生命早期食物过敏对幼鼠内脏痛敏感性影响及结肠P物质异常表达

Effect of food allergy early in life on visceral hyperalgesia in the developing rats and role of substance P abnormal expression in colon

目的探讨食物过敏（FA）对幼鼠内脏痛觉敏感性影响及结肠黏膜P物质（SP）异常表达在FA幼鼠内脏痛觉高敏形成中作用。方法3周龄雌性SD大鼠20只，随机分FA组和对照组（NS组），各10只。FA组通过卯白蛋白（OVA）低剂量腹腔注射基础致敏及高剂量灌胃激发建立幼鼠OVA-FA模型；检测血清OVA—IgE含量、观察肠黏膜病理改变及嗜酸性粒细胞、肥大细胞浸润情况进行幼鼠FA模型评价。通过观察两组幼鼠在不同压力结直肠扩张（CRD）刺激后的腹外斜肌（EOMA）放电测量进行内脏痛觉敏感性评价。应用免疫组织化学染色及计算机图像分析系统对结肠sP进行积分吸光度值半定量分析。采用SPSS13．0软件包进行统计分析，α=0．05为显著性检验标准。结果与对照组比较，FA组幼鼠血清OVA—IgE含量、空肠黏膜嗜酸性粒细胞和肥大细胞数及肥大细胞脱颗粒百分率均明显升高，差异有统计学意义（P均〈0．01）。在CRD为0、15、30、45、60、75mmHg（1mmHg=0．133kPa）时，FA组腹外斜肌放电幅值分别为（17．7±0．72）、（18．63±1．72）、（22．55±1．70）、（28．63±7．00）、（33．97±7．34）、（37．26±8．40）μV，NS组分另0为（17．43±1．18）、（17．27±1．16）、（17．73±1．42）、（19．55±3．54）、（23．29±5．46）、（25．20±4．75）μV。随CRD增加，腹外斜肌放电幅值较前明显升高，CRD30、45、60、75mmHg时，两组差异有统计学意义（P均〈0．01）。FA组、Ns组幼鼠结肠sP积分吸光度值分别为（247．12±90．83）、（103．90±58．94），差异有统计学意义（t=4．183，P〈0．01）。结论OVA低剂量腹腔注射基础致敏及高剂量灌胃激发可建立幼鼠FA模型。生命早期FA可导致幼鼠腹外斜肌放电幅值明显升高并伴有结肠sP表达增加，出现慢性内脏痛觉高敏感。生命早期FA导致的内脏痛觉高敏感性发生可能与肠黏膜肥大细胞表达增多、脱颗粒及肠道sP的异常表达有关。

Objective To explore the effect of food allergy （FA） on the development of viseeralgia sensibility and the substance P （SP） expression in colon of developing rats with FA. Method Three-week old female Sprague-Dawley （SD） rats were randomly divided into two groups （n = 10 in each）. The rats in FA group were sensitized with ovalbumin （OVA） 40 μg and Al（OH） 3 1 mg suspension solution （0. 2 ml） intraperitoneal （i. p. ） injection on day 0, only OVA 40 μg solution i.p. on day 2, 4, 7, 9, 11, respectively, and the rats were challenged by garage with OVA solution 30 mg （2 ml） on day 20, 24, 28, 30. The rats in non-sensitized （NS） group were not challenged except handled in the same ways. The serum OVA-IgE were determined by enzyme-linked immuno sorbent assay （ELISA） on day 0, 30. Jejunum segments were used to observe morphological structure, the expression of eosinophils, and the density and the percentage of degranulation of mast cells （ MC ）. The rats were appraised for the pain sensibility of intestinal tract under eolorectal distension irritation by the electrophysiological method on external oblique in the 18-24 hr after the last challenge. Colons were used to analyze the expression of SP through immunohistochemieal staining and computer image analyzing system. Result The serum OVA-IgE concentration and the eosinophils, mast cell, the percentage of mast cells degranulation in FA group were more than NS group （ P 〈 0. 01 ）. The amplitudes of spike external oblique muscle of abdomen （ EOMA, μV） of the FA group under the colorectal distension （CRD） pressures at 0, 15, 30, 45, 60, 75 mm Hg were （17.74±0.72）, （18.63＋1.72）, （22. 55±1.70）, （28.63 ±7.00）,（33.97±7.34）, （37.26± 8.40）, and （17.43±1.18）, （17.27±1.16）,（17.73±1.42）, （19.55±3.54）, （23.29±5.46）, （25.20 ± 4. 75 ） in NS group. With the CRD pressure increased, the amplitudes of spike EOMA increased significantly. There were significant differences between groups under the CRD pressures at 30, 45, 60, 75 mm Hg （ F = 47. 470, 13. 367, 13. 317, 15. 390, P 〈 0. 01 ）. The expressions of colons SP in FA group and NS group are 247. 12 ± 90. 83 and 103.90 ± 58.94, respectively （ t = 4. 183, P 〈 0.01 ）. Conclusion Sensitization through i.p. pathway and challenge by gavage with OVA in early life could result in FA in young SD rats. FA in early life enabled the amplitudes of spike EOMA and the expression of colons SP increase significantly. It may be related to increase in amount and degranulation of MC and SP abnormal expression in colon, which could lead to the development of visceralgia sensibility.