马齿苋多糖调节TLR4/NF-κB信号通路对大鼠溃疡性结肠炎的影响

Effect of portulaca oleracea polysaccharide on ulcerative colitis in rats by regulating TLR4/NF-κB signaling pathway

关键词:  溃疡性结肠炎  马齿苋多糖  炎症  氧化应激  凋亡  TLR4/NF-κB信号通路

Key words:  ulcerative colitis  portulaca oleracea polysaccharides  inflammation  oxidative stress  apoptosis  TLR4/NF-κB signaling pathway

摘要:

[目的] 探讨马齿苋多糖（POP）调节Toll样受体4（TLR4）/核转录因子-κB（NF-κB）信号通路对大鼠溃疡性结肠炎（UC）的影响。[方法] 将40只大鼠随机分为正常（Normal）组、模型（Model）组、POP组、POP+TLR4抑制剂瑞沙托维（TAK-242）组和POP+TLR4激动剂脂多糖（LPS）组，每组8只。除正常组外，其他4组采用连续7 d自由饮用5%葡聚糖硫酸钠溶液的方法制备UC大鼠模型，灌胃给药治疗2周。观察大鼠一般状态并进行疾病活动指数（DAI）评分，取结肠组织标本测量长度、行黏膜损伤指数（CMDI）评分，通过苏木精-伊红（HE）、原位末端标记法（TUNEL）染色观察结肠组织病理变化和细胞凋亡，酶联免疫吸附法（ELISA）检测结肠组织中炎症因子[干扰素（IFN）-γ、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-10、髓过氧化物酶（MPO）]水平和氧化应激指标[活性氧（ROS）、丙二醛（MDA）水平和超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性]，蛋白免疫印迹（Western blot）法、实时聚合酶链式反应（RT-PCR）检测结肠组织中TLR4、NF-κB p65及细胞凋亡调控相关蛋白和mRNA表达。[结果] 与Model组比较，POP组和POP+TAK-242组大鼠一般状态明显改善，DAI评分、CMDI评分和IFN-γ、TNF-α、IL-1β、MPO、ROS、MDA水平降低，结肠长度、IL-10水平和SOD、CAT活性升高（P＜0.05）；结肠组织病理变化和细胞凋亡状况明显改善，细胞凋亡指数降低（P＜0.05）；TLR4、NF-κB p65、B淋巴细胞瘤-2（Bcl-2）相关X蛋白（Bax）、剪切型半胱氨酸蛋白酶-3（Cleaved Caspase-3）蛋白和mRNA表达水平降低，Bcl-2蛋白和mRNA表达水平升高（P＜0.05）。与POP组比较，POP+TAK-242组对UC大鼠各项指标的作用明显增强（P＜0.05）；POP+LPS组对各项指标的作用则明显减弱（P＜0.05）。[结论] POP具有减轻UC大鼠结肠组织损伤的作用，可能与下调TLR4/NF-κB信号通路，抑制结肠组织炎症、氧化应激和细胞凋亡有关。

Abstract:

[Objective] To investigate the effect of portulaca oleracea polysaccharides（POP） on ulcerative colitis（UC） in rats by regulating toll type receptor 4（TLR4）/nuclear transcription factor-κB（NF-κB） signaling pathway. [Methods] The 40 rats were randomly divided into normal group，model group，POP group，POP+TLR4 inhibitor resatorvid（TAK-242） group and POP+TLR4 agonist lipopolysaccharide（LPS） group，with 8 rats in each group. The UC rat models were prepared by continuously drinking 5% sodium gluconate sulfate solution for 7 days. And the rats were treated by gavage for 2 weeks. The rats general status were observed and the disease activity index（DAI） scores were measured. The colon tissue samples were taken and measured its length，and then performed the mucosal injury（CMDI） scoring. The pathological changes and cell apoptosis of colon tissue was observed through hematoxylin-eosin（HE） or TdT-mediated dUTP nick end labeling（TUNEL） staining. The level of inflammatory factors [interferon（IFN）-γ，tumor necrosis factor（TNF）-α，interleukin（IL）-1β，IL-10，myeloperoxidase（MPO）] and oxidative stress indicators [the level of reactive oxygen species（ROS），malondialdehyde（MDA） and activity of superoxide dismutase（SOD），catalase（CAT）] in colon tissue were detected by enzyme linked immunosorbent assay（ELISA）. The expression of TLR4，NF-κB p65 and apoptosis related protein and mRNA in colon tissue were detected by Western blot or real-time fluorescence quantitative polymerase chain reaction（RT-PCR） method. [Results] Compared with model group，the general state of the rats in POP group and POP+TAK-242 group were significantly improved. The DAI score，CMDI score and the level of IFN-γ，TNF-α，IL-1β，MPO，ROS，MDA were decreased（P＜0.05）；the colon length，IL-10 level and the activity of SOD，CAT were increased（P＜0.05）. The pathological changes and cell apoptosis of colon tissue were significantly improved，and the apoptosis index was decreased（P＜0.05）. The protein and mRNA expression of TLR4，NF-κB p65，B lymphomatoma-2（Bcl-2） related X protein（Bax），Cleared Caspase-3 in colon tissue were decrease，while the protein and mRNA expression of Bcl-2 was increase（P＜0.05）. Compared with POP group，the effect of POP+TAK-242 group on various indicators of UC rats were significantly enhanced（P＜0.05）；the effect of POP+LPS group on various indicators were significantly weakened（P＜0.05）. [Conclusion] POP has the effect of alleviating colon tissue damage in UC rats，which may be related to downregulating the TLR4/NF-κB signaling pathway，inhibiting inflammation，oxidative stress and cell apoptosis of colon tissue.