Thinking on Functional Mechanism of Acupuncture for Inflammatory Bowel Diseases Based on Metabolomics

Dou Chuan-zi, Feng Hui2, Zheng Xiao3, Liu Xiao-xu4, Zhu Xi-fa3, Liu Shi-min4, Wu Lu-yi4, Yang Ling, Lu Yuan, Zhang Wei-wei4, Liu Hui-rong

1 Shanghai Research Institute of Acupuncture and Meridian, Shanghai 200030, China

2 Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai 200052, China

3 Taizhou Hospital of Traditional Chinese Medicine, Zhejiang Province, Taizhou 318000, China

4 Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

SPECIAL TOPIC FOR 973 PROGRAM

Thinking on Functional Mechanism of Acupuncture for Inflammatory Bowel Diseases Based on Metabolomics

Dou Chuan-zi1, Feng Hui2, Zheng Xiao3, Liu Xiao-xu4, Zhu Xi-fa3, Liu Shi-min4, Wu Lu-yi4, Yang Ling1, Lu Yuan1, Zhang Wei-wei4, Liu Hui-rong1

1 Shanghai Research Institute of Acupuncture and Meridian, Shanghai 200030, China

2 Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai 200052, China

3 Taizhou Hospital of Traditional Chinese Medicine, Zhejiang Province, Taizhou 318000, China

4 Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Author: Dou Chuan-zi, M.D., research assistant

Acupuncture has a good therapeutic effect in treatment of inflammatory bowel diseases (IBD), but its functional mechanism has not been systematically explained. Metabolomics is the scientific study of dynamic chemical processes involving metabolites as well as metabolic response of living organisms. Metabolomics, a research method with integrity and dynamics, corresponds to the overall regulatory effect of acupuncture and is in line with the overall concept of traditional Chinese medicine (TCM)and the concept of homeostasis. In the recent years, metabolomics has been extensively applied to the clinical and experimental study of IBD, and its potential applied value has been unanimously acknowledged by the researchers. In this article, the application status of metabolomics in acupuncture is summarized, and the research ideas to study the mechanism of acupuncture in the regulation and control of IBD by metabolomics are preliminarily explored.

Inflammatory Bowel Diseases; Metabolomics; Acupuncture-moxibustion Therapy; Acupuncture Therapy; Moxibustion Therapy

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), belong to intractable bowel diseases. In the recent years, good clinical therapeutic effects have been achieved in treatment of this disease by acupuncture therapy, but its functional mechanism has not been systematically explained. With the progressive development of the life science, people transferred their focus from the primary local study to the systematic biological study, so a series of ‘omics’ studies have emerged. In particular, metabolomics possesses the integrity and dynamics, because of its high-throughput techniques, high sensitivity, and high accuracy and its ability to detect qualitatively or quantitatively the changes of smallmolecule metabolites in the human body[1-2]. This research method of metabolomics corresponds to the overall regulatory effect of acupuncture and is in line with the overall concept of traditional Chinese medicine (TCM) and the concept of homeostasis, laying a good foundation for study of the regulatory mechanism in acupuncture.

1 Research Methods and Application of Metabolomics

The concept of metabolomics was pioneered by the team of Nicholson JK in 1999. It is defined as the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiologicalstimuli or genetic modification[3]. The metabolomics is an omics method to research the biological system, developed after genomics and proteomics, and belongs to an important component part of the system biology.

The expression of biological gene and proteins are closely related, and metabolites reflect mostly the changing environment where cells and organisms stay, but are also related to the impact from the cellular and nutritional status of the organism, as well as administration of medications and other external factors. By collecting various types of metabolites, such as urine, serum, feces, including local pathological tissues, metabolomics is supposed to analyze all small molecule metabolites of the biological systems (cells, tissues, or organisms) at a given time and conditions qualitatively or quantitatively, by using the techniques of nuclear magnetic resonance (NMR), mass spectrum (MS), high performance liquid chromatography (HPLC), gas chromatography (GC), and chromatograph-mass spectrometry. Its main process is divided into four parts: sample collection, data detection, optimized statistics, and results validation. The data of the results are generally managed by multivariate data analysis, such as principal component analysis and partial least-squares discriminant analysis. Currently, metabolomics has been extensively applied in the studies of IBD, severe chronic diarrhea, hepatic encephalopathy, hepatitis C, and various malignant gastrointestinal tumors[4-7].

2 Current Status of Metabolomics Application in Acupuncture Studies

The metabolomics can be used to analyze the general regulatory function of acupuncture at metabolites levels. Its applied study of acupuncture regulatory mechanism mainly emerges in the recent years. The feasibility in the application of metabolomics in the study of acupuncture regulatory mechanism and assessment of the therapeutic effects is confirmed by the preliminary research results, and its potential applied value has been unanimously acknowledged by the researchers.

It has been found out in the preliminary study on functional dyspepsia (FD) by domestic researchers that acupuncture is obviously influential to serum macromolecular metabolites and can remarkably change the levels of serum leucine/isoleucine, lactate and glucose in FD patients, which is different from the adjusting mode of Itopride in mainly reducing the levels of choline, leucine/isoleucine, indicating that acupuncture may have the metabolic regulatory mechanism different from Western medication[8-10]. It is also found out in the mechanism study of acupuncture in improving the rapidly-aging mice by metabolomics that the main impact of electroacupuncture stimulation on SAMP8 mice is to elevate the saturated fatty acids and triglycerides, and to reduce choline, phosphatidylcholine and unsaturated fatty acids. In the study on the acupuncture mechanism in the treatment of acute gouty arthritis by metabolomics[11-12]. Some researchers found out that acupuncture can restore the metabolites network in the rats with acute gouty arthritis, so as to produce the therapeutic effect[13]. Acupuncture possesses the extensive regulation on metabolites of cardiovascular diseases, such as endothelin (ET), calcitonin gene-related peptide (CGRP), nitric oxide (NO), thromboxane A2 (TXA2), prostacyclin (PGI2), interleukin (IL-1, IL-2, IL-6), tumor necrosis factor (TNF)-α, atrial natriuretic peptide (ANP), indicating that metabolomics has the extensive application prospects in acupuncture treatment of diseases[14].

The study on the specificity of acupoints is the hot spot in the modern study of acupuncture, and metabolomics based upon the nuclear magnetic resonance (NMR) and mode identification technology offers a new angle to this study. Ten randomlyselected healthy young males were respectively punctured at Zusanli (ST 36), Liangqiu (ST 34), Juliao (ST 3), Yanglingquan (GB 34), and Weizhong (BL 40), to test the urine metabolites of the subjects after continuous acupuncture for five days. Besides, ten cases were selected for control study. The influence on urine metabolites of the organism by puncturing the acupoints of Yangming Meridian is almost same, and can increase urine hippuric acid and trimethylamine oxide content, and reduce glycine. The influence on the urine metabolites by puncturing the acupoints of Yangming Meridian and Yanglingquan (GB 34) is not very different, but is obviously different from those after puncturing Weizhong (BL 40). After Weizhong (BL 40) was punctured, creatinine was increased significantly in urine metabolites, indicating that the impact on the metabolites by puncturing the acupoints of Yangming Meridian is in certain similarity, but the exact mechanism needs further study[15].

More and more scholars understand the advantage of metabolomics in the study of the overall regulatory mechanism of acupuncture. The dynamic and holistic feature of metabolomics can be well agreed with the functional regularity of acupuncture. But, because metabolites are influenced by many factors, in acupuncture study it is necessary to control the experimental condition strictly and standardize the storage and management of the collected metabolites,in order to avoid damaging the samples from pollution and long-term storage[16-17].

3 Current Status of Metabolomics Application in IBD

IBD is a group of chronic non-specific intestinal inflammatory diseases of unknown etiology and is a commonly encountered disease in the North America and Europe. In the past over 30 years, the incidence rate of IBD was in progressive tendency in Japan. There has no epidemiological data in China. But, in the recent ten years, the number of the patients with this disease has been gradually increased[18]. The etiology of IBD is unclear, and its incidence is closely related to genetic, environmental, food, and other immune factors[4-7,19], among which the intestinal mucosal immune response induced by intestinal flora with the regulation of the genetic, environmental and immune factors is thought to be important pathogenesis of IBD[20-23]. Currently, the treatment of this disease in Western medicine is mainly supposed to induce and maintain clinical relief, prevent and treat the complications, and improve the life quality of the patients. It has been proven by the study that Chinese medicine, especially acupuncture, is advantageous for treatment of this disease[24-26].

Up to today, there are fewer studies on IBD. But, judging from the research results, we can see that the analyzing method of metabolomics with high flux can offer new viewpoints and evidence to the study on IBD pathogenesis and offer new method to the treatment of IBD. In the study on IBD, the differences in sample type, research methods and lack of unified approval standard will lead to differences of final metabolites. Therefore, the further study is needed to prove the differences of those metabolites and enhance the accuracy of metabolites labeling.

3.1 Clinical study

It was found out in the study of Balasubramanian K, et al[27]on colonic mucosal tissue extract of the confirmed IBD patients and healthy adults by1H magnetic resonance spectroscopy (1H-MRS) that in comparison with the healthy adults, in UC and CD patients of the active statge, the concentration of amino acid (isoleucine, leucine, valine, alanine, glutamic acid and glutamate), choline, glycerol phosphate, choline, inositol, lactate, succinic acid decreased significantly (P≤0.05), and alpha-glucose concentration was elevated. In UC and CD patients of the remission stage, except lactate, the concentration of glycerol phosphate choline and inositol was lower than that in the healthy adults. The concentration of rest metabolites was not different from that in the healthy control group. Bezabeh T, et al[28]used1H-MRS in the differential diagnosis of UC and CD. It was found in the study on colonic mucosa metabolites spectrum in 45 UC patients and 31 CD patients that the classification accuracy of UC and CD by MRS was as high as 98.6%, indicating that MRS can be used for accurate diagnosis of undifferentiated colitis. In addition to the colon mucosa, methanol, mannose, acid, 3-methyl-2-oxo acid, amino acids, such as isoleucine, and urine mannitol, allantoin, wood sugar, carnitine in serum and plasma of UC patients were significantly higher than those in the normal adults, and trimethyl glycine, and hippurate were decreased remarkably[19]. It was obtained from calculation by using the quadrature signal cross that serum lipoproteins (especially high-density lipoprotein), bile pigment, acetyl glycoproteins, amino acids, etc, in UC patients were different from those in the normal adults[20]. In the analysis of feces of UC patients by multivariate discriminant analysis based upon NMR data, taurine and cadaverine levels were higher than those in the normal adults, showing the correlation of the intestinal flora composition and metabolite components, with high sensitivity and specificity for differentiation of UC and normal control[21].

In addition, it was found out in the metabolomics study on the pathological tissues of UC patients in the active stage and remission stage that the antioxidants and amino acids levels were higher in UC patients of the active stage than those in the remission stage, but lipids and choline contents were lower than those in UC patients of the remission stage. However, the metabolites in 20 percent of UC patients in the remission stage were similar to those of UC patients in the active stage, suggesting that metabolomics was still not enough for processing staging diagnosis of UC[23], and it is still needed to find more specific and sensitive biomarkers.

3.2 Animal model study

IBD pathological and effective mechanism was studied from animal models. Zhang XJ, et al[29]analyzed UC model rats by ultra performance liquid chromatography and electrospray ionization coupled with quadrupole time-of-flight mass spectrometry and detected respectively 7 kinds and 5 kinds of metabolites in plasma and urine related to intestinal barrier function, microbial population homeostasis, and immune regulation and inflammatory response, providing the new methods and channels to the physiological and pathological study on UC. Schicho R, et al[30]conducted more extensive study on 53 kinds of serum metabolites and 69 kinds of urine metabolites. In urine of UC mice induced by dextran sulfate sodium, the creatine, carnitine, and methylamine were increased, and anti-oxidative metabolites were decreased. And the contents ofketone bodies, hypoxanthine and tryptophan in serum were increased, and glucose and citric acid cycle intermediates were reduced. In the combined application of the pathological analysis of the intestinal tissue, plasma metabolomics analysis, and the test of plasma inflammatory markers and other methods, Martin FP, et al[31]found out that IL-10 was significantly influential to the relevant indexes of the etiology of the IBD, and could reduce the very low density lipoprotein level and increase the concentration of low density lipoprotein and polyunsaturated fatty acids. High levels of lactic acid, pyruvic acid, citric acid, and low levels of glucose could increase the oxidation and glycolysis of low-level fatty acid, while high levels of free amino acids showed that the muscles could be converted to produce energy by the decomposition of proteins and amino acid. Metabolomics could provide a noninvasive method for the evaluation of the level of inflammation in IBD. Meanwhile, the combined application of multiple modern detective methods could provide a more comprehensive analysis. In the study on the pathogenesis of UC by using serum metabolomics and by using high-level stearoyl lysolecithin and low-level oleoyl lysolecithin contained in serum of the mice induced by dextran sulfate sodium, Chen C, et al[32]confirmed that dextran sulfate sodium could inhibit the expression of stearoyl coenzyme A desaturase 1 in the liver, and these changes occurred before the presence of UC symptoms and were closely related to the expression of pro-inflammatory cytokines. Stearoyl coenzyme A desaturase 1 and lipids could be used as the indexes to interfere with the therapeutic effects in the treatment of IBD. It was found out in the study on the effect mechanism by analyzing the metabolomics principal components of mouse serum that rifaximin could activate the human pregnane X receptor to play a therapeutic role in the treatment of IBD, but there was no difference in the serum metabolomics of human pregnane X receptor mice and non-activated mice, indicating that the functional target of rifaximin is limited in the colon, instead of the whole body[33]. Metabolomics method could also be applied in the pharmacokinetic study in the relation to UC. It was found out by analyzing the influence of broadspectrum antibiotic enrofloxacin to the metabolites that there were ten and eight metabolites changes respectively in mouse urine and feces[34]. Organic cation transporters (OCTN1) were associated with IBD, and OCTN1 gene knockout could affect the distribution of antioxidant ergothioneine amino acids inside the body. It was found out in metabolomics that ergothioneine amino acids were significantly lower in the kidneys and small intestine of OCTN1 knockout mice than in the normal mice, suggesting that OCTN1 play a key role in the balancing process of ergothioneine amino acids inside the body[35].

Domestically, metabolomics has been less used in the study of IBD. It was found out in the study on metabolites with Varian spectrometer by the research group of Xinjiang Medical University, in comparison with the rats in the normal control rats, that the contents of branched-chain amino acids of leucine, isoleucine acid and valine and the contents of small molecules compounds of phenylalanine, pyruvic acid, hydroxybutyric acid, and creatine were significantly reduced, while VLDL contents were increased in rat plasma of the model group. In the rat colon tissue of UC model group, propionic acid, lactic acid, the contents of small molecule metabolites of taurine, acetic acid, proline, and alanine were significantly increased. These significant differences in metabolites could be used as the specific marker of UC[36-37].

4 Thinking on Study of Acupuncture in Regulating and Controlling IBD Based on Metabolomics

The better effects were achieved in acupuncture treatment of IBD by this research group. It was found out in the corresponding studies that acupuncture has the regulatory functions of multiple levels and multiple targets, and the effective mechanism of acupuncture was explained from the angles of morphology, gene levels, immune and inflammatoryrelated factors as the evaluation indexes[38-41], but the effective mechanism and overall regulatory features of acupuncture treatment for IBD have not yet been revealed fully.

Metabolomics technology shows its advantages in the explanation of the holistic concept, thinking of treatments based upon pattern identification in TCM, and in the efficacy, safety, and functional mechanism of herbal drugs, and new ideas have been formed for the study of herbal formulae in TCM[42-43]. Acupuncture treatment is advantageous for IBD, but the previous studies were just focused mostly on the research level of certain substance, certain positions or certain channels, without referring to the overall regulatory feature of acupuncture in the treatment of this disease. By using metabolomics technology in the study of acupuncture treatment of IBD, for analyzing the acupuncture-regulated metabolites, acupuncture mechanism can be revealed from more extensive levels. The following issues should be paid attention to.

4.1 Strictly screening subjects for acupuncture study

The patients with IBD must be strictly selected for acupuncture study. In enrolling the subjects, the subjects of same gender and specific age should be selected, in order to reduce the bias caused by genderand age. When the experimental conditions are permitted, the patients with IBD should be selected from the same race and from same area. It has been proven by evidence that metabolites may be associated with genetic, environmental, cultural and other factors. Besides, it is necessary to set up the strict exclusion criteria, in order to exclude the influence of metabolic diseases, such as thyroid disease, hyperlipidemia, diabetes and endocrine disorders, to the biochemical metabolism of the body. The patients with the history of common cold and fever, drug administration and alcohol drinking two weeks before the test should be excluded. Experimental animals should be selected based upon the standard of same breed and same sex, and similar week age and body weight, in order to reduce the differences between individuals.

4.2 Control diet, exercise, and circadian rhythm of the patients with IBD

Before the collection of blood or urine, the diet within 24 h must be strictly controlled in the patients with IBD. For instance, 24 h before the collection of samples, it must be prohibited to take tobacco and alcohol, to drink fruit juice, ingest foods with cheese and preservatives, as well as coffee, tea, chocolate, cola and other caffeine-contained food or beverages. At the ordinary time, the patients must avoid gluttony and excessive spicy food. Besides, the patient’s variety and number of the foodstuffs at the ordinary time and 24 h before the collection of samples should be recorded for the retrospective analysis and for excluding the interference from food factors. The sports and circadian rhythms will also bring about differences to the results of metabolomics. Therefore, IBD patients recruited for acupuncture study should avoid the strenuous sports of swimming and running 72 h before sampling. The regularity and consistency of the circadian rhythm should be maintained in the research process, and the irregular circadian rhythms of staying up overnight should be avoided[44-45]. For the experimental animals, the animal breeding room can be processed by the uniform rhythm method of illumination for 12 h and darkness for 12 h of darkness alternatively.

4.3 Explain the effective mechanism of acupuncture

On this basis, for specific metabolites, the biological specimens and information database of IBD should be built up, in order to analyze the expression changes in the metabolites spectrum of colon mucosa, blood and urine of IBD of different stages and different TCM patterns during acupuncture interference, for seeking the relevant metabolites spectrum or specific biological markers of IBD of different stages and different TCM patterns, so as to better reveal the pathological process and abnormal metabolic channels. Metabolites are mostly produced by hydrolysis of protein encoded by the gene. When mutations happen, the nature of protein produced by encoding and the activity of enzyme would also change, so as to affect the characteristics of the metabolites. Besides, some other factors, such as intake of food organisms and intestinal flora, also can affect the type of final metabolites. By metabolomics analysis, the further study could be expected to research the pathogenic factors, and occurring and developing mechanism of IBD from the perspective of metabolites, so as to reveal the overall regulatory mechanism of acupuncture intervention for IBD.

Metabolomics is also beneficial to highlight the advantages of acupuncture therapy. By analyzing the influence of the combination of different acupoints on the patients with IBD and on the expression of metabolites of colonic mucosa, blood, and urine of the modeled animals, it is beneficial to the selection of the effective acupoints combination and to the enhancement of acupuncture effect. On this basis, in the comparison of acupuncture in relieving the clinical symptoms of the patients with IBD of different stages and in improving the pathology of the colon mucosa, and its influence on the overall regulatory effect of blood and urine metabolite spectrum or specific biomarker, the scientific connotation can be discovered to reveal the cooperative effect of acupuncture for metabolomics of IBD.

To interpret TCM by modern technology has always been a challenge. The introduction of metabolomics technology into the study of acupuncture mechanism in the diagnosis and treatment of IBD is conducive to highlight the advantages of acupuncture, so as to promote better popularization and application of Chinese medicine.

Conflict of Interest

The authors declare that there is no potential conflict of interest in this article.

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 81303033); Outstanding Academic Leaders Training Plan of Shanghai Health Care System (No. XBR2013106); National Basic Research Program of China (973 Program, No. 2009CB522900); Natural Science Foundation of Shanghai (No. 11ZR1434300).

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Translator:Huang Guo-qi

Wu Lu-yi, on-job doctorate candidate, research assistant.

E-mail: luyitcm@163.com

R2-03

: A

Date:December 8, 2013