黄芪多糖对肠炎雏鸡小肠黏膜损伤的保护作用

Protective Effect of Astragalus Polysaccharide on Intestinal Mucosal Injury in Chicks with Enteritis

【目的】探究黄芪多糖(Astragalus polysaccharide,APS)对雏鸡肠道形态和局部黏膜免疫的影响,阐明APS减轻肠炎雏鸡肠黏膜损伤的作用机制。【方法】在构建LPS诱导肠炎模型试验中,选取15只14日龄SPF雏鸡随机分为3组:对照组(Con)、低剂量脂多糖(lipopolysaccharide,LPS)模型组(DL)和高剂量脂多糖模型组(DH),每组5只。对照组灌喂生理盐水,DL和DH组分别灌喂1和2 mg/kg BW LPS,连续处理3 d,取小肠组织,采用HE染色法观察小肠病理变化,采用实时荧光定量PCR检测白介素-1β(interleukin 1β,IL-1β)和肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)mRNA表达量,筛选构建肠炎模型的最佳LPS剂量。在APS对肠黏膜损伤的保护试验中,将20只7日龄雏鸡分为4组:对照组(C)、脂多糖炎症组(L)、黄芪多糖组(A)和黄芪多糖抑制炎症组(A+L),每组5只。A和A+L组从7日龄到试验结束每天自由饮用APS溶液(1.0 g/L),C组在此期间自由饮水;L和A+L组雏鸡14日龄时灌喂筛选所得剂量的LPS,连续3 d。取各组雏鸡胸腺、脾脏、法氏囊和小肠组织,计算雏鸡免疫器官指数;采用HE染色法观察雏鸡小肠黏膜形态,糖原PAS染色法检测杯状细胞数量,实时荧光定量PCR检测咬合蛋白-1(Occludin-1)、闭合蛋白-1(Claudin-1)和闭合小环蛋白-1(ZO-1)mRNA表达量。【结果】在构建LPS诱导肠炎模型试验中,DL、DH组雏鸡小肠组织均出现肠黏膜固有层充血、肠绒毛损伤等现象,且IL-1β和TNF-αmRNA表达量显著高于对照组(P<0.05),因此确定雏鸡灌喂1 mg/kg BW LPS建立肠炎模型。在APS对肠黏膜损伤的保护试验中,与对照组相比,L组雏鸡小肠绒毛破碎,固有层充血,免疫器官指数显著下降(P<0.05),小肠隐窝深度显著升高(P<0.05),绒腺比(V/C)显著下降(P<0.05),杯状细胞数量显著减少(P<0.05),紧密连接蛋白Occludin-1、Claudin-1和ZO-1的mRNA表达量均显著下降(P<0.05);与L组相比,A+L组雏鸡免疫器官指数显著增加(P<0.05),肠黏膜损伤程度减轻,肠绒毛高度和绒腺比等指标显著升高(P<0.05),隐窝深度显著降低(P<0.05),杯状细胞数量显著增多(P<0.05),空肠和回肠紧密连接蛋白mRNA表达量均显著升高(P<0.05);A组雏鸡杯状细胞数量相比于其他组明显增多,空肠和回肠紧密连接蛋白表达量也显著升高(P<0.05)。【结论】雏鸡灌喂1 mg/kg BW LPS可成功建立肠炎模型,1.0 g/L APS可优化肠道组织形态,促进雏鸡局部黏膜免疫系统的发育,可以保护肠炎雏鸡小肠组织及肠黏膜免受损伤,最终达到预防雏鸡细菌性肠炎的效果。

【Objective】 The study was aimed to investigate the effects of Astragalus polysaccharide(APS) on the intestinal morphology and local mucosal immune in chicks, in order to clarify the mechanism of APS in reducing intestinal mucosal injury in chickens with enteritis.【Method】 Fifteen 14-day-old SPF-grade chicks were randomly divided into three groups: Control group(Con),low-dose lipopolysaccharide(LPS) model group(DL) and high-dose LPS model group(DH),5 chicks in each group.Control group was gavaged with normal saline, DLand DHgroups were gavaged with 1 and 2 mg/kg BW LPS,respectively.After 3 days of continuous treatment, the small intestine were taken.HE staining method was used to observe the pathological changes of small intestine, and Real-time quantitative PCR was used to detect the changes in the expression of interleutin 1β(IL-1β) and tumor necrosis factor α(TNF-α),and screen the best LPS dose for constructing LPS-induced enteritis model.In the protection test of APS against intestinal mucosal injury, twenty 7-day-old chicks were divided into four groups: Control group(C),LPS inflammation group(L),APS group(A) and APS inhibited inflammation group(A+L),5 chicks in each group.A and A+L groups were treated with APS(1.0 g/L) from 7-day-old to the end of this experiment, C group was fed water during this period.Chicks in L and A+L groups were given the selected optimal dose of LPS,after 3 consecutive days, the thymus, spleen, bursa of Fabricius and small intestine tissues were collected, the immune organ indexes of chicks were calculated in each group.HE staining was used to detect the pathological changes of chick small intestine, PAS staining was used to observe the number changes of goblet cells, Real-time quantitative PCR was used to detect the mRNA expression of tight junction proteins Occludin-1,Claudin-1 and ZO-1.【Result】 In the experiment of constructing LPS-induced enteritis model, the small intestinal mucosa lamina propria of chicks in DLand DHgroups were congested and intestinal villi were injured, and the expressions of IL-1β and TNF-α mRNA were significantly higher than that of control group(P<0.05),which indicated that the intestinal tissues in DLgroup had obvious pathological changes.Therefore, it was determined to feed 1 mg/kg BW LPS to establish enteritis model.In the protective test of APS against intestinal mucosal injury, compared with control group, for the chicks in L group, the villus of small intestine were broken, the lamina propria was congested, and the immune organ indexes were significantly decreased(P<0.05),the crypt depth of small intestine was significantly increased(P<0.05),the V/C was significantly decreased(P<0.05),and the goblet cells of small intestine were significantly decreased(P<0.05),the mRNA expression of tight junction protein Occludin-1,Claudin-1 and ZO-1 were all significantly decreased(P<0.05).Compared with L group, the immune organ indexes of chicks in A+L group were significantly increased(P<0.05),the intestinal mucosal damage was decreased, the intestinal villus height and V/C were significantly increased(P<0.05),the crypt depth was significantly decreased(P<0.05),the goblet cells were increased significantly(P<0.05),and the expression of tight junction proteins mRNA in jejunum and ileum were significantly increased(P<0.05).Compared with other groups, the number of goblet cells and the expression of tight junction protein in jejunum and ileum of chicks in A group were significantly increased(P<0.05).【Conclusion】 The enteritis model could be successfully established by feeding 1 mg/kg BW LPS to chicks.1.0 g/L APS could optimize the intestinal morphology, promote the development of local mucosal immune system of chicks, protect the intestinal tissue and intestinal mucosa of chicks with enteritis from damage, and finally achieve the effect of preventing bacterial enteritis in chicks.