[Effect of electroacupuncture on intestinal mucosal barrier in IBS-D rats via NGF/TrkA pathway mediated by mast cells].

Abstract

<p>OBJECTIVE: To observe the effect of electroacupuncture (EA) on the activation of mast cells (MCs) in the colon of rats with diarrhea-predominant irritable bowel syndrome (IBS-D) and to explore the underlying mechanisms of its protective effect on the intestinal mucosal barrier. METHODS: Of 74 offsprings from 6 pregnant SD rats, 14 offsprings were randomly collected as a normal group, and the remaining 60 offsprings underwent IBS-D model prepared by maternal separation combined with acetic acid enema and chronic restraint stress. Fifty-six successfully modeled rats were randomly divided into a model group, a ketotifen group, an EA group, and an EA+ketotifen (combination) group, 14 rats in each one. In the ketotifen group, the rats received the intragastric administration of ketotifen solution (10 mL/kg). In the EA group, acupuncture was delivered at bilateral "Tianshu" (ST25) and "Shangjuxu" (ST37) with disperse-dense wave, a frequency of 2 Hz/100 Hz and a current of 0.3 mA. In the combination group, the intervention measures of the above two groups were operated in combination. In each group, the intervention was delivered once daily for 7 consecutive days. Separately, before modeling, after modeling and after intervention completion, diarrhea score and the minimum injection volume for the abdominal withdrawal reflex (AWR) in rats were evaluated in each group. After intervention, HE staining was adopted to observe the morphology of colonic mucosa; scanning electron microscopy to examine mucosal ultrastructure; Toluidine blue staining to observe MCs degranulation in the colonic mucosa; ELISA to detect serum levels of calcitonin gene-related peptide (CGRP), diamine oxidase (DAO), D-lactate (D-LA), and lipopolysaccharide (LPS); fluorescence microplate assay to detect the serum concentration of 4 kDa fluorescein isothiocyanate-dextran (FD4); Western blot, real-time quantitative PCR (RT-qPCR), and immunofluorescence to detect the expression of nerve growth factor (NGF), tyrosine kinase receptor A (TrkA), and transient receptor potential vanilloid type 1 (TRPV1) in colon tissue; and Pearson correlation coefficient to analyze the relationship between CGRP level and intestinal barrier function. RESULTS: Compared with the normal group, the model group showed the decrease in AWR minimum injection volume (P&lt;0.01), the increase in diarrhea score and MC degranulation rate (P&lt;0.01), the damage of colonic mucosal structure, obvious inflammatory cell infiltration, and the increase in serum levels of CGRP, DAO, D-LA, LPS, FD4 mean fluorescence intensity (MFI), as well as the protein, mRNA, and positive expression levels of NGF, TrkA, and TRPV1 in colon tissue (P&lt;0.01, P&lt;0.05). Compared with the model group, the ketotifen, EA, and combination groups exhibited the increase in AWR minimum injection volume (P&lt;0.01), the decline in diarrhea score and MC degranulation rate (P&lt;0.01), relatively intact intestinal mucosal structure, reduced inflammatory cell infiltration, and the decrease in serum levels of CGRP, DAO, D-LA, LPS, FD4 MFI, as well as the protein, mRNA, and positive expression levels of NGF, TrkA, and TRPV1 in colon tissue (P&lt;0.01). When compared with the ketotifen group, the EA group demonstrated lower FD4 MFI in serum and reduced protein expression of NGF, TrkA, and TRPV1 in colon (P&lt;0.01), and no statistically significant differences were presented in the other indicators (P&gt;0.05). In comparison with both the ketotifen and EA groups, the combination group showed the improvements in all the aforementioned indicators (P&lt;0.01, P&lt;0.05). Serum level of CGRP was positively correlated with the levels of DAO, D-LA, LPS, and FD4 (P&lt;0.001). CONCLUSION: Electroacupuncture may ameliorate intestinal mucosal barrier function and alleviate symptoms in IBS-D rats by inhibiting MC activation, modulating the NGF/TrkA pathway, and reducing serum levels of DAO, D-LA, LPS, and FD4. 目的：观察电针干预对腹泻型肠易激综合征（IBS-D）大鼠结肠肥大细胞（MCs）激活的影响，探讨其保护肠黏膜屏障的机制。 方法：从6只SD孕鼠所产下的74只幼鼠中，随机选取14只为正常组，其余60只通过母子分离联合醋酸灌肠及慢性束缚应激建立IBS-D模型。将造模成功的56只大鼠随机分为模型组、酮替芬组、电针组和针药组，每组14只。酮替芬组予酮替芬溶液灌胃（10 mL/kg）；电针组大鼠针刺双侧“天枢”和“上巨虚”，予疏密波，频率2 Hz/100 Hz，电流0.3 mA；针药组联合上述两法。各组干预均每天1次，连续7 d。于造模前、造模后及干预后，评估各组大鼠稀便率及腹部回撤反射（AWR）最小注水量。干预后，HE染色观察结肠组织形态；扫描电镜观察结肠黏膜超微结构；甲苯胺蓝染色观察结肠黏膜MCs脱颗粒情况；ELISA法检测血清降钙素基因相关肽（CGRP）、二胺氧化酶（DAO）、D-乳酸（D-LA）及脂多糖（LPS）含量；荧光酶标分析检测血清 4 kDa异硫氰酸荧光素葡聚糖（FD4）浓度；Western blot法、实时荧光定量PCR法及免疫荧光法检测结肠神经生长因子（NGF）、酪氨酸激酶受体A（TrkA）及瞬时受体电位香草酸亚型1（TRPV1）表达水平；采用Pearson相关系数分析CGRP水平与肠屏障功能的相关性。 结果：与正常组比较，模型组大鼠AWR最小注水量降低（P&lt;0.01），稀便率及MCs脱颗粒率升高（P&lt;0.01），结肠黏膜结构破坏，炎症细胞浸润明显，血清CGRP、DAO、D-LA、LPS含量与FD4浓度，以及结肠组织NGF、TrkA和TRPV1蛋白、mRNA与阳性表达均升高（P&lt;0.01，P&lt;0.05）。与模型组比较，酮替芬组、电针组和针药组大鼠AWR最小注水量升高（P&lt;0.01），稀便率及MCs脱颗粒率降低（P&lt;0.01），肠黏膜结构相对完整，炎症细胞浸润减少，血清CGRP、DAO、D-LA、LPS与FD4浓度，以及结肠组织NGF、TrkA和TRPV1蛋白、mRNA与阳性表达均降低（P&lt;0.01）。与酮替芬组和电针组比较，针药组以上全部指标均改善（P&lt;0.01，P&lt;0.05）。与酮替芬组比较，电针组大鼠血清FD4浓度及结肠组织NGF、TrkA、TRPV1蛋白表达水平降低（P&lt;0.01），其余指标差异无统计学意义（P&gt;0.05）。血清CGRP水平与DAO、D-LA、LPS、FD4均呈正相关（P&lt;0.001）。 结论：电针可能通过抑制MCs激活，下调NGF/TrkA通路，降低血清DAO、D-LA、LPS和FD4含量，改善大鼠肠黏膜屏障功能，从而缓解IBS-D症状。.</p>