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.

Alterations in microRNA expression profiles in inflamed and noninflamed ascending colon mucosae of patients with active Crohn's disease.

BACKGROUND AND AIM: The microRNA (miRNA) expression profiles of the terminal ileum, sigmoid colon, and rectal mucosa of adult patients with active Crohn's disease (CD) have been previously reported. The purpose of this study was to identify dysregulated miRNAs in the mucosa of the ascending colon. METHODS: Biopsy tissue samples were taken from the mucosae of inflammatory (iCD) or noninflammatory (niCD) areas of the ascending colons of adult patients with active CD. miRNA and mRNA expression profiles were detected using microarray analyses. miRNAs and messenger RNAs (mRNAs) demonstrating significant differences were validated via quantitative real-time polymerase chain reaction. Luciferase reporter genes were used to measure two miRNAs inhibition of potential target genes in human 293T cells in vitro. RESULTS: Compared with the healthy control group, the ascending colon miRNA expression profiles revealed that 43 miRNAs were significantly upregulated and 35 were downregulated in the iCD group. The mRNA expression profiles indicated that 3370 transcripts were significantly differentially expressed in the ascending colon, with 2169 upregulated and 1201 downregulated mRNAs in the iCD group, and only 20 miRNAs demonstrated significant differential expression in the niCD group. In contrast, nearly 100 miRNAs significantly varied between the iCD and niCD groups. Finally, luciferase reporter gene assays showed that hsa-miR-16-1 directly regulated the human C10orf54 gene and that they were negatively correlated. CONCLUSIONS: Our results indicated that the differentially expressed miRNAs and mRNAs were related to immune inflammation and intestinal flora. The data provide preliminary evidence that the occurrence of CD involves the inhibition of C10orf54 expression by hsa-miR-16-1.

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.

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.

Moxibustion treatment restoring the intestinal epithelium barrier in rats with Crohn's disease by down-regulating tumor necrosis factor alpha, tumor necrosis factor receptor 1, and tumor necrosis factor receptor 2.

OBJECTIVE: To investigate whether moxibustion regulates tumor necrosis factor alpha (TNF-α), tumor necrosis factor receptor 1 (TNFR1), and TNFR2 in the intestinal mucosa and to explore whether moxibustion could be used by means of this mechanism, to repair the intestinal epithelium barrier disruption in Crohn's disease (CD). METHODS: The CD rat models were established by trinitrobenzene sulfonic acid (TNBs), randomly divided into a model control (MC) group, an herb-partition moxibustion (HPM) group, a mild-warm moxibustion (MWM) group, and a salicylazosulfapyridine (SASP) group, and all were compared with a normal control (NC) group. The HPM and MWM groups were treated by moxibustion at Tianshu (ST25) and Qihai (RN6) for 14 days, and the SASP group obtained the SASP solution orally for the same period of time. The intestinal epithelium morphology and TNF-α, TNFR1, and TNFR2 contents were observed by the transmission electron microscopy and enzyme linked immunosorbent assay. RESULTS: The severity of morphological changes in CD intestinal epithelium was obviously improved, and the levels of TNF-α, TNFR1, and TNFR2 in the intestinal mucosa all significantly decreased in the HPM and MWM groups. However, there were no significant differences between the HPM and MWM groups. CONCLUSION: The moxibustion therapies (HPM and MWM) could reduce intestinal inflammation and restore intestinal epithelium barrier disruption in CD, which might be due to down-regulating TNF-α, TNFR1, and TNFR2 in intestinal mucosa and improving intestinal epithelium morphology.

Electro-acupuncture relieves visceral sensitivity and decreases hypothalamic corticotropin-releasing hormone levels in a rat model of irritable bowel syndrome.

Previous studies into electro-acupuncture (EA) treatment of irritable bowel syndrome (IBS) have principally focused on the peripheral effects of EA in a rat model of IBS. It is not known whether EA exerts central effects in this rat model. We have examined the effects of EA on hypothalamic corticotropin-releasing hormone (CRH) levels in a rat model of IBS provoked by colorectal distension (CRD) and forelimb immobilization. EA was administered once daily to IBS model rats over a period of 7 d; untreated IBS rats and controls were also studied. The behavioral response to distension was rated according to the abdominal withdrawal reflex (AWR) score; hypothalamic CRH levels were measured by radioimmunoassay. We report that EA treatment significantly decreased visceral sensitivity to CRD in this rat model. In treated animals, EA also decreased hypothalamic CRH to control levels. Reduced hypothalamic CRH levels may mediate the beneficial effects of EA in this rat IBS model.