安肠组方对放射性肠炎小鼠抗炎作用及肠道屏障功能的影响

Effect of Anchang Group formula on anti-inflammatory effect and intestinal barrier function in mice with radiation enteritis

关键词:  安肠组方  放射性肠炎  炎症  肠道屏障

Key words:  Anchang Group formula  radiation enteritis  inflammation  intestinal barrier

摘要:

[目的] 探讨安肠组方对放射性肠炎小鼠结肠保护作用及其机制。[方法] 选用50只雄性C57BL/6小鼠[体质量（20±2） g]，随机分为5组：正常组、模型组以及安肠组方低（2.1 g/mL）、中（4.2 g/mL）、高（8.4 g/mL）剂量的治疗组。除正常组外，其他各组小鼠经X线照射后连续7 d进行灌胃给药，正常组和模型组小鼠灌胃水。在灌胃过程中，每日观察小鼠的体质量、存活率、腹泻和便血情况以及毛发和精神状态。灌胃结束后处死小鼠，取血，结肠及粪便，测量小鼠的结肠长度，并采用苏木精-伊红（HE）和马松（Masson）染色评估结肠组织形态学变化和胶原纤维的积累情况。通过酶联免疫吸附实验（ELISA）测定小鼠血清中的白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、白细胞介素-10（IL-10）和肿瘤坏死因子-α（TNF-α）含量。利用免疫组化技术检测IL-1β、IL-6、IL-10和TNF-α在小鼠结肠组织中的表达。通过蛋白免疫印迹（Western blot）技术分析结肠中紧密连接蛋白ZO-1、claudin-1的表达水平。[结果] 与正常组相比，模型组小鼠体质量下降（P＜0.01），存活率降低，结肠缩短（P＜0.01）；结肠组织中观察到炎性细胞浸润，组织结构紊乱，部分细胞形态消失，胶原纤维增多；血清中促炎细胞因子TNF-α、IL-1β、IL-6含量增加（P＜0.01），抗炎细胞因子IL-10含量下降（P＜0.01），免疫组化结果显示结肠组织中促炎细胞因子TNF-α、IL-1β、IL-6的表达增加（P＜0.01），抗炎细胞因子IL-10的表达降低（P＜0.01）；结肠中紧密连接蛋白 ZO-1、claudin-1的表达下降（P＜0.01）。与模型组小鼠相比，安肠组方低、中、高组小鼠体质量增加（P＜0.05），存活率上升，结肠长度增加（P＜0.01）；尽管结肠中仍有炎性细胞浸润，但组织结构较为清晰，胶原纤维减少；血清中TNF-α、IL-1β、IL-6含量下降（P＜0.01），IL-10含量明显上升（P＜0.01），免疫组化结果显示结肠组织中TNF-α、IL-1β、IL-6的表达降低（P＜0.01），IL-10的表达增加（P＜0.01）；结肠组织中紧密连接蛋白ZO-1、claudin-1的表达上升（P＜0.01）。[结论] 安肠组方对C57BL/6小鼠放射性肠炎的改善作用可能主要通过减轻炎症反应和修复损伤的肠黏膜屏障，从而促进组织纤维化的改善和肠道屏障功能的提升。因此，安肠组方具有调控炎症反应并改善肠道屏障功能的潜力。

Abstract:

[Objective] Objective To investigate the protective effect of Anchang Group formula on colon of radiation enteritis mice and its mechanism. [Methods] Fifty male C57BL/6 mice[body weight(20±2) g] were randomly divided into five groups:normal group,model group,and low(2.1 g/mL),medium(4.2 g/mL),high(8.4 g/mL) Anchang Group prescription treatment group. In addition to the normal group,mice in other groups were given intragastric administration for 7 days after X-ray irradiation,and mice in normal group and model group were given intragastric water. During intragastric administration,the mice were observed daily for weight,survival,diarrhea and blood in stool,as well as hair and mental status. After the end of intragastric administration,the mice were killed,blood,colon and feces were collected,and the colonic length was measured. Hematoxylin-Eosin(HE) and Masson staining were used to evaluate the colonic histomorphologic changes and the accumulation of collagen fibers. Serum levels of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10) and tumor necrosis factor-α(TNF-α) were determined by enzyme-linked immunosorbent assay(ELISA). The expression of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10) and tumor necrosis factor-α(TNF-α) in colon tissues of mice was detected by immunohistochemical technique. The expression levels of tight junction proteins ZO-1 and claudin-1 in colon were analyzed by Western blot. [Results] Compared with the normal group,the weight of mice in the model group decreased(P＜0.01),the survival rate decreased,and the colon shortened(P＜0.01). Inflammatory cell infiltration,tissue structure disorder,partial cell morphology disappeared,and collagen fibers increased were observed in colon tissue. Serum contents of pro-inflammatory cytokines TNF-α,IL-1β and IL-6 were significantly increased(P＜0.01),and the content of anti-inflammatory cytokines IL-10 was significantly decreased(P＜0.01). Immunohistochemical results showed that the expressions of pro-inflammatory cytokines TNF-α,IL-1β and IL-6 in colon tissues were increased(P＜0.01). The expression of anti-inflammatory cytokine IL-10 was decreased(P＜0.01). The expressions of tight junction proteins ZO-1 and claudin-1 in colon were significantly decreased(P＜0.01). Compared with model group,the body weight,survival rate and colon length of mice in low,medium and high Anchang Group were increased(P＜0.05). Although there is still inflammatory cell infiltration in the colon,the tissue structure is clear,and the collagen fibers are reduced. The contents of TNF-α,IL-1β and IL-6 in serum were significantly decreased(P＜0.01),and the content of IL-10 was significantly increased(P＜0.01). Immunohistochemical results showed that the expressions of TNF-α,IL-1β and IL-6 in colon tissues were decreased(P＜0.01),and the expression of IL-10 was increased(P＜0.01). The expressions of tight junction proteins ZO-1 and claudin-1 in colon tissues were significantly increased(P＜0.01). [Conclusion] The ameliorative effect of Anchang Group formula on C57BL/6 mice radiation enteritis may be mainly through reducing inflammatory response and repairing the damaged intestinal mucosal barrier,thus promoting the improvement of tissue fibrosis and intestinal barrier function. Therefore,Anchang Group formula has the potential to regulate inflammatory response and improve intestinal barrier function.