姬松茸多糖和姬松茸多糖铁对DSS诱导的大鼠溃疡性结肠炎预防效果研究

Study on the preventive effect of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron on dss-induced ulcerative colitis in rats

为了探究姬松茸多糖和姬松茸多糖铁预防葡聚糖硫酸钠(DSS)诱导的大鼠溃疡性结肠炎(UC)效果,试验将64只SD大鼠随机分成8组,即空白对照组、模型组及姬松茸多糖和姬松茸多糖铁低剂量(50 mg/kg)、中剂量(100 mg/kg)、高剂量(200 mg/kg)组,姬松茸多糖和姬松茸多糖铁各剂量组大鼠按低、中、高剂量每只灌胃给予50,100,200 mg/kg姬松茸多糖和姬松茸多糖铁溶液1 mL,每天1次,连续灌胃14 d,空白对照组和模型组灌胃等体积的生理盐水。从第15天起,停止灌胃,除空白对照组外,其余组饮水全部替换成含4.5%DSS的水溶液,自由饮用,连用7 d,建立溃疡性结肠炎大鼠模型。干预结束后,测定大鼠体重、结肠长度,进行疾病活动指数(DAI)评分;采用H.E.染色法观察结肠组织病理学变化;采用ELISA法测定血清中炎症因子白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α);荧光定量PCR方法检测结肠组织中紧密连接蛋白相关基因ZO-1、Occludin和Claudin-1 mRNA的表达,以及肠道菌群Mucispirillum schaedleri、普拉梭菌、大肠杆菌、乳酸杆菌和双歧杆菌16S RNA的相对表达量。结果表明:与空白对照组相比,模型组大鼠从试验第18天起体重开始持续下降,结肠长度极显著缩短(P<0.01),DAI评分增加,结肠病变严重;血清中IL-1β、IL-6、TNF-α水平极显著升高(P<0.01);结肠组织中紧密连接蛋白ZO-1、Occludin和Claudin-1基因mRNA的相对表达量均极显著下降(P<0.01);肠道菌群Mucispirillum schaedleri和大肠杆菌16S RNA相对表达量极显著升高(P<0.01),普拉梭菌、乳酸菌和双歧杆菌16S RNA相对表达量极显著下降(P<0.01)。与模型组相比,试验第18,21天姬松茸多糖和姬松茸多糖铁各剂量组的大鼠体重有所回升,结肠长度缩短较少,DAI评分和血清中IL-1β、IL-6、TNF-α水平显著或极显著降低(P<0.05或P<0.01);姬松茸多糖和姬松茸多糖铁中、高剂量组紧密连接蛋白ZO-1、Occludin和Claudin-1基因的mRNA相对表达量升高;姬松茸多糖各剂量组中普拉梭菌、乳酸菌及双歧杆菌16S rRNA基因相对表达量显著或极显著升高(P<0.05或P<0.01),Mucispirillum schaedleri和大肠杆菌16S rRNA基因相对表达量显著或极显著降低(P<0.05或P<0.01);当姬松茸多糖铁浓度增大时,Mucispirillum schaedleri和大肠杆菌16S rRNA基因相对表达量表现为先下降后升高,并在高浓度时显著高于中剂量组(P<0.05),随姬松茸多糖铁浓度的增加,双歧杆菌和乳酸菌的16S rRNA相对表达量表现出先升高后降低趋势。说明姬松茸多糖和姬松茸多糖铁对DSS诱导的大鼠急性UC均具有预防效果,但姬松茸多糖铁在纠正肠道菌群平衡方面表现出不足,提示其作为结肠炎预防药物还需要进一步的研究。

In order to explore the effect of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron in preventing dextran sodium sulfate(DSS)-induced ulcerative colitis(UC) in rats, 64 SD rats were randomly divided into eight groups, namely the blank control group, the model group, Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron low(50 mg/kg), medium(100 mg/kg) and high(200 mg/kg) groups. Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron dosage groups were given 1 mL of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron by gavage of 50, 100, 200 mg/kg each at low, medium and high doses;one time a day, continuous gastric gavage for 14 days. The blank control group and the model group were given an equal volume of normal saline. From the 15 th day, the gavage was stopped, except for the blank control group, all the drinking water of the remaining groups was replaced with 4.5% DSS-containing aqueous solution, which could be drunk for seven days;a rat model of ulcerative colitis was established. At the end of the intervention, the body weight and colon length of rats were measured, and disease activity index(DAI) scoring was carried out;the histopathological changes of colon were observed by H.E. staining;the inflammatory factors interleukin(IL)-1β, IL-6 and tumor necrosis factor α(TNF-α) in serum were determined by ELISA;fluorescence quantitative PCR was used to detect the expression of tight junction protein-related genes ZO-1, Occludin and Claudin-1 mRNA in colonic tissues, and the relative levels of 16 S RNA gene of intestinal flora Mucispirillum schaedleri, Faecalibacterium prausnitzii, Escherichia coli, Lactobacillus and Bifidobacterium. The results showed that compared with the blank control group, the rats in the model group started to lose weight continuously since the 18 th day of the test, and the colon length was shortened very significantly(P<0.01);the DAI score increased and the colon lesions were severe;the serum levels of IL-1β, IL-6 and TNF-α were increased very significantly(P<0.01);the relative expression of tight junction proteins ZO-1, Occludin and Claudin-1 mRNA in colon tissues was very significantly decreased(P<0.01);the relative levels of 16 S RNA gene of intestinal flora Mucispirillum schaedleri and Escherichia coli were very significantly increased(P<0.01);and the relative levels of 16 S RNA gene of Faecalibacterium prausnitzii, Lactobacillus and Bifidobacterium were very significantly decreased(P<0.01). Compared with the model group, on the 18 th and 21 st day of the test, the rats in the Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron dose groups had regained body weight, and had less shortened colon length;DAI scores, and the serum levels of IL-1β, IL-6, TNF-α were significantly or very significantly lower(P<0.05 or P<0.01);the relative expression of mRNA of tight junction proteins ZO-1, Occludin and Claudin-1 in Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron medium and high groups was increased. In the Agaricus blazei polysaccharide dose groups, the relative expression of 16 S rRNA gene of Faecalibacterium prausnitzii, Lactobacillus and Bifidobacterium was significantly or very significantly increased(P<0.05 or P<0.01), and the relative expression of 16 S rRNA gene of Mucispirillum schaedleri and Escherichia coli was significantly or very significantly decreased(P<0.05 or P<0.01). When the concentration of iron agaricus polysaccharide increased, the relative expression of 16 S rRNA gene of Mucispirillum schaedleri and Escherichia coli showed a decrease and then an increase, and was significantly higher at the high concentration than that in the medium dose group(P<0.05), and with the increase of the concentration of Agaricus blazei polysaccharide iron, the relative expression of 16 S rRNA gene of Bifidobacterium and Lactobacillus showed a trend of increasing and then decreasing. The results suggested that both Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron had preventive effect on DSS-induced acute UC in rats, but Agaricus blazei polysaccharide iron showed deficiency in correcting intestinal flora, which inferred that further study would be needed for its use as a preventive drug for colitis.