Objective: To observe the effect of moxibustion on the expression of miR-345-3p, miR-216a-5p and nuclear factor-κB p65(NF-κB p65) in colonic tissue of rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its anti-inflammatory mechanism in relieving IBS-D.
Methods: SD rats were randomly divided into normal control (n=12), model (n=12), moxibustion (n=12) and ammonium pyrrolidine dithiocarbamate (PDTC,n=12) groups. The IBS-D model was established by neonatal mother-child separation combined with acetic acid enema stimulation and chronic binding methods. The rats in the moxibustion group received moxibustion stimulation of "Tianshu"(ST25) and "Shangjuxu"(ST37) for 20 min, once a day, for 7 days, and those of the PDTC group received intraperitoneal injection of PDTC (50 mg·kg-1·d-1) once daily for 7 days. After the intervention, the body weight, loose stool rate and the minimum volume threshold of abdominal withdrawal reflex (AWR) were observed, and histopathological changes of colonic mucosa were observed by HE staining. The contents of interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6) and tumor necrosis factor α (TNF- α) in serum were measured by ELISA. The expression of miR-345-3p, miR-216a-5p and NF-κB p65 mRNA in the colon tissue were detected by quantitative real-time PCR, and the immunoactivities of IL-1β, IL-6, TNF-α and NF-κB p65 in the colon tissue were determined by immunofluorescence histochemistry.
Results: Compared with the normal control group, the loose stool rate, contents of IL-1β, IL-6 and TNF-α, experssion of NF-κB p65 mRNA and the immunoactivities of IL-1β, IL-6, TNF-α and NF-κB p65 were significantly increased (P<0.01), whereas the body weight, minimum volume threshold of AWR, content of IL-4, and the relative expression of miR-345-3p and miR-216a-5p were remarkably decreased in the model group (P<0.01). In comparison with the model group, the loose stool rate, contents of IL-1β, IL-6, TNF-α, expression of NF-κB p65 mRNA and the immunoactivities of IL-1β, IL-6, TNF-α and NF-κB p65 were considerably down-regulated (P<0.01), while the content of IL-4 and the relative expressions of miR-345-3p and miR-216a-5p were obviously up-regulated in both moxibustion and PDTC groups (P<0.01, P<0.05). The content of IL-6 in serum was significantly lower in the PDTC group than in the moxibustion group (P<0.01).
Conclusion: Moxibustion can reduce the level of intestinal inflammation and visceral hypersensitivity in IBS-D rats, which may be related to its functions in increasing the expression levels of miR-345-3p and miR-216a-5p and in inhibiting the expression of NF-κB p65, thus reducing the levels of inflammatory factors.
目的：观察艾灸对腹泻型肠易激综合征(IBS-D)大鼠结肠组织miR-345-3p、miR-216a-5p、核因子-κB p65(NF-κB p65)表达的影响，探讨艾灸治疗IBS-D的抗炎机制。方法：SD幼鼠随机分为正常组、模型组、艾灸组、吡咯烷二硫代甲酸铵盐(PDTC)组，每组12只。采用新生仔鼠母子分离联合醋酸灌肠刺激联合慢性束缚法复制IBS-D大鼠模型。艾灸组艾灸“天枢”“上巨虚”，每次20 min，每天1次，治疗7 d。PDTC组腹腔注射PDTC(50 mg·kg-1·d-1)，共治疗7 d。观察各组幼鼠体质量、稀便率和腹部回缩反射(AWR)最小容量阈值；HE染色法观察各组幼鼠结肠黏膜层病理形态；ELISA法检测各组幼鼠血清中白细胞介素(IL)-1β、IL-4、IL-6、肿瘤坏死因子(TNF)-α含量；荧光定量PCR法检测各组幼鼠结肠组织中miR-345-3p、miR-216a-5p、NF-κB p65 mRNA表达水平；免疫荧光法检测各组幼鼠结肠组织中IL-1β、IL-6、TNF-α、NF-κB p65表达水平。结果：与正常组比较，模型组稀便率升高(P<0.01)，体质量、AWR最小容量阈值降低(P<0.01)，结肠组织黏膜存在轻度炎性反应，血清IL-1β、IL-6、TNF-α含量升高(P<0.01)，IL-4含量降低(P<0.01)，结肠组织miR-345-3p、miR-216a-5p相对表达量降低(P<0.01)，NF-κB p65 mRNA相对表达量升高(P<0.01)，结肠组织IL-1β、IL-6、TNF-α、NF-κB p65免疫荧光平均吸光度值升高(P<0.01)。与模型组比较，艾灸组和PDTC组稀便率降低(P<0.01)，体质量、AWR最小容量阈值升高(P<0.01)，结肠组织黏膜炎性反应减轻，血清IL-1β、IL-6、TNF-α含量降低(P<0.01)，IL-4含量升高(P<0.01)，结肠组织miR-345-3p、miR-216a-5p相对表达量升高(P<0.05)，NF-κB p65 mRNA相对表达量降低(P<0.01)，结肠组织IL-1β、IL-6、TNF-α、NF-κB p65免疫荧光平均吸光度值降低(P<0.01)。与艾灸组比较，PDTC组血清IL-6含量降低(P<0.01)。结论：艾灸可以减轻IBS-D大鼠肠道炎性反应水平，降低内脏高敏感性，其机制可能与提高结肠组织miR-345-3p、miR-216a-5p表达水平，抑制NF-κB p65表达水平，从而降低炎性因子水平有关。.
Keywords: Diarrhea-predominant irritable bowel syndrome; Inflammation; Moxibustion; NF-κB p65; miR-216a-5p; miR-345-3p.