Objectives: To observe the effect of mild moxibustion on stem cell factor (SCF)/tyrosine kinase receptor (c-kit) signaling pathway and visceral hypersensitivity of diarrhea predominant irritable bowel syndrome (IBS-D) model rats with liver-qi stagnation and spleen deficiency syndrome, so as to explore its mechanisms underlying improvement of IBS-D.
Methods: A total of 24 male Wistar rats were randomly divided into normal, IBS-D model, medication and mild moxibustion groups, with 6 rats in each group. The IBS-D model was established by glacial acetic acid (4%) enema plus restraint stress stimulation once daily for 14 days. Rats of the medication group were treated by gavage of pivamium bromide (15 mg/kg) once a day for 14 days. Mild moxibustion was applied to bilateral "Tianshu"(ST25), "Shangjuxu"(ST37) and "Taichong"(LR3) for 20 min, once daily for 14 consecutive days. After the intervention, the rats' general state of each group were observed. The rate of loose stools (LSR), and the minimum volume threshold for abdominal withdrawal reflex(AWR) were observed, and the open field test was used to assess the state of rats' motor activities (including rearing times, grooming times and total number of square-crossings in 5 min). Morphological changes of the colon tissue were observed by hematoxylin-eosin (H.E.) staining, The count of mast cells (MC) in the colon tissues was determined by toluidine blue staining. Contents of serum 5-hydroxytryptamine (5-HT) and substance P (SP) were determined by enzyme-linked immunosorbent assay (ELISA). The relative expression levels of SCF and c-kit mRNAs and proteins in the colon tissues were detected by real-time quantitative PCR and Western blot, respectively.
Results: Compared with the normal group, the body weight, minimum volume threshold of AWR, total numbers of square-crossing, rearing times and grooming times were significantly decreased (P<0.05), and the LSR, number of MC, contents of 5-HT and SP, and the expression levels of SCF and c-kit mRNAs and proteins were considerably increased in the model group (P<0.01). In comparison with the model group after interventions, the body weight, minimum volume threshold of AWR, total numbers of square-crossing, rearing times and grooming times were apparently increased in both medication and moxibustion groups (P<0.05, P<0.01), and the LSR, number of MC, 5-HT and SP contents in both medication and moxibustion groups, and the expression levels of SCF and c-kit mRNA and protein in the moxibustion group (not in the medication group) were obviously decreased (P<0.05, P<0.01). H.E. staining showed that in the model group, a small amount of inflammatory cells in the mucosal layer of colon tissue could be seen. in the medication group, a small number of lymphocytes in colon tissue were observed, while in the mild moxibustion group, a small amount of neutrophils in colon tissue were observed.
Conclusions: Mild moxibustion can reduce visceral hypersensitivity and improve abdominal pain, diarrhea and locomotion state in IBS-D rats with liver-qi stagnation and spleen deficiency syndrome, which may be associated with its functions in reducing the number of MC and the levels of 5-HT and SP and down-regulating the activities of SCF/c-kit signaling pathway.
目的: 观察温和灸对肝郁脾虚证腹泻型肠易激综合征（IBS-D）模型大鼠干细胞生长因子（SCF）/酪氨酸激酶受体（c-kit）信号通路及内脏高敏感的影响，探讨温和灸治疗肝郁脾虚证IBS-D大鼠的可能作用机制。方法: 将24只Wistar雄性大鼠随机分为正常组、模型组、西药组、温和灸组，每组6只。采用冰醋酸灌肠+束缚应激方式建立肝郁脾虚证IBS-D大鼠模型。西药组予匹维溴铵灌胃（15 mg/kg），每日1次；温和灸组选取双侧“天枢”“上巨虚”“太冲”予温和灸治疗，每次20 min，每日1次。两组均连续干预14 d。干预结束后，观察各组大鼠一般情况，检测稀便率（LSR）；检测引起腹部回缩反射（AWR）达3分时的最小容量阈值；旷场实验检测大鼠直立次数、修饰次数及总穿格数；HE染色法观察各组大鼠结肠组织病理形态变化；甲苯胺蓝染色法检测结肠组织中肥大细胞（MC）计数；ELISA法检测大鼠血清5-羟色胺（5-HT）和P物质（SP）含量；实时荧光定量PCR法检测结肠组织SCF、c-kit的mRNA相对表达量；Western blot法检测大鼠结肠组织SCF、c-kit的蛋白相对表达量。结果: 与正常组比较，模型组大鼠体质量、AWR达3分时的最小容量阈值降低（P<0.05），LSR升高（P<0.01），总穿格数、直立次数、修饰次数均减少（P<0.05），结肠组织中MC数量明显增加（P<0.01），血清中5-HT、SP含量明显升高（P<0.01），结肠组织SCF、c-kit的mRNA、蛋白的表达明显升高（P<0.01）。治疗后，与模型组比较，西药组、温和灸组大鼠体质量增加（P<0.05），AWR达3分时的最小容量阈值明显增加（P<0.01），LSR降低（P<0.05），总穿格数、直立次数、修饰次数均增加（P<0.05），结肠组织中MC计数减少（P<0.05），血清中5-HT、SP含量降低（P<0.01）；与模型组比较，温和灸组大鼠结肠组织SCF、c-kit的mRNA、蛋白表达降低（P<0.05）。正常组大鼠结肠组织结构完整，无糜烂及水肿；模型组大鼠结肠组织黏膜层可见少量炎性细胞浸润；西药组大鼠结肠组织可见少量淋巴细胞聚集；温和灸组大鼠结肠组织可见少量中性粒细胞聚集。结论: 温和灸可降低肝郁脾虚证IBS-D模型大鼠的内脏高敏感性，改善其腹痛、腹泻及精神状态等症状，其作用机制可能与下调SCF/c-kit信号通路的表达，减少MC数量，降低5-HT、SP水平有关。.
Keywords: Diarrhea predominant irritable bowel syndrome (IBS-D); Liver- qi stagnation and spleen deficiency syndrome; Mild moxibustion; Stem cell factor/tyrosine kinase receptor (SCF/c-kit) signaling; Visceral hypersensitivity.