白术炮制前后对溃疡性结肠炎小鼠肠黏膜屏障及肠道菌群的影响

Effects of Atractylodes macrocephala Koidz on intestinal mucosal barrier and intestinal flora in mice with ulcerative colitis before and after processing

关键词:  白术  麸炒  肠黏膜屏障  肠道菌群

Key words:  Atractylodes macrocephala Koidz  Bran-fried Atractylodes macrocephala Koidz  Intestinal mucosal barrier  Intestinal flora

摘要:

目的 探究白术炮制前后对溃疡性结肠炎小鼠肠黏膜屏障功能的影响。 方法 Balb/c小鼠随机分为空白对照组、模型组、阳性药组、生白术组、麦麸组、清炒白术组和麸炒白术组,每组10只。除空白对照组外,其余各组小鼠均采用2.5%的葡聚糖硫酸钠(DSS)水溶液自由饮用方法复制溃疡性结肠炎小鼠模型；造模成功后使用灌胃法给予各组小鼠相应的治疗药物,空白组与模型组灌胃等量的生理盐水；模型复制及治疗过程中记录各组小鼠病理活动指标并进行小鼠疾病活动指数(DAI)评价；采用HE染色观察结肠组织形态并评分；应用ELISA法测定血清中sIgA,Thl和Th2型细胞因子IL-4、IL-18、TNF-α、IFN-γ与Th17/Treg型细胞因子IL-10、IL-17和TGF-β的水平；采用PCR技术检测各组小鼠结肠组织中紧密连接蛋白ZO-1、Occludin、闭锁蛋白Claudin-1、FFAR3及黏蛋白MUC2的mRNA表达水平；采用Western blot技术检测各组动物结肠组织中紧密连接蛋白ZO-1、Occludin、闭锁蛋白Claudin-1、FFAR3及黏蛋白MUC2的蛋白表达量；使用16S rRNA测序技术检测各组小鼠粪便肠道菌群的多样性情况,并使用GC-MS技术测定各组小鼠粪便中短链脂肪酸含量。结果 各给药组均可减轻DSS诱导的UC小鼠DAI升高、结肠长度缩短等症状(P＜0.05或P＜0.01),能够降低模型动物体内IFN-γ、IL-17、IL-18、TNF-α等促炎因子水平(P＜0.05或P＜0.01),升高SIgA、IL-10、IL-4、TNF-β等抗炎因子水平(P＜0.05或P＜0.01),还能影响结肠黏膜组织中ZO-1、Occludin 1、Claudin-1、MUC2等蛋白及基因表达水平(P＜0.05或P＜0.01),重塑模型动物的肠道菌群多样性情况(P＜0.05或P＜0.01),可增加肠道中有益菌群的丰度、降低有害菌群的丰度(P＜0.05或P＜0.01),并调整肠道SCFAs的水平(P＜0.05或P＜0.01),且白术经麸炒后上述作用增强。结论 麸炒可增强白术改善溃疡性结肠炎小鼠肠黏膜屏障及肠道菌群多样性的作用,从而提高对溃疡性结肠炎的治疗作用。

Abstract:

Objective In order to explore the effect of Atractylodes macrocephala Koidz on intestinal mucosal barrier and intestinal flora in mice with ulcerative colitis before and after processing. Methods The Balb/c mice were randomly divided into blank control group, model group, positive drug group, raw Atractylodes macrocephala Koidz group, wheat bran group, fried Atractylodes macrocephala Koidz group and bran-fried Atractylodes macrocephala Koidz group, with 10 mice in each group. Except for the blank control group, the mice in the other groups were used to replicate the mouse model of ulcerative colitis by free drinking of 2.5% dextran sulfate sodium (DSS) aqueous solution. After successful preparation of the model, the therapeutic drugs were given to each group of mice by gavage. The blank group and the model group were given the same amount of normal saline by gavage. During the replication and treatment of the model, the pathological activity indexes of mice in each group were recorded and the disease activity index (DAI) of mice was evaluated. HE staining was used to observe the morphology of colon tissue and score. The levels of sIgA, Thl and Th2 cytokines IL-4, IL-18, TNF-α, IFN-γ and Th17/Treg cytokines IL-10, IL-17, TGF-β in serum tissue were measured by ELISA. The mRNA expression levels of tight junction proteins ZO-1, Occludin 1, Claudin-1, FFAR3 and mucin MUC2 in the colon tissue of each group were detected by PCR. Western blot was used to detect the protein expression of tight junction proteins ZO-1, Occludin, Claudin-1, FFAR3 and mucin MUC2 in the colon tissue of each group. The diversity of intestinal flora in feces of mice was detected by 16S rRNA sequencing technology. The content of short-chain fatty acids in feces of mice was determined by GC-MS technology. Results The each administration group could alleviate the symptoms of DSS-induced UC mice such as increased DAI and shortened colon length (P<0.05 or P<0.01), reduce the levels of pro-inflammatory factors such as IFN-γ, IL-17, IL-18 and TNF-α in model animals (P<0.05 or P<0.01), and increase the levels of anti-inflammatory factors such as SIgA, IL-10, IL-4 and TNF-β (P<0.05 or P<0.01). It could also affect the protein and gene expression levels of ZO-1, Occludin 1, Claudin-1 and MUC2 in colonic mucosa (P<0.05 or P<0.01). It could also remodel the diversity of intestinal flora in model animals (P<0.05 or P<0.01) that increase the abundance of beneficial flora and reduce the abundance of harmful flora (P<0.05 or P<0.01). It could also adjust the level of intestinal SCFAs (P<0.05 or P<0.01). Furthermore, the above effects of Atractylodes macrocephala Koidz were enhanced after bran-fried. Conclusion The bran-fried can enhance the therapeutic effect of Atractylodes macrocephala Koidz on ulcerative colitis by improving intestinal mucosal barrier and intestinal flora diversity.