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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 2 | Page : 131-140
Effects of acupuncture at Tianshu (ST25) and Shangjuxu (ST37) on the intestinal mucosa and metabolites of local skin tissues at Tianshu (ST25) in 2,4,6-Trinitrobenzene sulfonic acid and ethanol-induced rats
Jing-Ying Zhou1, Yi-Tian Lai2, Ling Ren2, Lei Lan3, Guo-Shan Zhang2, Mi Liu1
1 Department of Acupuncture and Moxibustion, College of Acupuncture, Moxibustion and Tuina, Hunan University of Chinese Medicine, Changsha, Hunan, China
2 Department of Professional Acupuncture and Moxibustion, College of Acupuncture, Moxibustion and Tuina, Hunan University of Chinese Medicine, Changsha, Hunan, China
3 Michael G. DeGroote Institute for Pain Research and Care, McMaster University, Hamilton, Ontario, Canada
Correspondence Address:
Prof. Guo-Shan Zhang
College of Acupuncture, Moxibustion and Tuina, Hunan University of Chinese Medicine, Changsha, Hunan
China
Prof. Mi Liu
College of Acupuncture, Moxibustion and Tuina, Hunan University of Chinese Medicine, Changsha, Hunan
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.372729
Objective: To observe the effect of acupuncture on local skin tissue metabolites of Tianshu (ST25) in rats with Crohn's disease (CD) using metabolomics and to explore the mechanism of acupuncture treatment metabolites and pathway on local skin tissue of Tianshu (ST25). Materials and Methods: Thirty-eight Sprague − Dawley rats were randomly assigned to a normal group (n = 14) and a CD modeling group (n = 24). Rats in the CD modeling group were administered with 2,4,6-trinitrobenzene sulfonic acid and ethanol enema, in addition to the normal group. Four normal rats and four CD modeling rats were selected at random after model identification was established. Furthermore, CD modeling group rats were randomly assigned to two groups of 10 rats: the model group and acupuncture group. Rats in the acupuncture group underwent acupuncture of Tianshu (ST25) and Shangjuxu (ST37) for 7 days. After the intervention, the colon tissue was collected from each group of rats and the pathological changes were observed through hematoxylin and eosin (HE) staining. The local skin tissues at Tianshu (ST25) of the rats in each group were taken, and the proton nuclear magnetic resonance technique was used to detect the metabonomics of the local skin tissues of Tianshu (ST25) in rats. Results: Compared with the normal group, the following were observed in the model group after HE staining: irregular colon morphology and other pathological changes such as intestinal mucosal hyperemia, edema, ulcers, polyps, and inflammatory cell infiltration. In the acupuncture group, colon tissue structure was relatively complete and layered, the colon gland structure was restored, and inflammatory cell infiltration was significantly improved. The choline, glycerin, glycine, guanidoacetic acid, and proline levels were significantly higher in the model group than in the normal group (P < 0.05 or P < 0.01). Compared with the model group, the acupuncture group had significantly increased contents of alanine, leucine, L-phenylalanine, and tyrosine in the skin (P < 0.05 or P < 0.01). Valine, leucine, and L-isoleucine biosynthesis and L-phenylalanine metabolism were the main metabolic pathways involved in the changes in the local skin tissues of the rats in each group, biosynthesis of tyrosine, and tryptophan. Conclusion: The change in the metabolites in the local area of Tianshu (ST25) may be related to intestinal disease. The acupuncture of Tianshu (ST25) can improve intestinal inflammatory reaction in rats with CD, and this finding may be related to the regulation of amino acid metabolites and their pathways in the local acupoint tissues of Tianshu (ST25) by acupuncture.
Keywords: Acupuncture, Crohn's disease, metabonomics, point, Tianshu (ST25)
Croshn's disease (CD) is a chronic intestinal infection of unknown etiology and often involves the terminal ileum and adjacent colonic ends. Patients with CD often have diarrhea, abdominal pain, abdominal lumps, fistula formation, intestinal obstruction, and so on.[1] In clinical practice, the treatment of CD mainly includes traditional drugs and biological agents such as aminosalicylic acid, immunosuppressants, and intestinal flora inhibitors. However, there are no specific drugs that can be used for treatment. As a result, most patients eventually require surgery. Acupuncture, which is an important part of traditional Chinese medicine, is safe, convenient, low cost, and environmentally friendly. Acupuncture has been widely used in clinics and has made great progress in the treatment of chronic digestive diseases. Studies have shown that acupuncture can dynamically regulate CD with multiple targets and links, thereby reducing the symptoms of abdominal pain, diarrhea, and bloating.[2] By quantitatively and qualitatively analyzing metabolites, metabolomics comprehensively evaluates the functional state of life at that time and its changes as a whole; this process is consistent with the idea of acupuncture, which is based on the holistic view of traditional Chinese medicine. The effect of acupuncture comes from the exogenous stimulation and biofeedback of human tissues and organs and ultimately plays a multilink, multilevel, multitarget regulatory role overall.[3] The meridians–internal organs theory of acupuncture indicates that acupuncture points are one of the most important links in the body. Diseases of the internal organs can cause various abnormal changes in some acupuncture points on the body surface through the qi of meridians, and this pathological reaction of acupuncture points has important implications for disease diagnosis and treatment. As the Front-Mu point of the large intestine, Tianshu (ST25) is a pathologic reaction point to intestinal diseases and is an important part of treating intestinal diseases. In the current study, metabolomics was applied to analyze the metabolites quantitatively and qualitatively in the local skin tissues of Tianshu (ST25). Metabolomics was also combined with the histopathological morphological observation of the colon to systematically evaluate the effect of acupuncture on metabolites in the local skin tissues of Tianshu (ST25) in CD rats. This study aimed to investigate the mechanism of acupuncture in the treatment of CD in terms of local skin metabolites and pathways.
Materials and MethodsAnimals
Thirty-eight specific-pathogen free Sprague−Dawley rats weighing 150–180 g were obtained from the Laboratory Animal Center of Xiamen University (experimental animal license no. SYXK [Min] 1216-0006; animal approval no. 20170208). The temperature of the breeding room was controlled at 21°C–25°C, and the humidity was controlled at 45%–70%. After 7 days of adaptation feeding, the rats were completely randomized into 14 normal groups and 24 modules for model preparation. After the model was completed, four normal rats and four model rats were randomly selected for model evaluation. When the models were developed, all the remaining model rats were divided into the model group and acupuncture group, with each group having 10 rats. The operation and treatment methods in the experimental research were performed according to the guiding opinions of the Guide for the Care and Use of Laboratory Animals approved by the ethics committee of the Hunan University of Chinese Medicine.
Major reagents and devices
2, 4, 6-Trinitrobenzene sulfonic acid (TNBS) was obtained from Sigma Corporation (USA). The BA110S 1/10,000 electronic balance was obtained from Sartorius AG (Germany). The RMB235 paraffin pathology microtome was acquired from Leica (Germany). The BM-II pathological tissue embedding machine was from the Anhui Institute of Electronic Sciences (China). The Bruker AVANCE III HD 850 MHz magnetic resonance imaging instrument was obtained from Bruker AG (Germany). The nuclear magnetic resonance (NMR) suite was from Chenomx (Canada).
Model development and evaluation criteria
TNBS was applied for modeling intestinal fibrosis in CD.[4],[5] To create TNBS enema, 5% (W/V) of TNBS was mixed with 50% ethanol at a ratio of 2:1. The rat enema dose was 3 ml/kg TNBS enema solution in the model group, and the normal group was injected 0.9% sodium chloride. Enema administration was repeated once every 7 days for four times. The model was successfully prepared if the rat has pebble-like changes in the lining of the colon, thickening of the intestinal wall, localized ulcers and polyps, microdeletion of the local mucosal epithelial layer, formation of cleft ulcers, damage to the mucosal glands, tissue edema, and inflammatory cell infiltration in autologous stroma and submucosal tissues.
Intervention methods
According to Experimental Acupuncture Science[6] and the anthropomorphic comparison method, Tianshu (ST25) is located 40 mm below the synchondrosis xiphosternalis, 5 mm next to the anterior midline, and at the rectus abdominis muscle and its sheath. Shangjuxu (ST37) is located approximately 10 mm below the fibula of the capitulum fibulae of the rat outer knee.
After successful modeling, rats in the acupuncture group were fixed in the supine position on a rat plate. The abdomen and limbs were exposed, and the rat acupuncture points were partially disinfected with 75% alcohol. A No. 25 Huatuo stainless steel millineedle (1 in) was inserted into the bilateral sky hub and upper giant void at a depth of 3–5 mm. During the treatment, a 20 s twist was performed, and the needle was retained for 15 min. Another 20 s twist was performed when the needle was taken. The intervention was performed once a day for 7 days. Normal and model rats ate normally, and both groups only grasped and bundled for 7 d without any intervention.
Observation indicators and detection methods
General behavioral observations: During the whole experiment, the mental condition, reaction ability, mobility, food intake, water intake, urine characteristics, and survival rate of rats were observed every dayObservation of colon pathological morphology: After intervention, 10% chloral hydrate was administered intraperitoneally in rats. The rats were immobilized on a rat plate and dissected with a 10 cm incision. Upper intestine tissue was dissected lengthwise, washed with ice saline, and pre-cooled at 0°C. Morphological changes were observed with the naked eye, such as those in intestinal tissues that were approximately 2 cm in size. The most obvious pathological lesions were stained with formaldehydePreparation and detection of local acupoint tissue NMR samples: After hair removal at the Tianshu (ST25) of the skin of each group, a small amount of Tianshu (ST25) skin tissue (approximately 5 mm × 5 mm × 5 mm) was removed and placed in a 1.5 mL centrifuge tube. The tissue was then stored in a -20°C refrigerator for testing. A total of 100–150 mg of skin tissue was obtained from the acupoint and 1.5 mL of sterile eppendorf (EP) tube was inserted. The skin tissue was cut completely, and 300 and 600 μL of ultrapure water and methanol were added, respectively. The mixture was vortexed and stirred for 30 s before adding 300 μL of chloroform. The mixture was vortexed again and then placed in an ultrasonic centrifuge. A total of 650 μL of supernatant was taken and transferred to a new 1.5 mL EP tube. The supernatant was concentrated in a nitrogen blowing instrument to remove methanol and then completely dried. A total of 600 μL of heavy water (D2O) solution containing 0.015% total suspended particles was added and mixed well. A total of 500 μL of solution was transferred to a dry 5 mm NMR tube and then sent to a Bruker 600 MHz spectrometer for proton NMR (1H-NMR) detection. NMR spectra were acquired using Bruker AVANCE III HD (600 MHz) at an experimental temperature of 25°C, and 1H-NMR was acquired using a Carr–Purcell–Meiboom–Gill pulse of cpmgpr1d [RD-90°-(τ-180°-τ) n-acq]. The experimental parameters were as follows: spin echo, t = 20 s (parameter τ =80 ms, n = 126 times); relaxation delay time, D1 = 4 s; Tacq = 2.7 s; spectral width, 16 ppm; acquisition point, 64 K; accumulation number, NS = 256 times; air sweep number, DS = 16 times. MestReNova V9.0.1 software was used for the baseline correction and phase adjustment of the 1H-NMR spectra of the tissue samples.Data processing methods
Metabolomic analysis
The computers were used to obtain 1H-NMR hydrogen spectra and integrate the metabolite peaks, and normalized data were fed into SIMCA-P software (V14, Umetrics, Sweden) for multivariable statistical analysis. Principal component analysis (PCA) was used to visually separate samples, and partial least squares–discriminant analysis (PLS-DA) and orthogonal PLS-DA (OPLS-DA) were used to maximize the differences between the samples. At the same time, the underlying variables were analyzed by using the OPLS-DA model and S-plot. Finally, differential metabolites were obtained on the basis of the importance of the sample variables in the OPLS-DA model (VIP > 1) and t-test (P < 0.05). Metabolic pathway analysis was performed using MetaboAnalyst 5.0 (https://www. metaboanalyst. ca/). After the screened peaks, metabolite recognition information was extracted from the self-built database of the laboratory, the common libraries were integrated, and the metDNA method was used. KEGG or HMDB codes for endogenous metabolites identified in the database were then entered in MetaboAnalyst 5.0 for metabolic pathway analysis.
Statistical analysis
All the data were analyzed using SPSS 20.0 statistical software (IBM, Chicago, USA) and measured in standard deviation (x ± s). For data conforming to normal distribution and variance homogeneity, one-way analysis of variance was performed. Least significant difference assays were used for two-by-two comparison, and Dunnet T3 assays were used for nonvariance homogeneity. Rank sum tests were performed on data that do not conform to normal distributions. The differences were statistically significant at P < 0.05.
Results and AnalysisComparisons of general behavioral changes in rats
During the whole experiment, the rats in the normal group had shiny coats, had good mental health, and were more active. Their food intake and stool were normal. After modeling, the model group had sparse and brittle hair; sleepiness; light-yellow, pasty feces in the anus; slow response to external stimuli; anorexia and emaciation; significant weight loss; and mucopurulent bloody stool. After the intervention, the mental state, mobility, hair gloss, and stool condition of the rats in the acupuncture group were significantly improved. During the modeling and needle intervention, no rats died.
Comparison of the histopathological morphology of the colon in rat groups
After hematoxylin and eosin (HE) staining, the normal group of rats had clear colonic tissue level, neatly arranged epithelial cells, complete colon gland, no obvious hyperemia, edema, and thin intestinal mucosa. The model group had shedding of colonic mucosal epithelial cells, formation of cleft ulcer, destruction of the mucosal gland, edema of interlamina propria and submucosal tissues, and massive infiltration of inflammatory cells that occasionally form granuloma-like lesions. The colonic tissue structure of rats in the acupuncture group was relatively complete, the structure of the colon gland was restored, and the infiltration of inflammatory cells was obvious. It is shown in [Figure 1].
Figure 1: Comparison of the HE staining of colon tissues in rat groups (H and E, ×200). (a) Normal group; (b) Model group, (c) Acupuncture group. HE: Hematoxylin and eosin3.3 1H-NMR analysis of metabolites in local skin tissues at Tianshu (ST25) of rats
The figure below is numbered as the skin sample signal attribution fragment. The attribution range is 0.0–10.0ppm, and the attributes of metabolites in 1H-NMR were obtained by consulting relevant literature. The 1H-NMR spectrum of metabolites in local skin tissues at the Tianshu (ST25) of rats is shown in [Figure 2]. The attributes of metabolites are shown in [Table 1].
Figure 2: 1H-NMR pattern metabolite and main metabolite peak attribution in local skin tissues of Tianshu (ST25)
Table 1: Metabolite attribution and chemical shift in the local skin tissues of Tianshu (ST25)Comparison of metabolites at the local skin tissues of Tianshu (ST25) in the normal group and model group
PCA, PLS-DA, and OPLS-DA were performed on each set of NMR hydrogen spectrum data to evaluate the data quality and understand the contribution of endogenous small molecule metabolites.
The PCA of the experimental data from quality control samples revealed that the metabolic patterns of the local acupoint tissues in the normal group and model group were significantly separated by focusing on the first principal component.
The results showed that the two groups partially coincided, but the differences between the groups were obvious. The cross-validation (R2 = 0.98, Q2 = 0.80) showed that the PLS-DA modeling had good stability, high predictive ability, and low risk of overfitting.
To further screen for differential metabolites, OPLS-DA established statistical models between the normal and model groups, and differential variables between the two groups were screened on the basis of VIP >1, with significant contributions observed at both ends of the corresponding S plots. Thereafter, the VIP value and P value were further compared (VIP >1, P < 0.05). Compared with the normal group, the model group showed increased levels of metabolites choline, glycerol, glycine, guanidinoacetic acid, and proline (P < 0.05 or P < 0.01) and decreased formic acid (P < 0.05) in the local skin tissues of the Tianshu (ST25) of the model group [Figure 3] and [Table 2].
Figure 3: Metabolite pattern of local skin tissues at the Tianshu (ST25) of the normal group and model group. Note: (a) PCA analysis; (b) PLS-DA analysis; (c) OPLS-DA analysis; (d) VIP value plot in OPLS-DA mode; (e) S-plot plot in OPLS-DA mode. PCA: Principal component analysis, PLS-DA: Partial least squares–discriminant analysis, OPLS-DA: Partial least squares–discriminant analysis, VIP: Variable important in projection
Table 2: Comparison of differential metabolites at the local skin tissues of the Tianshu (ST25) of the normal group and model groupComparison of metabolites at the local skin tissues of the Tianshu (ST25) of the model group and acupuncture group
The PCA results of the model group and acupuncture group showed that the metabolic patterns of the local acupoint tissues of the two groups overlapped, and intuitive separation and differentiation between groups could not be performed.
The results show that the differences between the two groups of rats are obvious, and the cross-validation (R2 = 0.98 and Q2 = 0.70) were cross-validated, thus indicating that the models are reliable.
The OPLS-DA score plot showed significant differences in the metabolic patterns in local acupuncture areas between the two groups of rats. OPLS-DA showed that under CD, the levels of alanine, formic acid, leucine, phenylalanine, and tyrosine increased in the local skin of the Tianshu (ST25) of rats in the acupuncture intervention group after acupuncture (P < 0.05 or P < 0.01). This information is shown in [Figure 4] and [Table 3].
Figure 4: Metabolites pattern of local skin tissues at the Tianshu (ST25) of the model group and acupuncture group. Note: (a) PCA analysis; (b) PLS-DA analysis; (c) OPLS-DA analysis; (d) VIP value plot in OPLS-DA mode; (e) S-plot plot in OPLS-DA mode. VIP: Variable important in projection
Table 3: Comparison of differential metabolites at the local skin tissues of the Tianshu (ST25) of the model group and the acupuncture groupAnalysis of the metabolic pathways of differential metabolites
Normal group and model group: Metabolic pathway analysis was conducted for screened differential metabolites by using MetaboAnalyst 5.0, combined with the KEGG database. Potential key metabolic pathways were also screened. Compared with the normal group, the model group had three main metabolic pathways for the disordered local skin tissues of Tianshu (ST25), namely, glycine, serine, and threonine metabolism; valine, leucine, and isoleucine biosynthesis; and phenylalanine, tyrosine, and tryptophan biosynthesis [Figure 5]a and [Figure 6].Model group and acupuncture group: After acupuncture intervention, Compared with the model group, the acupuncture group had five main metabolic pathways for the differential metabolites produced by the local skin tissues of rats, namely alanine, aspartic acid, and glutamate metabolism; valine, leucine, and isoleucine biosynthesis; phenylalanine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; glutamine; and glutamate metabolism [Figure 5]b and [Figure 6].
Figure 5: Path influence values of metabolite metabolic pathways altered at the local skin tissues of Tianshu (ST25) in each group.(a) Normal Group and Model Group; (b) Model Group and Acupuncture Group. Note: (a) rno00260 biosynthesis of glycine, serine, and threonine; rno00290 biosynthesis of valine, leucine, and isoleucine; rno00400 biosynthesis of phenylalanine, tyrosine, and tryptophan; (b) rno00250 biosynthesis of alanine, aspartic acid, and glutamate; rno00290 biosynthesis of valine, leucine, and isoleucine; rno00360 phenylalanine metabolism; rno00400 biosynthesis of phenylalanine, tyrosine, and tryptophan; rno00471 glutamine and glutamate metabolism
Figure 6: Related metabolites and metabolic pathway networks regulated by the Tianshu (ST25) in the CD state and after the acupuncture of CD. Note: The relevant metabolites and metabolic pathway networks of changes and regulation in the skin tissues of the rats at Tianshu (ST25) in each group.(a) rno00290 (valine, leucine, and isoleucine biosynthesis); (b) rno00400 (biosynthesis of phenylalanine, tyrosine, and tryptophan). CD: Crohn's disease Discussion In this experiment, the CD rat model was replicated by TNBS combined with ethanol mixture enema.[7] TNBS penetrates into colonic tissues, can covalently bind to lysine of intestinal epithelial cells, change surface proteins, and eventually activate intestinal immune response. Ethanol can disrupt the tight junction of rat intestinal epithelial cells, alter intestinal permeability, and lead to damage to the intestinal mucosal epithelial barrier.[8] The modeling method of TNBS combined with ethanol mixture is consistent with the pathogenesis and pathological manifestations of human CD; therefore, it is often used in the study of CD. Zhang et al.[5] and Wang et al.[7] successfully reproduced the CD rat model by using TNBS combined with ethanol mixture enema. The results showed that the intestinal wall of the rats in the model group was irregular, hyperemic, and red; had thickened adhesions; and had locally visible ulcers and polyps. These findings were consistent with the diagnosis of CD, and the CD rat model was successfully replicated.
CD belongs to the categories of “abdominal pain,” “diarrhea,” and “periappendicular abscess” in traditional Chinese medicine, and acupuncture therapy has significant efficacy in alleviating the development of CD and improving the quality of life for patients.[9] Studies show that acupuncture improves the inflammatory damage of CD intestinal tissues, reduce intestinal wall fibrosis, and inhibit the occurrence of intestinal luminal stenosis and intestinal obstruction, thereby playing a therapeutic role.[10],[11],[12] The study found that the improvement of intestinal fibrosis in CD rats by acupuncture was associated with the downregulation of colonic TGF-β protein, Smad4 protein, and mRNA expression.[13]Jing Yue Shu Diarrhea stated the following: “The root of diarrhea is all due to the spleen and stomach.” Thousand Gold Essentials stated the following: “stool leakage, and moxibustion of Tianshu (ST25).” The Complete Book of Acupuncture and Moxibustion stated the following: “Diarrhea does not stop, tenesmus is heavy, and uses the two acupoints of Tianshu.” According to traditional Chinese medicine, acupuncture points are perfused into the specific parts of the body's surface through the qi of the internal organs and meridians, and the stomach meridian belong to the stomach and spleen. Gastrointestinal diseases are usually treated by targeting acupoints. Tianshu (ST25) belongs to the stomach meridian, is the Front-Mu point of the large intestine, and regulates intestine and qi stagnation; it is an important acupoint for treating intestinal diseases. Zhang et al.[14] used a tenderness tester to measure the positive acupoints of abnormal meridians in ulcerative colitis, and found that Zusanli (ST36), Shangjuxu (ST37), and Tianshu (ST 25) were closely related to ulcerative colitis. It can be seen that the Tianshu acupoint is closely related to intestinal diseases.
As a new technology in the post-genomic era, metabolites are the final products of cellular regulatory processes at levels that can be considered the ultimate response of organisms to genetic or environmental changes, which can reflect the changes in the genome, transcriptome, and proteome.[15],[16] Early diagnosis can be performed on the basis of metabolomics research; the changes of metabolites such as amino acids, proteins, carbohydrates, and cytokines at the time of onset; and the interpretation of abnormal metabolic maps; it may even be possible to screen people at high risk for preventive effects.[17] At the same time, in terms of treatment, acupuncture can regulate the level of metabolites in the organism and cause changes in the “neuroendocrine–immune system” by stimulating the transmission of signal substances produced in acupoints so that the metabolite spectrum returns to normal.[18],[19],[20] Therefore, the current study used metabolomics to observe the local skin reaction of Tianshu (ST25) in the CD state and confirmed that Tianshu (ST25) was closely related to CD. To explore the metabolic mode of the initial effect of acupuncture, this study provides a metabolite-level basis for revealing acupuncture for CD and provides an important basis for acupuncture target therapy.
Nuclear magnetic metabolomics experiments showed that compared with the normal group, the model group experienced a certain change in the local skin tissue metabolism mode at the Tianshu (ST25), and this change mainly manifested as amino acid metabolism and changes in the nervous system. Glycine is a major inhibitor of neurotransmitters in the human central nervous system, protects the intestinal mucosa from ischemia–reperfusion injury, and contributes to the regeneration of the intestinal mucosa after injury.[21],[22],[23],[24],[25] Guanidinoacetic acid, which is a natural derivative of glycine, stimulates hormone release and neuromodulation.[25],[26] Guanidinoacetic acid is absorbed by the gastrointestinal tract and is rapidly metabolized into creatine, which powers muscles and nerves.[26],[27] It is an important component of brain homeostasis and metabolism and serves as an antioxidant.[28] Choline is an important component of biological cell membranes and plays an important role in maintaining cell membrane integrity and in cell signals.[29] In addition, choline is involved in maintaining normal intestinal function and nutrient absorption.[30],[31] At the same time, choline is a component of lecithin, can be found in sphingomyelin, and is a precursor of acetylcholine, which is synthesized in neurons and is the main component of the enteric nervous system. Neurotransmission occurs at the level of the intestinal mucosa via acetylcholine.[32] Glycine, guanidineacetic acid, and choline are significantly elevated in the CD state, thus indicating that the nervous system of the model rat has been affected and that the conduction of neurotransmitter signals on the intestinal mucosa has changed to a certain extent. As a functional amino acid, proline is a key regulator of a variety of physiological and biochemical processes within cells.[33] When the body is under stress and injury, the endogenous synthesis of proline increases to regulate the synthesis of collagen and promote damage repair.[34],[35] In addition, proline and its metabolite pyrroline-5-carboxylic acid can regulate redox reactions, scavenge free radicals, and enhance the body's ability to resist stress.[36],[37] Some studies have proposed that “acupuncture points” are the “referring points” of the body surface of the pathological process of the target organ and can cause a secondary trigger of “self-repair” at the lesion.[38] The results showed that the increase of the proline and glycine in the skin of rat meridians was due to injury and inflammation and indicated that the body may have initiated self-repair function to maintain the integrity of intestinal function and intestinal mucosa. This finding also confirms to a certain extent the principle of “those who have inside, must be outside” in the Danxi Heart Method. Visceral disease states can be reflected in the corresponding meridians or acupoints on the body surface, with specific signs and symptoms appearing in certain parts of the body or on the surface of the skin.
The results showed that the contents of local skin metabolites alanine, leucine, phenylalanine, and tyrosine increased in the Tianshu (ST 25) of rats in the acupuncture group and mainly affected phenylalanine metabolism and valine, leucine, and isoleucine biosynthesis. Studies have found that amino acids and amino acid metabolism pathways are closely related to inflammatory factors.[39] This is largely consistent with the findings of the current study. Phenylalanine is one of the essential aromatic amino acids, and the control of phenylalanine metabolism is essential for cognitive development and executive ability.[40] Tyrosine is catalyzed by phenylalanine hydroxylase. Tyrosine aminotransferase, which is a rate-limiting enzyme associated with tyrosine decomposition, has the effect of promoting apoptosis.[41] Tyrosine can be converted by tyrosine hydroxylase through endogenous neuropeptide Y to form catecholamine hormones such as dopamine and norepinephrine. Dopamine receptors are found in the nerve endings of the intestinal wall and in the intestinal mucosa.[42] Norepinephrine can reduce mucosal macrophage–induced inflammatory states through muscular macrophages.[43] Phenylalanine and alanine respond to chemical stimuli through the basolateral membrane, thereby activating sensory nerve endings that innervate the mucosa.[44] The results showed that the elevated content of phenylalanine metabolites may be due to acupuncture stimulation, which can activate sensory endings of skin tissues and sensory nerve endings in mucosa, promote apoptosis, and reduce mucosal macrophage–induced inflammation, perform self-repair of the body, and reduce the inflammatory state of the intestinal mucosa. Valine, leucine, and isoleucine are branched-chain amino acids (BCAAs). The oxygenation by tricarboxylic acid cycle and biooxidation system produces adenosine triphosphate for energy.[45],[46],[47] BCAAs are indispensable nutrients for maintaining the body's immune system, and immune cells such as lymphocytes, eosinophils, and neutrophils use isoleucine to synthesize proteins. BCAAs also promote gut development, amino acid transport, and mucin production in the gut; it is involved in innate and adaptive immune responses.[48],[49],[50] The acupuncture of the Tianshu (ST25) point may inhibit the development of CD intestinal inflammation by improving BCAA synthesis, promoting intestinal development and intestinal amino acid transport, maintaining the intestinal immune system, and participating in intestinal immune response.
In summary, the acupuncture treatment of CD model rat at the Tianshu (ST25) acupoint can improve the integrity of the intestinal barrier and reduce intestinal damage and inflammation. By using metabolomics technology and analyzing differential metabolites, it is preliminarily detected that in the CD state, the content of amino acids such as choline, glycerol, guanidinoacetic acid, proline, and other amino acids in the local skin tissues of the Tianshu (ST25) is increased. This finding indicates that there may be a certain connection between the Tianshu (ST25) and intestinal diseases. According to the theory of internal organs–meridians, acupuncture treatment takes the physical and chemical stimulation of acupuncture points to regulate functions of the corresponding organs as the core mechanism, and the lesions of the internal organs can be treated by intervening in the corresponding acupoints via external stimuli such as acupuncture. The results showed that acupuncture intervention in CD increased the content of metabolites such as alanine, leucine, phenylalanine, tyrosine, and other metabolites and their synthetic and metabolic pathways in the local skin tissues of Tianshu (ST25). Local tissue metabolism in the acupoint area may affect neurotransmitter conduction and immune response changes and may play a regulatory role as a whole. Metabolomics is a technology aimed at studying metabolic end products. The metabolite changes caused by acupuncture stimulation may be more associated with disease signals, lack of fixed substance characteristics, and quantity–response relationship. Therefore, there may be certain differences in metabolic results when different research specimens are used. Researchers need to explore and analyze from multiple aspects and perspectives and provide more evidence-based basis for clinical and basic research.
Financial support and sponsorship
National Natural Science Foundation of China (No. 81603705, No.81774438); China Postdoctoral Science Foundation Project (No. 2107M612567); Natural Science Foundation of Hunan Province (No. 2021JJ30513, No. 2017JJ3245); Scientific Research Project of Hunan Provincial Department of Education (No.21A0235, No. 20B444); Hunan Traditional Chinese Medicine Research Project (No.C2022027); Hunan Province Youth Backbone Teacher Training Target Project (2021); Changsha Natural Science Foundation (No. kq2208183).
Conflicts of interest
There are no conflicts of interest.
References
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