银杏苦内酯B对内毒素血症幼鼠肠道的保护作用

目的 胃肠功能障碍与严重感染引起的肠屏障功能破坏密切相关.通过应用血小板活化因子(platelet activating factor,PAF)受体拮抗剂银杏苦内酯B(Ginkgolide B)对幼年大鼠内毒素血症小肠上皮细胞形态学及二胺氧化酶(diamine oxidase,DAO)的影响,探讨PAF在内毒素血症肠损伤中的作用.方法 18日龄Wistar大鼠,随机分为内毒素(lipopolysaccharide,LPS)组和PAF受体拮抗剂组(预防组和治疗组)及对照组(每一时相点n=8),注射LPS后1.5,3,6,24,48,72 h取回肠.LPS组和对照组分别腹腔注射内毒素5 mg/kg及生理盐水1 ml/kg;预防组和治疗组分别于每一时相点注射LPS前、后30 min腹腔注射PAF受体拮抗剂BN52021(Ginkgolide B)5 mg/kg.苏木精-伊红染色及透射电镜作形态学检查.采用分光光度计法分别测定回肠组织及血中DAO含量.根据数据的正态性和方差齐性,选择单因素方差分析ANOVA比较多组间差别,最小显著差异法LSD(least significant difference)-t检验比较两组间差别.所有实验在本院动物实验室、病理实验室、生化实验室及辽宁中医药科大学电镜室进行.结果 1.5,3,6,24 h LPS组可见绒毛水肿,固有层血管充血,间质淋巴管扩张,肠腔炎性渗出;拮抗剂组可见绒毛水肿.电镜下对照组肠微绒毛及细胞间紧密连接未见异常.实验组上皮细胞连接增宽,微绒毛变细、稀疏,部分断裂、脱落;细胞器受损.拮抗剂组改变较实验组减轻.LPS组回肠DAO水平明显低于对照组,6 h从对照组(0.172±0.004)15/mg降至(0.096±0.010)U/mg(F=13.372,P＜0.01);LPS组血浆DAO水平明显高于对照组.预防组及治疗组变化趋势同LPS组.结论 PAF在内毒素血症肠道屏障功能损伤中可能起一定作用,预防和治疗性应用PAF受体拮抗剂Ginkgolide B可减轻肠损伤.血浆和小肠组织中DAO活性同步变化,故测定血浆DAO活性可早期、敏感地反映小肠黏膜功能的破坏.

The roles of Ginkgolide B in the protection of intestine against endotoxemia in young rats

Objective Gastrointestinal dysfunction is closely correlated with the impairment of intestinal barrier caused by serious infection.We focused on the role of platelet activating factor(PAF)in the intestinal impairment caused by endotoxemia by studying on the morphology of intestinal epithelial ceils and diamine oxidase (DAO)levels with the application of Ginkgolide B(PAF receptor antagonist).Method Eighteen-day-old Wistar rats were randomized into lipopolysaccharide(LPS)(5 mg/kg),PAF receptor antagonist(pretreatment and treatment)and normal saline(Control)groups(n=8 at each time point).Ginkgolide B(PAF receptor antagonist BN52021)5 mg/kg was administered 30 minutes before(in pretreatment group)and after LPS injection(in treatment group).The ileum specimens(n=8)were harvested at 1.5,3,6,24,48 and 72 hours after LPS or NS injection.The ultrastructures of intestinal epithelial cells were studied by transmission electron microscopy (TEM)and with hematoxylin and erosin staining.The contents of DAO in ileum tissue and plasma were measured respectively with spectrophotometer.According to data Normality and Variance equality,ANOVA analysis and LSD (least significant difference)-t test were used for multiple group difference.The whole test Was performed in the animal laboratory,pathological laboratory,biochemical laboratory of our hospital and electron microscopy laboratory of Liao-ning University of Traditionary Chinese Medicine.Results Histologic examination of intestinal injury in LPS group showed the edema of intestinal villi,the capillary congestion in lamina propria,the dilation of interstitial lymphatic vessel.and the polymorphonuclear infiltration in enteric cavity in LPS group at 1.5,3,6,24 hours.The edema of the intestinal villi were shown in antagonist group.Ultrastructural study showed microvilli and tight junctions were intact in the control group.The tight junctions enlarged and the microvilli were thin,rare or disrupted in the experimental group.The pathological changes in PAF antagonist group were slightly lighter than that in the LPS group.The DAO content in the ileum tissue was obviously decreased in the LPS group compared with that in the control group.It reached to a nadir at 6 hrsfrom(0.172±0.004)U/mg to(0.096±0.010)U/mg,F=13.372,P＜0.01).The DAO content in plasma was obviously higher in the LPS group than that in the control group.The patterns of DAO changes in the PAF antagonist group were as the same as that in the LPS group at each time point.Conclusions PAF may play a certain role in the injury of intestinal barrier in endotoxemia.Preventive and remedial administration of Ginkgolide B may relieve intestinal injury.The activity changes of DAO in plasma synchronized with that in ileum tissue.It may be deduced that the alterations of DAO in plasma may indicate the destruction of intestinal mucosa in the early stage sensitively.