Mechanism of electroacupuncture and herb-partitioned moxibustion on ulcerative colitis animal model: A study based on proteomics.

BACKGROUND: Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease. Acupuncture and moxibustion is proved effective in treating UC, but the mechanism has not been clarified. Proteomic technology has revealed a variety of biological markers related to immunity and inflammation in UC, which provide new insights and directions for the study of mechanism of acupuncture and moxibustion treatment of UC. AIM: To investigate the mechanism of electroacupuncture (EA) and herb-partitioned moxibustion (HM) on UC rats by using proteomics technology. METHODS: Male Sprague-Dawley rats were randomly divided into the normal (N) group, the dextran sulfate sodium (DSS)-induced UC model (M) group, the HM group, and the EA group. UC rat model was prepared with 3% DSS, and HM and EA interventions at the bilateral Tianshu and Qihai acupoints were performed in HM or EA group. Haematoxylin and eosin staining was used for morphological evaluation of colon tissues. Isotope-labeled relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry were performed for proteome analysis of the colon tissues, followed by bioinformatics analysis and protein-protein interaction networks establishment of differentially expressed proteins (DEPs) between groups. Then western blot was used for verification of selected DEPs. RESULTS: The macroscopic colon injury scores and histopathology scores in the HM and EA groups were significantly decreased compared to the rats in the M group (P < 0.01). Compared with the N group, a total of 202 DEPs were identified in the M group, including 111 up-regulated proteins and 91 down-regulated proteins, of which 25 and 15 proteins were reversed after HM and EA interventions, respectively. The DEPs were involved in various biological processes such as biological regulation, immune system progression and in multiple pathways including natural killer cell mediated cytotoxicity, intestinal immune network for immunoglobulin A (IgA) production, and FcγR-mediated phagocytosis. The Kyoto Encyclopedia of Genes and Genomes pathways of DEPs between HM and M groups, EA and M groups both included immune-associated and oxidative phosphorylation. Network analysis revealed that multiple pathways for the DEPs of each group were involved in protein-protein interactions, and the expression of oxidative phosphorylation pathway-related proteins, including ATP synthase subunit g (ATP5L), ATP synthase beta subunit precursor (Atp5f), cytochrome c oxidase subunit 4 isoform 1 (Cox4i1) were down-regulated after HM and EA interventions. Subsequent verification of selected DEPs (Synaptic vesicle glycoprotein 2A; nuclear cap binding protein subunit 1; carbamoyl phosphate synthetase 1; Cox4i1; ATP synthase subunit b, Atp5f1; doublecortin like kinase 3) by western blot confirmed the reliability of the iTRAQ data, HM and EA interventions can significantly down-regulate the expression of oxidative phosphorylation-associated proteins (Cox4i1, Atp5f1) (P < 0.01). CONCLUSION: EA and HM could regulate the expression of ATP5L, Atp5f1, Cox4i1 that associated with oxidative phosphorylation, then might regulate immune-related pathways of intestinal immune network for IgA production, FcγR-mediated phagocytosis, thereby alleviating colonic inflammation of DSS-induced UC rats.

Gut microbiome alterations in colitis rats after moxibustion at bilateral Tianshu acupoints.

BACKGROUND: The pathogenesis of ulcerative colitis (UC) is closely related to the gut microbiota. Moxibustion has been used to improve the inflammation and gastrointestinal dysfunctions in gastrointestinal disorders such as UC. In this study, we investigated whether moxibustion could improve the gut microbial dysbiosis induced by dextran sulphate sodium. METHODS: Twenty-five male rats were randomly assigned into five groups. The UC rat model was established by administering DSS solution. The rats in the moxibustion and normal rats with moxibustion groups were treated with moxibustion at Tianshu (bilateral, ST25) points, and the mesalazine group rats were treated with mesalazine once daily for 7 consecutive days. Disease activity index (DAI) and haematoxylin and eosin staining were used to evaluate the effect of moxibustion. Gut microbiota profiling was conducted by metagenomic high throughput sequencing technology. The gut microbiota composition, diversity and function were analyzed and compared using metagenomics methodologies. RESULTS: The DAI scores and histopathology scores in the moxibustion and mesalazine groups were significantly decreased compared with the UC group (P < 0.01). Moxibustion treatment increased abundance levels of Bacteroidetes, Actinobacteria, Ascomycota, Synergistetes and decreased abundance of Firmicutes, Proteobacteria. At the genus level, the abundance of Bacteroides, Bacteroides_bacterium_M7, Prevotella, Bacteroidales_bacterium_H2, were increased and Bacteroides_bacterium_H3, Parabacteroides, Porphyromonas, Alistipes, Parasutterella were decreased in the UC group in comparsion with those in the NG group. Moxibustion increased the abundance of Bacteroides and Bacteroides_bacterium_H3 and decreased Bacteroides_bacterium_M7, Prevotella, Bacteroidales_bacterium_H2. In UC group, the specie Bacteroides_massiliensis was negatively (P < 0.05) correlated with IL-23, Bacteroides_eggerthii_CAG109 and Bacteroides_eggerthii were negatively (P < 0.05) correlated with TGF-ß. And the species Prevotella_sp_CAG1031 and Bacteroides_bacterium_H2 were significant positively (P < 0.05) correlated with IL-23. In addition, compare with the normal group, genes involved in certain metabolic pathways, such as energy production and conversion, amino acid transport and metabolism, carbohydrate transport and metabolism, were under-represented in the UC group, and these changes in the metabolic pathways could be reversed by moxibustion treatment and mesalazine treatment. CONCLUSIONS: Our findings suggest that moxibustion treatment may protect the host from mucosal inflammation by modulating the intestinal microbiota community.

Herb-partitioned moxibustion regulated the miRNA expression profile in the thyroid tissues of rats with experimental autoimmune thyroiditi.

OBJECTIVE: To observe the effect of herb-partitioned moxibustion (HPM) on the miRNA expression profile of thyroid tissue in experimental autoimmune thyroiditis (EAT) rats. METHODS: Rats were randomly divided into normal control (NC) group, EAT model (EAT) group, HPM group and western medicine (Med) group. EAT model rats were prepared by a combined immunization with complete and incomplete Freund's adjuvant emulsified with porcine thyroglobulin and iodine. Rats in the HPM group were treated with HPM, while rats in the Med group were treated with levothyrocine (1 µg/2 mL) by gavage. HE staining was used to observe the pathological morphological changes of thyroid tissue, ELISAs was uaed to detect the serum concentrations of TGAb, TPOAb, FT3, FT4, TSH. We then performed high-throughput miRNA sequencing to analyse the miRNA expression profiles in the thyroid tissues, followed by a bioinformatics analysis. RT-qPCR was used to verify the identified differentially expressed miRNAs. RESULTS: HPM improved the thyroid tissue morphology and reduced serum TPOAb, TGAb, TSH concentration in EAT rats (P < 0.05), but with no obvious effect on FT3 and FT4 concentration. While the TSH, FT3 and FT4 concentration was significantly changed in the Med group (P < 0.01 or P < 0.05) compared with that of EAT group. Sequencing results showed that a total of 17 miRNAs were upregulated, and 4 were downregulated in the EAT rats, in which the expression levels of miR-346 and miR-331-5p were reversed by HPM. The target genes of the miRNAs that regulated by HPM were associated with a variety of immune factors and immune signals. RT-qPCR verification showed that the expression of miRNA-346 and miRNA-331-5p was consistent with the sequencing results. CONCLUSIONS: HPM could regulate the the expression of miRNA-346 and miRNA-331-5p, then act on their target genes to immune and inflammation-related pathways, which may be one of the mechanisms of HPM on EAT rats.

Influence of herb-partitioned moxibustion at Qihai (CV6) and bilateral Tianshu (ST25) and Shangjuxu (ST37) acupoints on toll-like receptors 4 signaling pathways in patients with ulcerative coliti.

OBJECTIVE: To evaluate the efficacy of herb-partitioned moxibustion (HPM) at Qihai (CV6), Tianshu (ST25) and Shangjuxu (ST37) acupoints in relieving symptoms and the immune regulation of HPM on the toll-like receptors 4 (TLR4) signaling pathway in ulcerative colitis (UC) patients. METHODS: A randomized, single-blind study was conducted 63 patients to receive HPM or sham HPM treatment. The efficacy outcomes included scores of the Mayo, Baron, inflammatory bowel disease questionnaire (IBDQ), self-rating depression scale (SDS), self-rating anxiety scale (SAS). HE staining was used to observe the histopathological changes of the colon. The expression of inflammatory cytokines and TLR4 signaling pathway related molecules were determined by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Baron, SDS, SAS scores were significantly decreased in moxibustion group (P < 0.05), IBDQ score was significantly greater in the moxibustion group than in the sham moxibustion group (P < 0.05). Histopathology of mucosal biopsies showed that both two groups improved in mucosa after treatment. The expression levels of tumor necrosis factor-α, interleukin-2, interleukin-12, interferon-γ, and TLR4, lipopolysaccharide, myeloid differentiation factor 88, interleukin receptor associated kinase, tumor necrosis factor receptor associated factor 6 and nuclear factor kappa-B p65 were significantly lower in the moxibustion group than in the sham moxibustion group (P < 0.05). CONCLUSION: This study showed that HPM at Qihai?(CV6),?Tianshu?(ST25) and?Shangjuxu (ST37) acupoints is effective to relieve symptoms, anxiety, depression and improving life quality in UC patients, which may be related to the immune regulation of HPM on TLR4 signaling pathway.

Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn's disease via signaling involving distinct classes of phosphatidylinositol 3-kinases.

BACKGROUND: Autophagy is an evolutionarily conserved biological process in eukaryotic cells that involves lysosomal-mediated degradation and recycling of related cellular components. Recent studies have shown that autophagy plays an important role in the pathogenesis of Crohn's disease (CD). Herbal cake-partitioned moxibustion (HM) has been historically practiced to treat CD. However, the mechanism by which HM regulates colonic autophagy in CD remains unclear. AIM: To observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism. METHODS: Rats were randomly divided into a normal control (NC) group, a CD group, an HM group, an insulin + CD (I + CD) group, an insulin + HM (I + HM) group, a rapamycin + CD (RA + CD) group, and a rapamycin + HM (RA + HM) group. 2,4,6-trinitrobenzenesulfonic acid was administered to establish a CD model. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and the formation of autophagosomes was observed by electron microscopy. The expression of autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Insulin and rapamycin were used to inhibit and activate colonic autophagy, respectively. The mRNA expression levels of phosphatidylinositol 3-kinase class I (PI3KC1), Akt1, LC3B, sequestosome 1 (p62), and mammalian target of rapamycin (mTOR) were evaluated by RT-qPCR. The protein expression levels of interleukin 18 (IL-18), tumor necrosis factor-α (TNF-α), nuclear factor κB/p65 (NF-κB p65), LC3B, p62, coiled-coil myosin-like BCL2-interacting protein (Beclin-1), p-mTOR, PI3KC1, class III phosphatidylinositol 3-kinase (PI3KC3/Vps34), and p-Akt were evaluated by Western blot analysis. RESULTS: Compared with the NC group, the CD group showed severe damage to colon tissues and higher expression levels of IL-18 and NF-κB p65 in colon tissues (P < 0.01 for both). Compared with the CD group, the HM group showed significantly lower levels of these proteins (P IL-18 < 0.01 and P p65 < 0.05). There were no significant differences in the expression of TNF-α protein in colon tissue among the rat groups. Typical autophagic vesicles were found in both the CD and HM groups. The expression of the autophagy proteins LC3B and Beclin-1 was upregulated (P < 0.01 for both) in the colon tissues of rats in the CD group compared with the NC group, while the protein expression of p62 and p-mTOR was downregulated (P < 0.01 for both). However, these expression trends were significantly reversed in the HM group compared with the CD group (P LC3B < 0.01, P Beclin-1 < 0.05, P p62 < 0.05, and P m-TOR < 0.05). Compared with those in the RA + CD group, the mRNA expression levels of PI3KC1, Akt1, mTOR, and p62 in the RA + HM group were significantly higher (P PI3KC1 < 0.01 and P Akt1, mTOR, and p62 < 0.05), while those of LC3B were significantly lower (P < 0.05). Compared with the RA + CD group, the RA + HM group exhibited significantly higher PI3KC1, p-Akt1, and p-mTOR protein levels (P PI3KC1 < 0.01, P p-Akt1 < 0.05, and P p-mTOR < 0.01), a higher p62 protein level (P = 0.057), and significantly lower LC3B and Vps34 protein levels (P < 0.01 for both) in colon tissue. CONCLUSION: HM can activate PI3KC1/Akt1/mTOR signaling while inhibiting the PI3KC3 (Vps34)-Beclin-1 protein complex in the colon tissues of CD rats, thereby inhibiting overactivated autophagy and thus exerting a therapeutic effect.

Effect of mild moxibustion on intestinal microbiota and NLRP6 inflammasome signaling in rats with post-inflammatory irritable bowel syndrome.

BACKGROUND: About one-third of refractory irritable bowel syndrome (IBS) cases are caused by gastrointestinal (GI) infection/inflammation, known as post-infectious/post-inflammatory IBS (PI-IBS). Although it is known that intestinal microbiota and host NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammsome signaling are closely related to PI-IBS and moxibustion has a therapeutic effect on PI-IBS, whether moxibustion regulates the intestinal flora and host NLRP6 events in PI-IBS remains unclear. AIM: To examine the regulatory effect of moxibustion on intestinal microbiota and host NLRP6 inflammatory signaling in PI-IBS. METHODS: Sprague-Dawley rats were divided into a normal control group, a model control group, a mild moxibustion group, and a sham mild moxibustion group. PI-IBS rats in the mild moxibustion group were treated with moxibusiton at bilateral Tianshu (ST 25) and Zusanli (ST36) for 7 consecutive days for 10 min each time. The sham group rats were given the same treatment as the mild moxibustion group except the moxa stick was not ignited. Abdominal withdrawal reflex (AWR) score was measured to assess the visceral sensitivity, and colon histopathology and ultrastructure, colonic myeloperoxidase (MPO) activity, and serum C-reactive protein (CRP) level were measured to evaluate low-grade colonic inflammation in rats. The relative abundance of selected intestinal bacteria in rat feces was detected by 16S rDNA PCR and the NLRP6 inflammsome signaling in the colon was detected by immunofluorescence, qRT-PCR, and Western blot. RESULTS: The AWR score was significantly decreased and the low-grade intestinal inflammation reflected by serum CRP and colonic MPO levels was inhibited in the mild moxibustion group compared with the sham group. Mild moxibustion remarkably increased the relative DNA abundances of Lactobacillus, Bifidobacterium, and Faecalibacterium prausnitzii but decreased that of Escherichia coli in the gut of PI-IBS rats. Additionally, mild moxibustion induced mRNA and protein expression of intestine lectin 1 but inhibited the expression of IL-1ß, IL-18, and resistance-like molecule ß by promoting the NLRP6 and reducing the mRNA and protein expression of apoptosis-associated speck-like protein containing CARD (ASC) and cysteinyl-aspartate-specific proteinase 1 (Caspase-1). The relative DNA abundances of Lactobacillus, Bifidobacteria, Faecalibacterium prausnitzii, and Escherichia coli in each group were correlated with the mRNA and protein expression of NLRP6, ASC, and Caspase-1 in the colon. CONCLUSION: These findings indicated that mild moxibustion can relieve low-grade GI inflammation and alleviate visceral hypersensitivity in PI-IBS by regulating intestinal microbes and controlling NLRP6 inflammasome signaling.

Herbs-partitioned moxibustion alleviates aberrant intestinal epithelial cell apoptosis by upregulating A20 expression in a mouse model of Crohn's disease.

BACKGROUND: A20 inhibits intestinal epithelial cell apoptosis in Crohn's disease, and herbs-partitioned moxibustion (HPM) has been demonstrated to be an effective treatment for Crohn's disease. However, the mechanism by which HPM reduces intestinal epithelial cell apoptosis in Crohn's disease has not been thoroughly elucidated to date. AIM: To elucidate whether HPM exerts its effects by upregulating A20 to affect intestinal epithelial cell apoptosis in a Crohn's disease mouse model. METHODS: In this study, mice with A20 deletion in intestinal epithelial cells (A20IEC-KO) were utilized to establish a Crohn's disease mouse model with 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration, as well as wild-type mice. Mice were randomly divided into normal control (NC), model control (MC), mesalazine (MESA), and HPM groups. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and serum endotoxin and apoptosis of epithelial cells were evaluated by enzyme-linked immunosorbent assay and terminal dUTP nick-end labeling assay accordingly. The protein expression levels of A20 and tumor necrosis factor receptor 1 (TNFR1)-related signaling molecules were evaluated by Western blot, and co-expression of A20 and TNFR1-associated death domain (TRADD) and co-expression of A20 and receptor-interacting protein 1 (RIP1) were observed by double immunofluorescence staining. RESULTS: The intestinal epithelial barrier was noted to have an improvement in the HPM group of wild-type (WT) mice compared with that in A20IEC-KO mice. Compared with A20 IEC-KO HPM mice, serum endotoxin levels and apoptosis percentages were decreased (P < 0.01), A20 expression levels were increased (P < 0.01), and expression of TNFR1, TRADDD, and RIP1 was decreased in the HPM group of WT mice (P TNFR1 < 0.05, P TRADD < 0.01, P RIP1 < 0.01). Both of the co-expression of A20/TRADD and A20/RIP1 showed a predominantly yellow fluorescence in the HPM group of WT mice, while a predominantly red fluorescence was noted in the HPM group of A20IEC-KO mice. CONCLUSION: Our findings suggest that HPM in treating Crohn's disease functions possibly via upregulation of the A20 expression level, resulting in downregulation of TNFR1, TRADD, and RIP1 to alleviate increased cell apoptosis in the intestinal epithelial barrier in Crohn's disease.

Effect of Herb-Partitioned Moxibustion on Autophagy and Immune-Associated Gene Expression Profiles in a Rat Model of Crohn's Disease.

OBJECTIVE: To investigate the immune regulation mechanism of herb-partitioned moxibustion in rats with Crohn's disease (CD) focusing on autophagy. METHODS: Rats were randomly divided into normal (N) group, CD model (M) group, CD model with herb-partitioned moxibustion (MM) group, normal with herb-partitioned moxibustion (NM) group, CD model with mesalazine (western medicine, Med ) group, and normal saline (NS) group, with 10 rats in each group. The CD model rats were prepared by trinitrobenzene sulphonic expect for the N group and NM group. After the CD rats model were established, the rats in the MM and NM groups were treated with herb-partitioned moxibustion at Tianshu (ST25) and Qihai (CV6) acupoints once daily for 7 days, and rats in the Med and NS groups were respectively treated with mesalazine enteric coated tablet and normal saline once daily for 7 days. After intervention, hematoxylin-eosin staining was used to observe the histological changes of colon; RNA sequencing was used to observe the changes in autophagy- and immune-associated gene expression profiles. In addition, autophagy- and immune-associated cytokines and signaling pathways in CD rats were also screened. RESULTS: HPM significantly increased the body weight of CD rats (P<0.01) and improved the pathological injury of colon in CD rats (P<0.01). HPM also changed the expression of many autophagy- and immune-associated genes, especially downregulating the expression of autophagy-associated Nod2, Irgm genes as well as the receptor of immune-associated Il12b, Il22 (Il12rb1, Il22ra2) genes in the colon of CD rats. HPM also changed the enrichment levels of differentially expressed genes in the human T-cell leukemia virus type-1 infection pathway, the Epstein-Barr virus infection pathway, and the cell adhesion molecule pathway. In addition, the expression levels of Nod2, Irgm, IL-12b, and IL-22 mRNA were increased (all P< 0.01) in the M group compared to the N group, while the expression levels of Nod2, Irgm, IL-12b, and IL-22 mRNA were decreased (P<0.05 or P<0.01) in the MM and Med groups compared to the M group. CONCLUSION: Herb-partitioned moxibustion may effectively attenuate intestinal inflammation and promote the repair of colon mucosal injury of CD rats through the regulation of autophagy- and immune-associated gene expression and signaling pathways.

Comparison of Electroacupuncture and Mild-Warm Moxibustion on Brain-Gut Function in Patients with Constipation-Predominant Irritable Bowel Syndrome: A Randomized Controlled Trial.

OBJECTIVE: To compare the effects of electroacupuncture (EA) and mild-warm moxibustion (Mox) therapies for constipation-predominant irritable bowel syndrome (C-IBS) patients. METHODS: Sixty C-IBS patients were assigned to 2 groups by simple randomized method, i.e. EA group (30 cases) and Mox group (30 cases). Both EA and Mox treatments were performed on bilateral Tianshu (ST 25) and Shangjuxu (ST 37) for 30 min each time, 6 times per week, for 4 consecutive weeks. The gastrointestinal symptoms and psychological symptoms of the two groups were scored before and after treatment. The effects on the corresponding functional brain areas, namely the anterior cingulate cortex (ACC), insular cortex (IC) and prefrontal cortex (PFC) were observed by functional magnetic resonance imaging (fMRI) before and after treatment. RESULTS: Compared with the Mox group, greater improvements in abdominal distension, defecation frequency, diffificulty in defecation and stool features were observed in the EA group (all P<0.01), both Hamilton Anxiety Rating Scale and Hamilton Depression Rating Scale scores were signifificantly decreased in the EA group (all P<0.01). Finally, decreased activated voxel values were observed in the ACC, right IC and PFC brain regions of EA group with 150 mL colorectal distension stimulation (P<0.05 or P<0.01). CONCLUSIONS: Both EA and Mox could signifificantly improve some of the most intrusive symptoms of C-IBS patients, and EA was more effective than Mox. The therapeutic effect of these two therapies might through modulating of the brain-gut axis function. (Registration No. ChiCTRTRC-11001349).

Comparison of the analgesic effects between electro-acupuncture and moxibustion with visceral hypersensitivity rats in irritable bowel syndrome.

AIM: To observe whether there are differences in the effects of electro-acupuncture (EA) and moxibustion (Mox) in rats with visceral hypersensitivity. METHODS: EA at 1 mA and 3 mA and Mox at 43 °C and 46 °C were applied to the Shangjuxu (ST37, bilateral) acupoints in model rats with visceral hypersensitivity. Responses of wide dynamic range neurons in dorsal horns of the spinal cord were observed through the extracellular recordings. Mast cells (MC) activity in the colons of rats were assessed, and 5-hydroxytryptamine (5-HT), 5-hydroxytryptamine 3 receptor (5-HT3R) and 5-HT4R expressions in the colons were measured. RESULTS: Compared with normal control group, responses of wide dynamic range neurons in the dorsal horn of the spinal cord were increased in the EA at 1 mA and 3 mA groups (1 mA: 0.84 ± 0.74 vs 2.73 ± 0.65, P < 0.001; 3 mA: 1.91 ± 1.48 vs 6.44 ± 1.26, P < 0.001) and Mox at 43 °C and 46 °C groups (43 °C: 1.76 ± 0.81 vs 4.14 ± 1.83, P = 0.001; 46 °C: 5.19 ± 2.03 vs 7.91 ± 2.27, P = 0.01). MC degranulation rates and the expression of 5-HT, 5-HT3R and 5-HT4R in the colon of Mox 46 °C group were decreased compared with model group (MC degranulation rates: 0.47 ± 0.56 vs 0.28 ± 0.78, P < 0.001; 5-HT: 1.42 ± 0.65 vs 7.38 ± 1.12, P < 0.001; 5-HT3R: 6.62 ± 0.77 vs 2.86 ± 0.88, P < 0.001; 5-HT4R: 4.62 ± 0.65 vs 2.22 ± 0.97, P < 0.001). CONCLUSION: The analgesic effects of Mox at 46 °C are greater than those of Mox at 43 °C, EA 1 mA and EA 3 mA.

Review of Clinical Studies of the Treatment of Ulcerative Colitis Using Acupuncture and Moxibustion.

Background. Clinical studies suggest that acupuncture and moxibustion therapy in ulcerative colitis (UC) can regulate bowel inflammation, and these treatments have the advantages of low rates of adverse reactions and recurrence as well as good long-term efficacy. We reviewed the current status of clinical studies of the treatment. Methods. Randomized controlled trials (RCTs) using the therapy as the major intervention for treating UC were included from 1995 to 2015. The extracted data mainly included diagnostic standards, treatment methods, selection of acupoints, treatment times and courses, and efficacy determination criteria. Results. The use of diagnostic standards and efficacy criteria lacked unification and standardization. There were two main groups: acupuncture and moxibustion therapy combined with drug treatment and the use of all types of acupuncture and moxibustion therapy alone or in combination. The acupoint compositions included distal-proximal point combinations, back-shu point and front-mu point combinations, and acupuncture through meridians. The treatment courses in all the clinical trials had large variations. Conclusion. The treatment of UC in the examined articles was mainly based on the classical theory. However, many links of the clinical regimen design were still lacking, which affected the repeatability of the clinical studies and the accuracy of the clinical conclusions.

Comparison of Electroacupuncture and Moxibustion for Relieving Visceral Hypersensitivity in Rats with Constipation-Predominant Irritable Bowel Syndrome.

Aim. To compare whether there is different effect between electroacupuncture (EA) and moxibustion (Mox) on visceral hypersensitivity (their analgesic effects) in constipation-predominant irritable bowel syndrome (C-IBS). Methods. EA at 1 mA and 3 mA and Mox at 43°C and 46°C were applied to the Shangjuxu (ST37, bilateral) acupoint in rats with C-IBS and normal rats. An abdominal withdrawal reflex (AWR) score was used to assess visceral hypersensitivity. Toluidine blue staining was used to assess mast cell (MC) activity in colon of rats. Immunochemistry was used to measure 5-HT and 5-HT4 receptor expression in the colon. Results. AWR scores in all EA (1 mA and 3 mA) and Mox (43°C and 46°C) treatment groups after colorectal distention (CRD) stimulation pressure of 20, 40, 60, and 80 mmHg were significantly lower than those of the model (MC) group (P all < 0.01). The MC counts and degranulation rates in the colon of all EA and Mox treatment groups and the MC group were significantly higher than those of the NC group (P all < 0.01). MC degranulation rates in the colon of all EA and Mox treatment groups were lower than those of the MC group (P all < 0.05). 5-HT expression in colon of all EA and Mox treatment groups was significantly lower than that of the MC group (P all < 0.01), and 5-HT4R expression in colon of both EA groups was significantly higher than that of the MC group (P both < 0.01). Conclusion. EA and Mox treatments may both ameliorate visceral hypersensitivity at different degree in rats with C-IBS, and EA treatment was better than Mox.

[Effect of Electroacupuncture and Moxibustion on Visceral Pain and Expression of VR 1 and HSP 70 of "Tianshu" (ST 25) Region in Colorectal Distension-induced Visceral Hypersensitivity Rats].

OBJECTIVE: To compare the effects of electroacupuncture (EA) and moxibustion (Moxi) on visceral pain and expression of vanilloid receptor subtype 1 (VR 1) and heat shock protein (HSP)70 in "Tianshu" (ST 25) region in colorectal distension (CRD)-induced visceral hypersensitivity (VHS) rats. METHODS: Fifty male SD rats were randomly divided into normal control, VHS model, 43℃-moxi, 46℃-moxi, 1 mA-EA and 3 mA-EA groups (n=10 in each group). The VSH model was established by CRD once daily for 14 days. EA or Moxi stimulation was applied to bilateral "Tianshu" (ST 25) for 10 min, once daily for consecutive 10 days. The abdominal withdrawal reflex (AWR) scores (0-4 points) were rated according to Al-Chaer's and coworkers' standards (2000) and the expression levels of VR 1 and HSP 70 in bilateral ST 25 area tissues detected by immunohistochemistry. RESULTS: The AWR scores for 20, 40, 60 and 80 mmHg CRD pressures were significantly increased compared to the normal control group (P<0.01) and notably decreased after 43℃- and 46℃-moxi, and 1 mA- and 3 mA-EA stimulation of bila-teral ST 25 in comparison with the model group (P<0.05, P<0.01), and the effect of 46℃-moxi was apparently superior to those of 1 mA-EA at 40 and 80 mmHg, and 3 mA-EA at 40 mmHg (P<0.05). After modeling, the expression of both VR 1 and HSP 70 (percentages of area of positive-cells) in ST 25 region had no significant changes (P>0.05). Compared to the model group, the expression levels of VR 1 in the 43℃-moxi and 46℃-moxi groups, and HSP 70 in the 43℃-moxi and 46℃-moxi, 1 mA-EA and 3 mA-EA groups were significantly up-regulated (P<0.01), but without obvious changes in the expression of VR 1 in the 1 mA-EA and 3 mA-EA groups (P>0.05). The effects of 46℃-moxi were considerably better than those of 43℃-moxi, 1 mA-EA and 3 mA-EA in up-regulating VR 1 and HSP 70 expression (P<0.05, P<0.01). No significant differences were found among the 43℃-moxi, 1 mA-EA and 3 mA-EA groups in the expression of VR 1 and HSP 70 (P>0.05). CONCLUSIONS: Moxibustion at 43℃ and 46℃ and EA at 1 mA and 3 mA, especially the 46℃-moxi, can relieve visceral pain in visceral hypersensitivity rats, which may be related to their effects in up-regulating expression of VR 1 and HSP 70 in "Tianshu" (ST 25) area.

Effects of electroacupuncture on corticotropin-releasing hormone in rats with chronic visceral hypersensitivity.

AIM: To investigate the effect of electroacupuncture on corticotropin-releasing hormone (CRH) in the colon, spinal cord, and hypothalamus of rats with chronic visceral hypersensitivity. METHODS: A rat model of chronic visceral hypersensitivity was generated according to the internationally accepted method of colorectal balloon dilatation. In the 7(th) week after the procedure, rats were randomly divided into a model group (MG), electroacupuncture group (EA), and sham electroacupuncture group (S-EA). After treatment, the abdominal withdrawal reflex (AWR) score was used to assess the behavioral response of visceral hyperalgesia. Immunohistochemistry (EnVision method), ELISA, and fluorescence quantitative PCR methods were applied to detect the expression of CRH protein and mRNA in the colon, spinal cord, and hypothalamus. RESULTS: The sensitivity of the rats to the colorectal distension stimulus applied at different strengths (20-80 mmHg) increased with increasing stimulus strength, resulting in increasing AWR scores in each group. Compared with NG, the AWR score of MG was significantly increased (P < 0.01). After conducting EA, the AWR scores of the rats were decreased compared with MG rats. The relative expression of CRH mRNA in the colon, spinal cord, and hypothalamus of MG rats was significantly increased compared with NG rats (P < 0.01). CRH mRNA in the colon and spinal cord of EA and S-EA rats was decreased to varying degrees (P > 0.05) compared with normal rats (NG). However, the decrease in EA compared with MG rats was statistically significant (P < 0.01). The average optical density of CRH expression in the colon of the MG rats was significantly enhanced compared with NG (P < 0.05), while the average optical density of CRH expression in the EA and S-EA rats was significantly decreased compared with MG rats (P < 0.01, P < 0.05, respectively). Compared with MG rats, the CRH concentration in the spinal cord of EA rats was significantly reduced (P < 0.01), but there was no significant change in S-EA rats (P > 0.05). CONCLUSION: Electroacupuncture at the Shangjuxu acupoint was able to significantly reduce the visceral hypersensitivity in rats, and regulated the expression of CRH protein and mRNA in the colon, spinal cord and hypothalamus at different levels, playing a therapeutic role in this model of irritable bowel syndrome.

Moxibustion combined with acupuncture increases tight junction protein expression in Crohn's disease patients.

AIM: To investigate the effect of herb-partitioned moxibustion combined with acupuncture on the expression of intestinal epithelial tight junction (TJ) proteins. METHODS: Sixty patients diagnosed with mild to moderate Crohn's disease (CD) were allocated into the herb-partitioned moxibustion combined with acupuncture (HMA) group (n = 30) or the mesalazine (MESA) group (n = 30) using a parallel control method. There were 2 sets of acupoints used alternately for HMA treatment. The following points were included in Set A: ST25 (Tianshu), RN6 (Qihai), and RN9 (Shuifen) for herb-partitioned moxibustion and ST36 (Zusanli), ST37 (Shangjuxu), LI11 (Quchi), and LI4 (Hegu) for acupuncture. The points for Set B included BL23 (Shenshu) and BL25 (Dachangshu) for herb-partitioned moxibustion and EX-B2 of T6-T1 (Jiajixue) for acupuncture. The patients received the same treatment 6 times a week for 12 consecutive weeks. The MESA group received 1 g of mesalazine enteric coated tablets 4 times daily for 12 consecutive weeks. Intestinal tissues were stained and examined to compare the morphological and ultrastructural changes before and after the treatment session. Immunohistochemistry and in situ hybridization assays were used to detect the expression of intestinal epithelial TJ proteins zonula occludens-1 (ZO-1), occludin, and claudin-1. The mRNA levels were also evaluated. RESULTS: After the treatment, both herb-partitioned moxibustion combined with acupuncture and mesalazine improved intestinal morphology and ultrastructure of CD patients; the patients treated with HMA showed better improvement. HMA significantly increased the expression of ZO-1 (P = 0.000), occludin (P = 0.021), and claudin-1 (P = 0.016). MESA significantly increased the expression of ZO-1 (P = 0.016) and occludin (P = 0.026). However, there was no significant increase in the expression of claudin-1 (P = 0.935). There was no statistically significant difference between the two groups for the expression of occludin and claudin-1 (P > 0.05). The HMA group showed a significant improvement in ZO-1 expression compared to the MESA group (2333.34 ± 352.51 vs 2160.38 ± 307.08, P = 0.047). HMA significantly increased the expression of ZO-1 mRNA (P = 0.000), occludin mRNA (P = 0.017), and claudin-1 mRNA (P = 0.017). MESA significantly increased the expression of ZO-1 mRNA (P = 0.000), occludin mRNA (P = 0.042), and claudin-1 mRNA (P = 0.041). There was no statistically significant difference between the two groups in the expression of occludin and claudin-1 mRNA (P > 0.05). However, the HMA group showed a significant improvement in ZO-1 mRNA expression compared with the MESA group (2378.17 ± 308.77 vs 2200.56 ± 281.88, P = 0.023). CONCLUSION: HMA can repair intestinal epithelial barrier lesions and relieve inflammation by upregulating the expression of TJ proteins and their mRNAs.

Comparison of electroacupuncture and moxibustion on brain-gut function in patients with diarrhea-predominant irritable bowel syndrome: A randomized controlled trial.

OBJECTIVE: To compare the effects of electroacupuncture (EA) and moxibustion therapies on patients with diarrhea-predominant irritable bowel syndrome (D-IBS). METHODS: A total of 60 D-IBS patients were randomly allocated to the EA group (30 cases) and moxibustion group (30 cases). Before and after treatment, the gastrointestinal symptoms and psychological symptoms were scored by Visual Analogue Scale, Bristol Stool Form Scale, Hamilton Anxiety Rating Scale (HAMA), and Hamilton Depression Rating Scale (HAMD); the expressions of 5-hydroxytryptamine (5-HT), 5-HT3 receptor (5-HT3R), and 5-HT4 receptor (5-HT4R) in the sigmoid mucosal tissue were measured by immunohistochemical staining. Additionally, the effects on the functional brain areas of the anterior cingulate cortex (ACC), insular cortex (IC) and prefrontal cortex (PFC) were observed by functional magnetic resonance imaging. RESULTS: Compared with before treatment, both EA and moxibustion groups reported significant improvements in abdominal pain and abdominal bloating after treatment (P<0.01 or P<0.05). The moxibustion group reported greater improvements in defecation emergency, defecation frequency, and stool feature than the EA group (P<0.01). Both HAMA and HAMD scores were significantly decreased in the moxibustion group than in the EA group (P<0.01). Both groups demonstrated significantly reduced expressions of 5-HT, 5-HT3R and 5-HT4R in the colonic mucosa after treatment (P<0.01), with a greater reduction of 5-HT in the moxibustion group (P<0.05). Finally, decreased activated voxel values were observed in the left IC, right IC and PFC brain regions of patients in the moxibustion group under stimulation with 150 mL colorectal distension after treatment (P<0.05 or P<0.01), while in the EA group only PFC area demonstrated a reduction (P<0.05). CONCLUSION: Moxibustion can significantly improve the symptoms of D-IBS, suggesting that moxibustion may be a more effective therapy than EA for D-IBS patients.

Factorial study of moxibustion in treatment of diarrhea-predominant irritable bowel syndrome.

AIM: To identify an appropriate therapeutic regimen for using aconite cake-separated moxibustion to treat diarrhea-predominant irritable bowel syndrome (D-IBS). METHODS: A factorial design was employed to examine the two factors of moxibustion frequency and number of cones. The two tested frequencies were three or six moxibustion sessions per week, and the two tested doses were one or two cones per treatment. A total of 166 D-IBS patients were randomly divided into four treatment groups, which included each combination of the examined frequencies and doses. The bilateral Tianshu acupoints (ST25) and the Qihai acupoint (RN6) were selected for aconite cake-separated moxibustion. Each patient received two courses of treatment, and each course had a duration of 2 wk. For each group, the scores on the Birmingham irritable bowel syndrome (IBS) symptom questionnaire, the IBS Quality of Life scale, the Self-Rating Depression Scale (SDS), the Self-Rating Anxiety Scale (SAS), the Hamilton Depression (HAMD) scale, and the Hamilton Anxiety (HAMA) scale were determined before treatment, after the first course of treatment, and after the second course of treatment. RESULTS: The symptom, quality of life, SDS, SAS, HAMD, and HAMA scores of the patients in all 4 aconite cake-separated moxibustion groups were significantly lower after the first and second courses of treatment than before treatment (P < 0.001 for all). The symptom, quality of life, SDS, SAS, HAMD, and HAMA scores of the patients in all four aconite cake-separated moxibustion groups were significantly lower after the second course of treatment than after the first course of treatment (P < 0.001 for all). Between-group comparisons after the second course of treatment revealed that the symptom scores for group 1 (1 cone, 3 treatments/wk) and group 3 (2 cones, 3 treatments/wk) were significantly lower than that for group 2 (1 cone, 6 treatments/wk) (5.55 ± 5.05 vs 10.45 ± 6.61, P < 0.001; 5.65 ± 4.00 vs 10.45 ± 6.61, P < 0.001). Regarding the two levels of the two examined factors for aconite cake-separated moxibustion, after the first course of treatment, the changes in HAMA scores were significantly different for the two tested moxibustion frequencies (P = 0.011), with greater changes for the "6 treatments/wk" groups than for the "3 treatments/wk" groups; in addition, there were interaction effects between the number of cones and moxibustion frequency (P = 0.028). After the second course of treatment, changes in symptom scores for the 2 tested moxibustion frequencies were significantly different (P = 0.002), with greater changes for the "3 treatments/wk" groups than for the "6 treatments/wk" groups. CONCLUSION: An aconite cake-separated moxibustion treatment regimen of 3 treatments/wk and 1 cone/treatment appears to produce better therapeutic effects for D-IBS compared with the other tested regimens.

Randomized controlled trial: moxibustion and acupuncture for the treatment of Crohn's disease.

AIM: To evaluate the clinical efficacy and safety of acupuncture and moxibustion for the treatment of active Crohn's disease (CD). METHODS: Ninety-two patients were equally and randomly divided into the treatment group and received herb-partitioned moxibustion combined with acupuncture, and the control group received wheat bran-partitioned moxibustion combined with superficial acupuncture. The patients received three treatment sessions per week for 12 wk and were followed up for 24 wk. The main outcome was evaluated using the CD Activity Index (CDAI) score, and the secondary outcomes were evaluated using laboratory indicators such as hemoglobin (HGB), C-reactive protein (CRP), erythrocyte sedimentation rate, quality-of-life, endoscopic ratings, and intestinal histology scores. RESULTS: The CDAI scores of both the treatment and control groups were significantly reduced after treatment compared with those measured before treatment. However, the degree of improvement in the treatment group was significantly greater than that of the control group. The improvement in symptoms in patients of the treatment group was sustained at follow-up, whereas that of the control group was not. The overall efficacy of the treatment was significantly greater than that of the control. Both groups demonstrated significant improvements in quality-of-life ratings after treatment, but the improvement was significantly greater in the treatment group than in the control group. In addition, the patients in the treatment group showed significantly increased HGB and significantly decreased CRP levels and histopathological scores at the end of treatment, whereas the control group did not exhibit significant changes. CONCLUSION: Moxibustion with acupuncture provided significant therapeutic benefits in patients with active CD beyond the placebo effect and is therefore an effective and safe treatment for active CD.

Warming Moxibustion Relieves Chronic Visceral Hyperalgesia in Rats: Relations to Spinal Dynorphin and Orphanin-FQ System.

As a twin therapy of acupuncture in traditional Chinese medicine, moxibustion has shown its effects in relieving abdominal pain in irritable bowel syndrome (IBS) patients and IBS rat models, but its mechanisms are largely unknown. In this paper, we determined the role of spinal dynorphin and orphanin-FQ system in analgesic effect of warming moxibustion (WM) on chronic visceral hyperalgesia (CVH) in IBS-like rat model. Here, we show that (1) repeated WM at bilateral ST25 and ST37 acupoints markedly attenuated the abdominal withdrawal reflex scores in CVH rats; (2) intrathecal administration of κ receptor antagonist prior to WM significantly attenuated the WM analgesia and dynorphinA (1-17) enhanced the WM analgesia. WM significantly reinforced the upregulation of spinal dynorphin mRNA/protein and κ receptor mRNA levels in CVH rats; (3) intrathecal administration of orphanin-FQ receptor antagonist prior to WM significantly attenuated the WM analgesia and orphanin-FQ enhanced the WM analgesia. WM reinforced the upregulation of spinal orphanin-FQ mRNA/protein and orphanin-FQ receptor mRNA levels in CVH rats. These results suggest that moxibustion may relieve CVH at least in part by activating spinal dynorphin and orphanin-FQ system.

Moxibustion inhibits interleukin-12 and tumor necrosis factor alpha and modulates intestinal flora in rat with ulcerative colitis.

AIM: To investigate the effect of moxibustion on intestinal flora and release of interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α) from the colon in rat with ulcerative colitis (UC). METHODS: A rat model of UC was established by local stimulation of the intestine with supernatant from colonic contents harvested from human UC patients. A total of 40 male Sprague-Dawley rats were randomly divided into the following groups: normal (sham), model (UC), herb-partition moxibustion (HPM-treated), and positive control sulfasalazine (SA-treated). Rats treated with HPM received HPM at acupuncture points ST25 and RN6, once a day for 15 min, for a total of 8 d. Rats in the SA group were perfused with SA twice a day for 8 d. The colonic histopathology was observed by hematoxylin-eosin. The levels of intestinal flora, including Bifidobacterium, Lactobacillus, Escherichia coli (E. coli), and Bacteroides fragilis (B. fragilis), were tested by real-time quantitative polymerase chain reaction to detect bacterial 16S rRNA/DNA in order to determine DNA copy numbers of each specific species. Immunohistochemical assays were used to observe the expression of TNF-α and IL-12 in the rat colons. RESULTS: HPM treatment inhibited immunopathology in colonic tissues of UC rats; the general morphological score and the immunopathological score were significantly decreased in the HPM and SA groups compared with the model group [3.5 (2.0-4.0), 3.0 (1.5-3.5) vs 6.0 (5.5-7.0), P < 0.05 for the general morphological score, and 3.00 (2.00-3.50), 3.00 (2.50-3.50) vs 5.00 (4.50-5.50), P < 0.01 for the immunopathological score]. As measured by DNA copy number, we found that Bifidobacterium and Lactobacillus, which are associated with a healthy colon, were significantly higher in the HPM and SA groups than in the model group (1.395 ± 1.339, 1.461 ± 1.152 vs 0.045 ± 0.036, P < 0.01 for Bifidobacterium, and 0.395 ± 0.325, 0.851 ± 0.651 vs 0.0015 ± 0.0014, P < 0.01 for Lactobacillus). On the other hand, E. coli and B. fragilis, which are associated with an inflamed colon, were significantly lower in the HPM and SA groups than in the model group (0.244 ± 0.107, 0.628 ± 0.257 vs 1.691 ± 0.683, P < 0.01 for E. coli, and 0.351 ± 0.181, 0.416 ± 0.329 vs 1.285 ± 1.039, P < 0.01 for B. fragilis). The expression of TNF-α and IL-12 was decreased after HPM and SA treatment as compared to UC model alone (4970.81 ± 959.78, 6635.45 ± 1135.16 vs 12333.81 ± 680.79, P < 0.01 for TNF-α, and 5528.75 ± 1245.72, 7477.38 ± 1259.16 vs 12550.29 ± 1973.30, P < 0.01 for IL-12). CONCLUSION: HPM treatment can regulate intestinal flora and inhibit the expression of TNF-α and IL-12 in the colon tissues of UC rats, indicating that HPM can improve colonic immune response.