【摘要】 目的:鉴定柿叶提取物中黄酮类成分，从TLR4/NLRP3炎症信号通路和肠道菌群角度探究对溃疡性结肠炎(ulcerative colitis, UC)的作用机制。方法:利用HPLC法对柿叶(persimmon leaf, PL)提取物中5个黄酮类成分进行含量测定。采用3%葡聚糖硫酸钠(dextran sulfate sodium, DSS)构建小鼠UC模型。每天记录小鼠体质量和饮水量。Elisa法检测血清IL-6,TNF-α,IL-1β,IL-10水平，HE染色观察结肠组织的变化，Western blot法检测结肠组织中TLR4,IL-1β,NF-κB p65和NLRP3蛋白的表达，16S rDNA扩增子测序法检测粪便中肠道菌群的变化。结果:与Control组相比，DSS组小鼠体质量、结肠长度、IL-10水平显著降低(P<0.01);疾病活动指数和IL-6,TNF-α,L-1β水平升高(P<0.01)。与DSS组相比，给药组小鼠体质量下降缓慢，结肠长度延长，疾病活动指数降低；IL-6,TNF-α,IL-1β水平显著降低(P<0.01),IL-10水平显著升高(P<0.01);TLR4/NLRP3相关蛋白表达显著上升(P<0.01);PL给药组TLR4,IL-1β,NF-κB p65,NLRP3蛋白表达降低(P<0.01);16S rDNA扩增子测序法结果显示鼠乳杆菌、约氏乳杆菌、罗姆布茨菌、艾克曼菌种均有增加的趋势。病理结果显示DSS组小鼠可见结肠组织有严重的黏膜溃疡、隐窝丧失、炎性细胞浸润和杯状细胞耗竭，给药后小鼠结肠组织病变得到改善。HPLC法分离度好、准确度高。结论:柿叶提取物对DSS诱导的UC小鼠肠道损伤有保护作用，可能与抑制TLR4/NLRP3炎症信号通路降低炎症反应从而改善肠道微环境有关。更多还原

【Abstract】 Objective: To identify the flavonoids in persimmon leaf(PL) extract, and to explore the mechanism of the action on ulcerative colitis(UC) from the perspective of TLR4/NLRP3 inflammatory signal pathway and intestinal flora. Methods: HPLC was used to determine the five flavonoids in persimmon leaf extract. The animal model of UC mice was established by 3% dextran sulfate sodium(DSS). The body weight and drinking volume of the mice were measured daily. The detection of IL-6, TNF-α, IL-1β and IL-10 in the serum of the mice were measured by Elisa kit, the changes of colon tissue were observed by HE staining, the expression levels of TLR4, IL-1β, NF-κB p65 and NLRP3 proteins in the colon tissues were determined by western blot, and the changes of intestinal flora in feces were detected by 16S rDNA amplification sequencing. Results: The body weight, colon length and IL-10 level in DSS group were significantly lower than that in control group(P<0.01). The disease activity index and the levels of IL-6, TNF-α and IL-1β in DSS group were significantly increased(P<0.01) compared with the control group. Compared with the DSS group, the body weight decreased slowly, the colon length prolonged and the disease activity index decreased in the treated group, the levels of IL-6, TNF-α and IL-1β significantly reduced(P<0.01) and IL-10 significantly increased(P<0.01). The expression levels of TLR4/NLRP3-related proteins significantly increased(P<0.01), the expression levels of TLR4, IL-1β, NF-κB p65 and NLRP3 proteins significantly decreased(P<0.01). The results of 16S rDNA amplification sequencing showed that Lactobacillus murinus, Lactobacillus johnsonii, Romboutsia and Akkermansia muciniphila had an increasing trend in the treated group. The pathological results showed that there were severe mucosal ulcer, crypt loss, inflammatory cell infiltration and goblet cell depletion in colon tissue of mice in DSS group and the pathological changes of colon tissue in mice were improved after administration. The HPLC method has good separation degree and high accuracy. Conclusions: Persimmon leaf extract has protective effect on intestinal injury in DSS-induced UC mice. It may improve the intestinal microenvironment by inhibiting TLR4/NLRP3 inflammatory signal pathway and relieving inflammation.更多还原