病因相关肠易激综合征动物模型研究进展

肠易激综合征(irritable bowel syndrome, IBS)属于慢性功能性肠道疾病,其发病率在全球范围内呈逐年上升趋势.目前病因和发病机制尚不完全明确,较为公认的病因包括内脏高敏感、肠道感染、精神心理、胃肠激素、肠道菌群紊乱、基因遗传等因素.因此,研究者研发了多种与IBS病因相关的动物模型,取得了重大研究进展并普遍应用于基础实验研究.本文概括总结了目前与IBS病因相关的国内外通用的动物模型的制作方法,以期为IBS基础研究的动物模型选择提供参考.

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/postinflammatory 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,qRTPCR,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-1β,IL-18,and resistance-like moleculeβby 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.

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.

Moxibustion treatment modulates the gut microbiota and immune function in a dextran sulphate sodium-induced colitis rat model

AIM To investigate the effect and mechanism of moxibustion in rats with ulcerative colitis.METHODS A rat colitis model was established by administering 4% dextran sulphate sodium solution. Seventy male rats were randomly divided into seven groups: Healthy controls(HC), ulcerative colitis model group(UC), UC with 7 d of moxibustion(UC-7), UC with 14 d of moxibustion(UC-14), UC with mesalazine gavage(UC-W), HC with 7 d of moxibustion(HC-7), HC with 14 d of moxibustion(HC-14). Moxibustion was applied to the bilateral Tianshu(ST25). Gut microbiome profiling was conducted by 16 S r RNA amplicon sequencing, and PCR and ELISA determined the expression of inflammatory cytokines in colon mucosa and serum, respectively. RESULTS Moxibustion treatment restored the colonic mucosa and decreased submucosal inflammatory cell infiltration in colitis rats. Rats treated with moxibustion and mesalazine had significantly lower levels of the dominant phyla Proteobacteria and the genera Saccharibacteria, Sphingomonas and Barnesiella than colitis rats, and they could restore the microbiome to levels similar to those observed in healthy rats. UC rats had reduced alpha diversity, which could be alleviated by moxibustion therapy, and UC-7 had a higher alpha diversity than UC-14. This finding suggests that short-term(7 d) but no longer term(14 d) moxibustion treatment may significantly affect the gut microbiome. The potential bacterial functions affected by moxibustion may be ascorbate and aldarate metabolism, and amino acid metabolism. Compared with HC group, the levels of the cytokines interleukin-12(IL-12)(P < 0.05) and IL-6, IL-17, IL-23, interferon-γ, lipopolysaccharide, Ig A, tumour necrosis factor-α and its receptors 1(TNFR1) and TNFR2(P < 0.01) were all increased, whereas anti-inflammatory cytokine IL-2 and IL-10(P < 0.01) and transforming growth factor-β(P < 0.05) were decreased in UC rats. These changes were reversed by moxibustion.CONCLUSION Our findings suggest that moxibustion exerts its therapeutic effect by repairing mucosal tissue damage and modulating the gut microbiome and intestinal mucosal immunity.

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)fo r acupuncture.The patients received the same treatment6 times a week for 12 consecutive weeks.The MESA group received 1 g of mesalazine enteric coated tablets4 times daily for 12 consecutive weeks.Intestinaltissues 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 m RNA 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 m RNA(P=0.000),occludin m RNA(P=0.017),and claudin-1 m RNA(P=0.017).MESA significantly increased the expression of ZO-1 m RNA(P=0.000),occludin m RNA(P=0.042),and claudin-1 m RNA(P=0.041).There was no statistically significant difference between the two groups in the expression of occludin and claudin-1 m RNA(P>0.05).However,the HMA group showed a significant improvement in ZO-1 m RNA 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 m RNAs.

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 decreasedCRP 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.

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 m A and 3 m A and Mox at 43?℃ and 46?℃ 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-HT4Rexpressions 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 m A and 3 m A groups(1 m A: 0.84 ± 0.74 vs 2.73 ± 0.65, P < 0.001; 3 m A: 1.91 ± 1.48 vs 6.44 ± 1.26, P < 0.001) and Mox at 43?℃ and 46?℃ groups(43?℃: 1.76 ± 0.81 vs 4.14 ± 1.83, P = 0.001; 46?℃: 5.19 ± 2.03 vs 7.91 ± 2.27, P = 0.01). MC degranulation rates and the expression of 5-HT, 5-HT3 R and 5-HT4 R in the colon of Mox 46?℃ 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?℃ are greater than those of Mox at 43?℃, EA 1 m A and EA 3 m A.

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 herbspartitioned 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(A20 IEC-KO) were utilized to establish a Crohn's disease mouse model with 2,4,6-trinitrobenzenesulfonic 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 A20 IEC-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(PTNFR1 < 0.05, PTRADD < 0.01, PRIP1 < 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 A20 IEC-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.

Acupuncture and moxibustion for irritable bowel syndrome:An umbrella systematic review

BackgroundIrritable bowel syndrome(IBS)is a functional bowel disease characterized by abdominal pain or discomfort associated with altered bowel habits.Several clinical studies have demonstrated the effectiveness of acupuncture and moxibustion for IBS.Many systematic reviews of acupuncture and moxibustion for IBS have been published in recent years,but their results are not entirely consistent.ObjectiveTo evaluate the methodological,reporting,and evidence quality of systematic reviews of acupuncture and moxibustion for IBS.Search strategySystematic reviews of acupuncture and moxibustion for IBS published before February 20,2023 were searched in eight databases:PubMed,Embase,Cochrane Library,Web of Science,China National Knowledge Infrastructure,Wanfang Data,VIP Database for Chinese Technical Periodicals,and China Biology Medicine.The keywords used for literature search were acupuncture,moxibustion,systematic review,meta-analysis,and irritable bowel syndrome.Inclusion criteriaSystematic reviews and meta-analyses of randomized controlled trials of acupuncture and moxibustion for IBS were included.Data extraction and analysisRelevant information was independently extracted by two investigators.The A MeaSurement Tool to Assess systematic Reviews 2(AMSTAR 2),Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA 2020),and Grading of Recommendations Assessment,Development and Evaluation(GRADE)were used to evaluate the methodological quality,reporting quality and evidence quality,respectively.ResultsA total of 342 studies were retrieved and 15 systematic reviews were included.The results of AMSTAR 2 showed low methodological quality in 2 studies and very low methodological quality in the remaining 13 studies,with main issues being failure to register a protocol,incomplete search strategy,not providing a list of excluded studies,incomplete consideration of the risk of bias in the included studies,and a failure to assess the publication bias.The results of PRISMA 2020 showed seriously deficient reporting quality of 2 studies,somewhat deficient reporting quality of 12 studies,and relatively complete reporting quality of 1 study,with the main problems being lack of a complete search strategy,non-availability of a list of excluded studies with justification for their exclusion,not conducting heterogeneity and sensitivity analyses,not evaluating the credibility of the evidence,and not registering the protocol.The results of GRADE showed that the quality of the evidence is low or very low.ConclusionMost included systematic reviews interpreted findings to suggest that acupuncture and moxibustion have benefits for IBS.However,there is a need to improve the methodological,reporting and evidence quality of the systematic reviews.Larger,multicenter,rigorously designed randomized controlled trials and high-quality systematic reviews are required to obtain more robust evidence.