补中益气丸对DSS诱导的结肠炎模型小鼠不同病理阶段NLRP3炎性体通路的影响

Effect of Buzhong Yiqiwan on NLRP3 Inflammasome Pathway of DSS-induced Colitis Model Mice at Different Pathological Stages

目的:探讨补中益气丸对结肠炎小鼠的干预作用及机制。方法:64只C57BL/6小鼠随机分为2周空白组、2周模型组、2周补中益气丸(12 g·kg^(-1))组、2周补中益气丸(6 g·kg^(-1))组、4周空白组、4周模型组、4周补中益气丸12 g·kg^(-1)组及4周补中益气丸6 g·kg^(-1)组。采用3%右旋葡聚糖硫酸钠(DSS)自由饮水7 d诱导结肠炎小鼠模型,于造模后第8天开始给予补中益气丸(12、6 g·kg^(-1))灌胃,每天1次。分别于第2、4周处死动物,测量小鼠结肠长度及质量,苏木素-伊红(HE)染色病理观察并进行结肠黏膜炎症评分,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测NOD样受体热蛋白结构域3(NLRP3)、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1(Caspase-1)的mRNA和蛋白表达,酶联免疫吸附测定法(ELISA)检测结肠组织中的炎性细胞因子白细胞介素(IL)-1β、IL-18和IL-33含量。结果:与2周空白组比较,2周模型组小鼠结肠长度明显缩短、质量明显增加(P<0.05),光镜下可见上皮细胞缺失、腺体结构破坏、黏膜及黏膜下层大量炎细胞浸润,局部出现隐窝脓肿,黏膜炎症评分显著增加(P<0.01),结肠组织中IL-1β、IL-18和IL-33含量明显增高(P<0.05),NLRP3、ASC、Caspase-1的mRNA及蛋白表达明显上升(P<0.05);与2周模型组比较,补中益气丸(12 g·kg^(-1))干预可恢复结肠长度,缓解黏膜损伤程度(P<0.05),下调炎性因子IL-18含量(P<0.05),降低NLRP3、ASC的mRNA表达和ASC、Caspase-1的蛋白表达水平(P<0.05)。与4周空白组比较,4周模型组小鼠结肠长度明显减少,质量明显增加(P<0.05),光镜下见黏膜层腺体减少、腺腔扩张,隐窝萎缩,局部结缔组织增生及淋巴细胞浸润,炎症评分明显升高,呈现慢性炎症阶段(P<0.01);炎性细胞因子IL-1β、IL-18和IL-33表达增高(P<0.05),NLRP3炎性体复合物的mRNA和蛋白表达水平增高(P<0.05);与4周模型组比较,两种剂量的补中益气丸干预均可改善结肠长度及质量(P<0.05),其中,补中益气丸(12 g·kg^(-1))干预还可改善结肠炎症评分(P<0.05)。与急性期不同的是,此时补中益气丸(两种剂量)干预提高肠黏膜IL-33含量并显著上调NLRP3炎性体复合物ASC、Caspase-1 mRNA及蛋白表达(P<0.05)。结论:补中益气丸可减轻DSS模型小鼠结肠炎症损伤,其机制与调节NLRP3炎性体介导的肠道免疫反应有关,且在急、慢性炎症阶段呈现不同的调节作用。

Objective:To explore the intervention effect and mechanism of Buzhong Yiqiwan(BZYQ) on colitis mice. Method: Sixty-four C57BL/6 mice were randomly divided into 2 weeks blank group,2 weeks model group,2 weeks high-dose BZYQ(12 g·kg^(-1))group,2 weeks low-dose BZYQ(6 g·kg;)group,4 weeks blank group,4 weeks model group,4 weeks high-dose BZYQ(12 g·kg^(-1))group,and 4 weeks low-dose BZYQ(6 g·kg;)group. The colitis model was induced in mice by feeding 3% dextran sodium sulfate(DSS)for 7days. The mice received BZYQ(12 and 6 g·kg;)by gavage on the 8thday after modeling,once per day,and sacrificed on the 2ndand 4thweeks,correspondingly. The colon length and weight of mice in each group were measured. Hematoxylin-eosin(HE) staining was used for pathological observation and colonic mucosal inflammation was scored. The mRNA and protein expression of NOD-like receptor thermoprotein domain 3(NLRP3),apoptosis-associated speck-like protein containing a CARD(ASC),and cysteinyl aspartate-specific protease-1(Caspase-1)was detected by real-time quantitative polymerase chain reaction(Real-time PCR)and Western blot,respectively. Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of inflammatory cytokines,such as interleukin(IL)-1β,IL-18,and IL-33 in colonic tissues. Result: Compared with the 2 weeks blank group,the 2 weeks model group showed shortened colon length,increased colon weight(P<0.05),loss of epithelial cells,destruction of gland structure,infiltration of a large number of inflammatory cells in mucosa and submucosa,local crypt abscess,and increase in mucosal inflammation score(P<0.01)as revealed by light microscopy,elevated levels of IL-1β,IL-18,and IL-33 in colonic tissues(P<0.05),and increased mRNA and protein expression of NLRP3,ASC,and Caspase-1(P<0.05). The intervention of BZYQ(12 g·kg^(-1))restored colon length,alleviated mucosal injury(P<0.05),down-regulated the content of IL-18(P<0.05),reduced the mRNA expression of NLRP3 and ASC as well as the protein expression of ASC and Caspase-1 compared with the conditions in the 2 weeks model group. Compared with the 4 weeks blank group,the 4weeks model group showed decreased colon length,increased colon weight(P<0.05),decreased glands in the mucosal layer, expansion of glandular cavity, atrophy of crypt, local connective tissue hyperplasia and lymphocyte infiltration,increased inflammation score(P<0.01)as revealed by the light microscopy,increased expression of IL-1β,IL-18,and IL-33(P<0.05),and elevated mRNA and expression of NLRP3 inflammasome complex(P<0.05). Compared with the conditions in the 4 weeks model group,the intervention of BZYQ(12and 6 g·kg^(-1))could improve colon length and weight(P<0.05),and the intervention of BZYQ(12 g·kg^(-1))could also improve the inflammation score of the colon(P<0.05). Different from the acute stage, the intervention of BZYQ(12 and 6 g·kg^(-1))increased the content of IL-33 in the intestinal mucosa and up-regulated the mRNA and protein expression of NLRP3 inflammasome complexes ASC and Caspase-1(P<0.05).Conclusion: BZYQ can relieve the injury of colitis induced by DSS in mice. The mechanism is related to the regulation of intestinal immune response mediated by NLRP3 inflammasome,and it has different regulatory effects in acute and chronic inflammation stages.