Effect of electro-acupuncture on substance P, its receptor and corticotropin-releasing hormone in rats with irritable bowel syndrome

AIM： To investigate the effect and mechanism of electro-acupuncture lEA） at ST25 and ST37 on irritable bowel syndrome （IBS） of rats. METHODS： A total of 21 male Sprague-Dawley rats were randomly divided into normal group, model group and EA group. A rat model of IBS was established by constraining the limbs and distending the colorectum of rats. Rats in EA group received bilateral EA at ST25 and ST37 with a sparse and intense waveform at a frequency of 2/50 Hz for 15 min, once a day for 7 d as a course. Rats in normal and model groups were stimulated by distending colorectum （CR）. An abdominal withdrawal reflex （AWR） scoring system was used to evaluate improvements in visceral hypersensitivity. Toluidine blue-improved method, immunohistochemistry and radioimmunoassay were used to observe mucosal mast cells （MC）, changes of substance P （SP） and substance P receptor （SPR） in colon and change of corticotropin-releasing hormone （CRH） in hypothalamus. RESULTS： The threshold of visceral sense was significantly lower in model group than in normal group,and significantly higher in EA group than in model group. The number of mucosal MC was greater in model group than in normal group and significantly smaller in EA group than in model group. The CRH level in hypothalamus of rats was significantly higher in model group than in normal group, which was remarkably decreased after electro-acupuncture treatment. The SP and SPR expression in colon of rats in model group was decreased after electro-acupuncture treatment. CONCLUSION： EA at ST25 and ST37 can decrease the number of mucosal MC and down-regulate the expression of CRH in hypothalamus, and the expression of SP and SPR in colon of rats with IBS.

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.

Moxibustion down-regulates colonic epithelial cell apoptosis and repairs tight junctions in rats with Crohn's disease

AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn's disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulphonic acid to induce intestinal inflammation. The rats in the HPM and MWM groups were treated at the Tianshu (ST25) and Qihai (CV6) acupoints once daily for 14 d, and the SASP group was fed SASP twice daily for 14 d. No additional treatment was given to the MC and NC groups. Themicrostructure of the colonic epithelium was observed under a transmission electron microscope, the transepithelial resistance was measured using a shortcircuit current, colonic epithelial cell apoptosis was determined by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labelling assay, and the expression of occludin, claudin-1 and zonula occludens-l (ZO-1) in the colonic epithelial junction was determined by Western blotting and immunofluorescence staining. RESULTS: Compared with the MC group, the microstructure of the colonic epithelial barrier was signifi-cantly improved in rats treated with HPM, MWM or SASP, meanwhile, the current flow was reduced signifi-cantly, with values of 168.20 ± 6.14 vs 99.70 ± 3.13, 99.10 ± 4.28 and 120.30 ± 3.65 mA, respectively (P = 0.001). However, the HPM and MWM groups had higher current flow rates than the SASP group (99.70 ± 3.13, 99.10 ± 4.28 vs 120.30 ± 3.65 mA, P = 0.001). The number of the apoptotic colonic epithelial cells in HPM, MWM and SASP groups was largely reduced (61.5 ± 16.91 vs 15.5 ± 8.89, 14.8 ± 6.27 and 24.7 ± 9.68, respectively (P = 0.001); and the expression of occlu- din, claudin-1 and ZO-1 in the MWM and HPM groups was signifi cantly enhanced (0.48 ± 0.10, 0.64 ± 0.09 vs 0.18 ± 0.05 for occludin, 0.12 ± 0.02, 0.17 ± 0.03 vs 0.05 ± 0.01 for claudin-1, and 0.08 ± 0.01, 0.11 ± 0.01 vs 0.02 ± 0.01 for ZO-1). And in SASP group, the expression of occludin and ZO-1 was also signifi cantly increased (0.27 ± 0.04 vs 0.18 ± 0.05 for occludin and 0.05 ± 0.01 vs 0.02 ± 0.01 for ZO-1), but there was no significant difference for claudin-1. The HPM and MWM groups had higher expression of occludin, claudin-1 and ZO-1 than the SASP group. CONCLUSION: HPM and MWM treatment can down-regulate apoptosis of colonic epithelial cells, repair tight junctions and enhance colonic epithelial barrier function in rats with CD.

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.

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&#x02019;s disease (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.

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, difficulty 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 significantly 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 significantly 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. ChiCTR- TRC-11001349）.

Mechanism of acupuncture on anti-neuronal apoptosis and the regulation of PI3K/AKT signaling pathway in heroin relapse rats

Objective:To explore the mechanism of acupuncture intervention on anti-neuronal apoptosis in heroindependent rats based on the PI3K/AKT signaling pathway.Methods:A total of 30 SD rats were randomly divided into a normal group,a model group and an acupuncture group,10 rats in each one.In the model group and the acupuncture group,the heroin relapse rat model was established by intramuscular incremental injection of heroin.In the model group,no any intervention was applied.In the acupuncture group,after modeling,acupuncture was applied at"Bǎihuì(百会GV20)"and"Dàzhuī(大椎GV14)".Transmission electron microscope(TEM)was adopted to observe neuronal apoptosis in the rats.The effect of acupuncture on anti-neuronal apoptosis was compared.Using reverse transcription-polymerase chain reaction(RT-PCR)and western blot methods,the mRNA and protein expressions of PI3 K and AKT were detected in ventral tegmental area(VTA).Results:It was found that edema was presented in VTA neuronal cytoplasm and organelles basically disappeared in the heroin relapse rats,nuclear chromatin aggregation,condensation and increased neuronal apoptosis were presented as well.After acupuncture at"Bǎihuì(百会GV20)"and"Dàzhuī(大椎GV14)",a small amount of mitochondria,rough endoiplasmic reticulum and glycogen granules were visible in the cytoplasm of VTA neurons.The nuclear membrane structure was clear and the chromatin in the nucleus was basically normal.The fluorescence quantitative polymerase chain reaction(PCR)and western blot methods were adopted to detect mRNA and protein expressions of PI3K and AKT.It was found that the mRNA and protein expressions of PI3K and AKT in the brain of the rats in the model group were lower than those in the normal group(all P<0.05).Compared with the model group,the mRNA and protein expressions of PI3K and AKT in the acupuncture group were increased(all P<0.05),tending to the levels as the normal group.Conclusion:The effect of acupuncture on anti-brain cell apoptosis in heroin relapse rats may be related to the inhibition of PI3K/AKT signaling pathway.