncture and model groups.There were 11 differential metabolites for model versus sham-operated, 115 differential metabolites for electro-acupuncture versus model, and 117 differential metabolites for electro-acupuncture versus sham-operated.There are two differential lipids common to all three groups.And 94 metabolites that overlap between electroacupuncture versus model and electro-acupuncture versus shamoperated (Figure 4), mainly included glycerol esters, phospholipids and sphingolipids, and the trend of response changes after electroacupuncture was the same.Further metabolic pathway analysis revealed that electroacupuncture mainly affected lipids associated with four pathways: cholesteryl esters, sphingolipids, glycerol esters and phospholipids, which was consistent with the results of differential metabolite analysis.Yeom M et al 18 have used electroacupuncture to treat hyperlipidaemia rats, and the results showed that it can significantly reduce the content of triglyceride and total cholesterol in serum and improve the level of serum lipid metabolism.In addition, the haemoperfusion volume and wound healing rate of the electroacupuncture group was faster than that of the model group.
Histological and immunohistochemical also revealed that the electroacupuncture group had faster skin repair than the model group.
These results suggest that electroacupuncture may promote skin study may provide a safer and more effective complementary and alternative therapy for skin wound healing and provide an experimental basis for the study of the mechanism of electroacupuncture to promote skin wound healing.
Phospholipids not only constitute an important component of cell membranes, but also participate in the regulation of various cellular functions and play an important role in skin wound repair.Transcriptomic and lipidomic data revealed that Narciclasine extensively regulated lipid metabolism-related genes, especially the Phospholipase A2 (PLA2) family, and increased anti-inflammatory lipid molecules. 19Lysophospholipids and FFA (especially arachidonic acid) are the main products of phospholipids catalysed by PLA2. 20Among them, lysophosphatidylcholine (lysoPC) stimulates leukocyte activation, Tlymphocyte chemotaxis and inflammatory cell accumulation. 21,22ostaglandins (PG) and leukotrienes (LT) produced by the PLA2mediated arachidonic acid cascade reaction can activate nuclear factor-kB through the tumour necrosis factor signalling pathway, thus effectively promoting the division and activation of keratin-forming cells. 23It has also been shown that exogenous phosphatidylcholine accelerates the wound healing process, allowing keratinocytes to complete proliferation and migration to form a new layer of keratin in a relatively short period of time. 24Oral administration of lactophospholipids to hairless mice exposed to UV-B radiation-induced photoaging resulted in downregulation of protein expression of nuclear factor kappa-B (NF-κB) and phosphorylated IκBα (κBα inhibitor), a significant decrease in the expression of pro-inflammatory cytokines, especially tumour necrosis factorα, and improved skin hydration and barrier function. 25The skin barrier-improving effect of lactoferrin appears to be associated with the activation of Nrf2-keap1, which is associated with ROS scavenging.Lactoferrin activates Nrf2 and increases the expression of the antioxidant enzyme HO-1, thereby reducing ROS produced by UV exposure. 26In addition, phosphatidylinositol also plays an important role in wound repair.Phosphatidylinositol is involved in the regulation of multiple signalling pathways in skin cells, including cell proliferation, apoptosis, and differentiation. 27,28Li J et al 29 have  This may be a metabolic mechanism of action of electroacupuncture to promote wound repair.
Sphingolipids are biologically active lipid molecules that are widely present in cell membranes and are involved in the regulation of a variety of life activities, being key components of skin barrier homeostasis and cellular processes such as apoptosis and stress response. 30,31Impaired sphingolipid metabolism underlies several common skin pathologies, including atopic dermatitis, psoriasis and aging. 32,33There are few studies on the effects of electroacupuncture on sphingolipid metabolism in skin repair.However, other studies have shown that electroacupuncture can regulate the changes of lipid metabolism in mice with post-traumatic stress disorder, especially the content changes of sphingolipids, glycerides and fatty acyl groups. 34In the sphingomyelinase pathway, CER is formed by hydrolysis of sphingomyelins in the granular layer of the skin by sphingomyelinase.CERs are considered to be one of the most important epidermal sphingolipids, accounting for approximately 50% of the intercellular lipids of the SC 35,36 The CERs are transported from the endoplasmic reticulum to the Golgi apparatus, where the conversion to glucosyl CERs or sphingomyelin occurs. 37Signalling pathways such as protein kinase B (Akt), protein kinase C (PKC), mitogenactivated protein kinase (MAPK), Jun amino-terminal kinase (JNK), or phospholipase D (PLD) are regulated during CER stimulation. 38Although the importance of CER in skin cell proliferation and differentiation has long been known, in recent years sphingosine 1-phosphate sphingosine (S1P) has also been found to be involved in processes such as the proliferation and differentiation of keratin-forming cells.The ability of FTY720 to inhibit lymphocyte efflux was significantly diminished as shown by using a rat model with internalization-resistant S1PR1. 39In S1PR1 knockout mice, enhanced macrophage migration is eliminated during inflammation regression. 40 search guidelines of the National Institutes of Health and the Animal Research Committee.And approved by the Experimental Animal Ethics Committee of Zhejiang University of Traditional Chinese Medicine (NO.IACUC-20220221-19).

Figure 1 (
Figure 1(A),1(B) show the QC Base Peak Chromatograms (BPC) in positive and negative ion modes.The Base Peak Chromatogram is a continuous depiction of the most intense ion intensity obtained at each time point, and the BPC contains the ion intensity as well as the retention time of the ion in the chromatogram.Figure 1(C), Figure 1(G) show the PCA plots of all samples in positive and negative ion mode, respectively.It can be seen that the QC samples are more tightly clustered, indicating that this experiment has good stability and reproducibility.The consistency of the samples

Figure 2 (
Figure 2 (J-O) shows the OPLS-DA plot for electro-acupuncture versus sham-operated, with a clear distinction between the two.

Figure 2 (
Figure 2 (P,Q) shows the volcano plot for electro-acupuncture versus sham-operated, with red colour indicating variables upregulated after electro-acupuncture, blue colour indicating variables downregulated after electro-acupuncture, and grey colour indicating variables with no difference.The 117 differential metabolites of electro-acupuncture versus sham-operated were screened and identified, and it was seen that cholesteryl esters and sphingolipids were elevated after electro-acupuncture, triglycerides were largely decreased after electro-acupuncture with some triglycerides upregulated, and phospholipids showed no significant change pattern, the results are shown in Table 2. Detailed response changes are shown in Figure 3.

3. 2 . 3 |
Figure 4 (A-F) shows the OPLS-DA plots for electro-acupuncture versus model, which shows that the two groups are clearly differentiated and the model is valid.Figure 4 (G-H) shows the volcano plot for electro-acupuncture versus model, with red colour indicating variables that were upregulated after electroacupuncture, blue colour indicating variables that were downregulated after electroacupuncture, and grey colour indicating variables with no difference.The 115 differential metabolites of the electro-acupuncture versus model were screened and identified, and the pattern of differential metabolite changes was consistent with the results of 3.2.2, and the results are shown in Table 3. Detailed response changes are shown in Figure 5.
The TIC diagram of QC samples in positive and negative ion mode.(A) Negative ion mode, (B) Positive ion mode.Figure 1 (C-F) PCA analysis of negative ions.(C) PCA analysis of all samples (R 2 X 0.770, Q 2 0.450).(D) PCA-class analysis of sham-operated group.(E) PCA-class analysis of model group.(F) PCA-class analysis of electro-acupuncture group.

TA B L E 2
(Continued) relatively close to the sham-operated group and the electroacupuncture group was more clearly distinguished from the other two groups, suggesting the overall lipidome migrated more after electroacupuncture, which is generally consistent with previous results.All differential metabolites involved in the three groups were summarized by venn diagram (Figure 6E): the red box surrounded by the number of differential lipids for model versus sham-operated, 11 in total; the blue box surrounded by the number of differential lipids for electro-acupuncture versus model, 115 in total; and the green box surrounded by the number of differential lipids for electro-acupuncture versus sham-operated was 117.There are only two differential lipids common to all three groups.And 94 metabolites that overlap between electro-acupuncture versus model and electro-acupuncture versus sham-operated (Table

F I G U R E 3
Bar chart of electro-acupuncture versus sham-operated differential metabolites.Red indicates an increase in the electroacupuncture group and green indicates a decrease in the electroacupuncture group.(A) Sphingolipids.(B) Cholesteryl esters.(C) Glycerides.(D) Glycerophospholipids.

TA B L E 3
wound repair in rats by mobilizing the lipid metabolism function of serum and improving local haemoperfusion volume.Therefore, our F I G U R E 4 (A-F) OPLS-DA: Electro-acupuncture versus Model; (A) Negative ions (R 2 X 0.736, R 2 Y 0.999, Q 2 0.949).(B) Positive ions (R 2 X 0.892, R 2 Y 1.000, Q 2 0.933).(C) Negative ion replacement test.(D) Positive ion replacement tests.(E) Negative ion S-plot.(F) Positive ion S-plot.Figure 5 (G, H).Volcano diagram.(G) Negative ions.(H) Positive ions.①Fold change (E/M) > 1.2 or <0.83, and ②p < 0.05 was the effective variable.Blue means down, red means up.Basic information of differential metabolites of electro-acupuncture versus model.
reported that electroacupuncture treatment can effectively decrease the over-expression of related factors of phosphatidylinositol system in rats with acute cerebral infarction, improve cerebral autonomy movement, and alleviate cerebral vascular spasm.Our study revealed that phospholipid serum metabolites and metabolic pathways were mobilized by electroacupuncture intervention.It suggests that electroacupuncture may promote the balance of phospholipid metabolism by inhibiting excessive oxidative stress, accelerating SC recovery, regulating cell proliferation and migration, and suppressing inflammatory responses.
Incubation of macrophages with S1P appears to have an anti-inflammatory effect, as the production of the pro-inflammatory cytokines TNFα, IL-6, IL-12 and CCL2 is significantly reduced after activation by lipopolysaccharide F I G U R E 5 Bar chart of electro-acupuncture versus Model differential metabolites.Red indicates an increase in the electroacupuncture group and green indicates a decrease in the electroacupuncture group.(A) Sphingolipids.(B) Cholesteryl esters.(C) Glycerides.(D) Glycerophospholipids.(LPS).41In addition, by increasing sphingolipid content and altering sphingolipid metabolic pathways, cell proliferation and differentiation can be promoted, which in turn promotes wound healing.42,43Dietary Sphingomyelin may improve skin barrier function by altering skin inflammation and covalently-bound ω-hydroxy CERs and may promote epidermal keratinized envelope formation by altering inflammation-associated gene expression.44Our study found that sphingolipids and their metabolic pathways migrated more after electroacupuncture intervention relative to the sham-operated and model groups, suggesting that electroacupuncture may have promoted skin wound healing by promoting intra-traumatic cell migration and regeneration, regulation of inflammatory response and immune response, enhancement of phagocytosis of immune cells, and improvement of the consequent skin barrier function.This may be another metabolic mechanism of action of electroacupuncture to promote skin wound healing.In conclusion, as shown in Figure9, our results suggest(that metabolites and metabolic pathways such as serum cholesteryl esters, sphingolipids, glycerides and phospholipids migrate more after electroacupuncture intervention relative to the sham and model groups and may play a key role in the mechanism of action of electroacupuncture to promote skin wound repair.In the process of skin wound healing in rats, electroacupuncture treatment helped restore the amount of wound perfusion, could promote the proliferation of epithelial cells and fibroblasts, attenuated the inflammatory response and lipid peroxidation in rat wounds, and improved serum lipid metabolism in rats.Although there are some limitations of our study, such as the urgent need for more experimental studies to verify the mechanism of action of electroacupuncture intervention on the regulation of serum lipid metabolism during skin wound repair, our results provide a new direction for the treatment of skin wound repair.AUTH O R CO NTR I B UTI O N S weibin du: Funding acquisition (lead); project administration (equal); writing -original draft (lead); writing -review and editing (lead).Lihong He: Conceptualization (equal); writing -original draft (equal); writing -review and editing (equal).Zhenwei Wang: Conceptualization (equal); data curation (equal); writing -original draft (equal).Yi Dong: Conceptualization (equal); data curation (equal); formal analysis (equal).Xiaofen He: Formal analysis (equal); resources (equal); supervision (equal).Jintao Hu: Funding acquisition (equal); methodology (equal); supervision (equal).Min Zhang: Methodology (lead); project administration (lead); supervision (lead); writing -review and editing (lead).

F
I G U R E 6 (A-D) PLS-DA diagram of all samples.(A) PLS-DA plot in negative ion mode (R 2 X 0.721, R 2 Y 0.963, Q 2 0.844), (B) PLS-DA plot in positive ion mode (R 2 X 0.777, R 2 Y 0.907, Q 2 0.741), (C) Negative ion replacement test, (D) Positive ion replacement test.Figure 7E Venn diagram of the number of differential metabolites.The red box Model versus sham-operated, the blue box electro-acupuncture versus Model and the green box electro-acupuncture versus sham-operated.| 3143 DU et al.

7
Lipid metabolism enrichment analysis.(A) Top lipid classification, enrichment analysis bar chart.(B) Top lipid classification, enrichment analysis dot plot.(C) Intermediate lipid classification, enrichment analysis bar chart.(D) Intermediate lipid classification, enrichment analysis dot plot.(E) The lowest lipid classification, enrichment analysis bar chart.(F) The lowest lipid classification, enrichment analysis dot plot.F I G U R E 8 (A) The wounds of the three groups at different time points after surgery, the corresponding blood perfusion imaging images and the histological examination results of the three groups at 7 days after operation (X200) were compared.(B) Comparison of wound blood perfusion volume in the three groups at different time points after surgery.(C) Comparison of wound healing rate between Model group and electro-acupunture at different time points after surgery.
Common differential lipid information after electroacupuncture.
TA B L E 4