金银花提取液通过调控NLRP3/ASC/caspase-1信号通路对溃疡性结肠炎大鼠肠道黏膜的影响

Effect of honeysuckle flower extract on intestinal mucosa of rats with ulcerative colitis by regulating NLRP3/ASC/caspase-1 signaling pathway

目的:探讨金银花提取液通过调控核苷酸结合寡聚化结构域样受体家族吡啉结构域蛋白3(NLRP3)/含CARD结构域的凋亡相关颗粒样蛋白(ASC)/半胱氨酸天冬氨酸蛋白酶1(caspase-1)信号通路对溃疡性结肠炎(UC)大鼠肠道黏膜的影响。方法:随机选取10只大鼠为对照组,另取55只大鼠在肛门约8 cm结肠处注射含5%TNBS和50%乙醇的混合液(4 ml/kg)构建大鼠UC模型,将造模成功的50只大鼠分为UC组、金银花低、中、高剂量组和金银花+NSS(NLRP3激活剂)组,每组10只。金银花低、中、高剂量组分别灌胃给予40、80、120 mg/(kg·d)金银花提取液,金银花+NSS组灌胃给予120 mg/(kg·d)金银花提取液和4 mg/(kg·d)NSS,对照组、UC组灌胃给予等体积生理盐水。观察大鼠一般情况,末次给药24 h后腹主动脉取血,处死大鼠,收集结肠组织,量取结肠长度。试剂盒检测大鼠血清超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、丙二醛(MDA)、TNF-α、IL-6水平;HE染色检测大鼠结肠黏膜病理学变化;RT-PCR和Western blot检测大鼠肠道黏膜NLRP3/ASC/caspase-1通路mRNA和蛋白表达。结果:与对照组比较,UC组大鼠疾病活动指数(DAI)、结肠黏膜损伤程度、血清LDH、MDA、TNF-α、IL-6水平及结肠黏膜组织NLRP3、ASC、caspase-1、IL-1β mRNA和蛋白表达显著升高,结肠长度、血清SOD水平显著降低(P<0.05);与UC组比较,金银花低、中、高剂量组DAI、结肠黏膜损伤程度、LDH、MDA、TNF-α、IL-6水平及NLRP3、ASC、caspase-1、IL-1β mRNA和蛋白表达显著降低,结肠长度、血清SOD水平显著升高(P<0.05),且呈剂量依赖性(P<0.05);与金银花高剂量组比较,金银花+NSS组DAI、结肠黏膜损伤程度、LDH、MDA、TNF-α、IL-6水平及NLRP3、ASC、caspase-1、IL-1β mRNA和蛋白表达显著升高,结肠长度、SOD水平显著降低(P<0.05)。结论:金银花提取液可能通过抑制NLRP3/ASC/caspase-1信号通路激活缓解UC大鼠肠道黏膜损伤。

Objective:To explore influence of honeysuckle flower extract on intestinal mucosa of rats with ulcerative colitis(UC)by regulating NOD-like receptor family pyrin domain containing 3(NLRP3)/apoptosis-associated speck-like protein containing CARD(ASC)/caspase-1 signal pathway.Methods:Ten rats were randomly selected as control group,another 55 rats were injected with a mixture containing 5%TNBS and 50% ethanol(4 ml/kg)into colon about 8 cm in anus to construct rat UC model,and 50 rats which successfully modeled were divided into UC group,honeysuckle flower low,medium and high doses groups,and honeysuckle flower+NSS(NLRP3 activator)group,with 10 rats in each group. Honeysuckle flower low,medium and high doses groups were given40,80,120 mg/(kg·d)honeysuckle flower extract by gavage,and honeysuckle flower+NSS group was given 120 mg/(kg·d)honeysuckle flower extract and 4 mg/(kg·d)NSS,control group and UC group were given an equal volume of normal saline by gavage. General conditions of rats were observed. 24 h after the last administration,blood was taken from abdominal aorta,and rats were sacrificed,colon tissues were collected and length of colon was measured. Kits were used to detect levels of serum superoxide dismutase(SOD),lactate dehydrogenase(LDH),malondialdehyde(MDA),TNF-α,IL-6;HE staining was used to detect pathological changes of rat colonic mucosa;mRNA and protein expressions of NLRP3/ASC/caspase-1 pathway in rat intestinal mucosa were detected by RT-PCR and Western blot.Results:Compared with control group,disease activity index(DAI),degree of colonic mucosal damage,serum LDH,MDA,TNF-α,IL-6 levels,NLRP3,ASC,caspase-1,IL-1β mRNA and protein expressions were increased significantly in UC group,colon length and serum SOD level were decreased significantly(P<0.05);compared with UC group,DAI,degree of colonic mucosal damage,serum LDH,MDA,TNF-α,IL-6 levels,NLRP3,ASC,caspase-1,IL-1β mRNA and protein expressions were decreased significantly in honeysuckle flower low,medium,and high doses groups,and colon length and serum SOD level were increased significantly(P<0.05),which was in dose-dependent manner(P<0.05);compared with honeysuckle flower high-dose group,DAI,degree of colonic mucosal damage,serum LDH,MDA,TNF-α,IL-6 levels,NLRP3,ASC,caspase-1,IL-1β mRNA and protein expressions were increased significantly in honeysuckle flower+NSS group,and colon length and serum SOD level were decreased significantly(P<0.05).Conclusion:Honeysuckle flower extract may inhibit activation of NLRP3/ASC/caspase-1 signaling pathway and reduce intestinal mucosal damage in UC rats.