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Title: Effect of moxibustion on colonic low-grade inflammatory response in rats with diarrhea-predominant irritable bowel syndrome based on mast cell degranulation.
Author: Fang M, Chu H, Song X, Ruan J, Zou L, Li K, Liao L, Ma W, Han X, Zhu J, Wang Z, Fang Y.
Journal: Zhongguo Zhen Jiu; 2024 Mar 12; 44(3):283-294. PubMed ID: 38467503.
Abstract:
OBJECTIVES: To observe the effects of moxibustion on colonic mast cell degranulation and inflammatory factor expression in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and explore the potential mechanism of moxibustion in treating IBS-D. METHODS: Forty-five rat pups born from 5 healthy SPF-grade pregnant SD rats, with 8 rats were randomly selected as the normal group. The remaining 37 rats were intervened with maternal separation, acetic acid enema, and chronic restraint stress to establish the IBS-D model. The successfully modeled 32 rats were then randomly assigned to a model group, a ketotifen group, a moxibustion group, and a moxibustion-medication group, with 8 rats in each group. The rats in the ketotifen group were intervened with intragastric administration of ketotifen solution (10 mL/kg); the rats in the moxibustion group were intervened with suspended moxibustion on bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the moxibustion-medication group were intervened with suspended moxibustion combined with intragastric administration of ketotifen solution. All interventions were administered once daily for 7 consecutive days. The diarrhea rate and minimum volume threshold of abdominal withdrawal reflex (AWR) were calculated before and after modeling, as well as after intervention. After intervention, colonic tissue morphology was observed using HE staining; colonic mucosal ultrastructure was examined by scanning electron microscopy; colonic mast cell ultrastructure was observed using transmission electron microscopy; mast cell degranulation was assessed by toluidine blue staining; serum and colonic levels of histamine, interleukin (IL)-1β, IL-6, IL-1α, trypsin-like enzyme, and protease-activated receptor 2 (PAR-2) were measured by ELISA; the Western blot and real-time quantitative PCR were employed to evaluate the protein and mRNA expression of colonic IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2; the immunofluorescence was used to detect the positive expression of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colonic tissue. RESULTS: Compared to the normal group, the rats in the model group exhibited extensive infiltration of inflammatory cells in colonic tissue, severe damage to the colonic mucosa, disordered arrangement of villi, reduced electron density, and a significant decrease in granule quantity within mast cells. The diarrhea rate and mast cell degranulation rate were increased (P<0.01), AWR minimum volume threshold was decreased (P<0.01); the serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were elevated (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all elevated (P<0.01). Compared to the model group, the rats in the ketotifen group, the moxibustion group, and the moxibustion-medication group exhibited significantly reduced infiltration of inflammatory cells in colonic tissue, relatively intact colonic mucosa, orderly arranged villi, increased electron density, and an augmented number of mast cell granules; the diarrhea rate and mast cell degranulation rate were decreased (P<0.01), and AWR minimum volume threshold was increased (P<0.01); the serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were reduced (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all decreased (P<0.01). Compared to the ketotifen group, the moxibustion group showed decreased serum levels of histamine, IL-6, and trypsin-like enzyme (P<0.01, P<0.05), as well as reduced colonic levels of IL-1β and IL-6 (P<0.01, P<0.05); the protein expression of colonic IL-1β, IL-1α, and PAR-2 was reduced (P<0.05), and the positive expression of colonic IL-1β and trypsin-like enzyme was reduced (P<0.01, P<0.05). Compared to both the ketotifen group and the moxibustion group, the moxibustion-medication group exhibited decreased diarrhea rate and mast cell degranulation rate (P<0.01), an increased AWR minimum volume threshold (P<0.01), reduced serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01), decreased protein expression of colonic IL-1β, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), reduced mRNA and positive expression of colonic IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), and decreased positive expression of colonic histamine (P<0.01). CONCLUSIONS: Moxibustion on "Tianshu" (ST 25) and "Shangjuxu" (ST 37) might inhibit low-grade inflammatory reactions in the colon of IBS-D model rats. The mechanism may be related to the inhibition of histamine and trypsin-like enzyme secreted by mast cell, thereby reducing the expression of related inflammatory factors. 目的: 观察艾灸对腹泻型肠易激综合征（IBS-D）模型大鼠结肠肥大细胞脱颗粒及炎性因子表达的影响，探讨艾灸治疗IBS-D的可能机制。方法: 从5只健康SPF 级 SD 孕鼠产下的 45只幼鼠中随机选取 8只作为正常组，其余37只采用母子分离+醋酸灌肠+慢性束缚应激的方法制备IBS-D模型，将造模成功的32只大鼠随机分为模型组、酮替芬组、艾灸组、灸药组，每组8只。酮替芬组给予酮替芬溶液（10 mL/kg）灌胃，艾灸组给予悬灸双侧“天枢”“上巨虚”，灸药组给予悬灸双侧“天枢”“上巨虚”结合酮替芬溶液灌胃，均每天 1 次，连续干预 7 d。分别于造模前后及干预后计算各组稀便率、检测腹壁回缩反射（AWR）最小容量阈值。干预后，采用HE染色观察各组大鼠结肠组织形态，扫描电镜观察结肠黏膜超微结构，透射电镜观察结肠黏膜肥大细胞超微结构，甲苯胺蓝染色观察结肠黏膜肥大细胞脱颗粒情况，ELISA法检测大鼠血清和结肠组胺、白细胞介素（IL）-1β、IL-6、IL-1α、类胰蛋白酶、蛋白酶激活受体 2 （PAR-2）含量，Western blot法和实时荧光定量PCR法检测大鼠结肠IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2蛋白和mRNA表达，免疫荧光法检测大鼠结肠组胺、IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2阳性表达。结果: 与正常组比较，模型组大鼠结肠组织被大量炎性细胞浸润，结肠黏膜严重破坏，绒毛排列散乱，肥大细胞内颗粒电子密度降低、颗粒数量明显减少；稀便率、肥大细胞脱颗粒率升高（P<0.01），AWR最小容量阈值降低（P<0.01）；血清和结肠组胺、IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2含量升高（P<0.01）；结肠组胺阳性表达升高（P<0.01），IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2蛋白、mRNA及阳性表达均升高（P<0.01）。与模型组比较，酮替芬组、艾灸组、灸药组大鼠结肠组织炎性细胞浸润明显减少，结肠黏膜较完整，绒毛排列较整齐，肥大细胞内颗粒电子密度升高、颗粒数量增多；稀便率、肥大细胞脱颗粒率下降（P<0.01），AWR最小容量阈值升高（P<0.01）；血清和结肠组胺、IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2含量降低（P<0.01）；结肠组胺阳性表达降低（P<0.01），结肠IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2蛋白、mRNA及阳性表达均降低（P<0.01）。与酮替芬组比较，艾灸组血清组胺、IL-6、类胰蛋白酶含量降低（P<0.01，P<0.05），结肠IL-1β、IL-6含量降低（P<0.01，P<0.05）；结肠IL-1β、IL-1α、PAR-2蛋白表达降低（P<0.05），结肠IL-1β、类胰蛋白酶阳性表达降低（P<0.01，P<0.05）。与酮替芬组和艾灸组比较，灸药组稀便率、肥大细胞脱颗粒率下降（P<0.01），AWR最小容量阈值上升（P<0.01）；血清和结肠组胺、IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2含量降低（P<0.01）；结肠IL-1β、类胰蛋白酶、PAR-2蛋白表达降低（P<0.01，P<0.05）；结肠IL-1β、IL-6、IL-1α、类胰蛋白酶、PAR-2 mRNA及阳性表达均降低（P<0.01，P<0.05）；结肠组胺阳性表达降低（P<0.01）。结论: 艾灸“天枢”“上巨虚”可抑制IBS-D模型大鼠的结肠低度炎性反应，其作用机制可能与抑制肥大细胞分泌组胺、类胰蛋白酶，从而降低相关炎性因子表达有关。.

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