Spirulina platensis aqueous extracts ameliorate colonic mucosal damage and modulate gut microbiota disorder in mice with ulcerative colitis by inhibiting inflammation and oxidative stress

Abstract

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease (IBD) that has become a major gastroenterologic problem during recent decades. Numerous complicating factors are involved in UC development such as oxidative stress, inflammation, and microbiota disorder. These factors exacerbate damage to the intestinal mucosal barrier. Spirulina platensis is a commercial alga with various biological activity that is widely used as a functional ingredient in food and beverage products. However, there have been few studies on the treatment of UC using S. platensis aqueous extracts (SP), and the underlying mechanism of action of SP against UC has not yet been elucidated. Herein, we aimed to investigate the modulatory effect of SP on microbiota disorders in UC mice and clarify the underlying mechanisms by which SP alleviates damage to the intestinal mucosal barrier. Dextran sulfate sodium (DSS) was used to establish a normal human colonic epithelial cell (NCM460) injury model and UC animal model. The mitochondrial membrane potential assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and Hoechst 33258 were carried out to determine the effects of SP on the NCM460 cell injury model. Moreover, hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), western blot, and 16S ribosomal DNA (rDNA) sequencing were used to explore the effects and underlying mechanisms of action of SP on UC in C57BL/6 mice. In vitro studies showed that SP alleviated DSS-induced NCM460 cell injury. SP also significantly reduced the excessive generation of intracellular reactive oxygen species (ROS) and prevented mitochondrial membrane potential reduction after DSS challenge. In vivo studies indicated that SP administration could alleviate the severity of DSS-induced colonic mucosal damage compared with the control group. Inhibition of inflammation and oxidative stress was associated with increases in the activity of antioxidant enzymes and the expression of tight junction proteins (TJs) post-SP treatment. SP improved gut microbiota disorder mainly by increasing antioxidant enzyme activity and the expression of TJs in the colon. Our findings demonstrate that the protective effect of SP against UC is based on its inhibition of pro-inflammatory cytokine overproduction, inhibition of DSS-induced ROS production, and enhanced expression of antioxidant enzymes and TJs in the colonic mucosal barrier.

摘要

目的

探究钝顶螺旋藻水提取物(SP)对葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎(UC)小鼠肠道菌群紊乱的调节作用,并阐明其减轻结肠黏膜屏障损伤的潜在机制。

创新点

(1)成功鉴定出钝顶螺旋藻粉末及SP的化学成分;(2)研究结果证实SP可改善UC小鼠的结肠黏膜损伤,并能够调节肠道菌群,SP对UC的保护作用机制是减少炎症因子的过量产生、抑制氧化应激和增强结肠黏膜屏障中紧密连接蛋白(TJ)的表达。(3)SP可作为一种治疗UC、肠道菌群紊乱或氧化应激相关疾病的天然药物,并为该药物临床应用提供理论依据。

方法

采用DSS建立正常肠上皮细胞(NCM460)损伤模型和UC动物模型。采用噻唑兰法(MTT)、细胞凋亡染色法(AnnexinV-FITC/PI)、细胞染色法(Hoechst 33258)和线粒体膜电位法(MMP)测定SP对NCM460细胞损伤模型的影响。并采用苏木精&伊红染色法(H&E)、透射电镜(TEM)、酶联免疫吸附测定法(ELISA)、实时荧光定量聚合酶链式反应(qPCR)、蛋白质印迹法(Western blot)和16S rDNA测序等方法,探讨SP对UC小鼠的结肠黏膜损伤和肠道菌群的影响及其潜在作用机制。

结论

体外研究表明,SP可减轻DSS诱导的NCM460细胞损伤;SP还能显著减少该细胞内活性氧(ROS)的过度产生,并抑制DSS刺激后引起的细胞线粒体膜电位降低。体内研究表明,SP可减轻DSS诱导的结肠黏膜损伤程度。SP治疗后,炎症和氧化应激水平随着抗氧化酶和TJ表达的升高而降低。此外,SP主要通过增加抗氧化酶活性和结肠中TJ的表达来改善肠道菌群紊乱。综上所述,SP对UC的保护作用是基于抑制促炎细胞因子的过量产生、抑制ROS产生、增强结肠黏膜屏障中抗氧化酶和TJ的表达实现。