PAF受体拮抗剂对内毒素血症幼年大鼠胃黏膜PGE_2含量及COX-2表达的影响

Effect of PAF receptor antagonist on PGE_2 content and COX-2 expression in gastric mucosa of young rats with endotoxemia

目的探讨前列腺素E2(PGE2)、环氧化酶-2(COX-2)、血小板活化因子(PAF)受体拮抗剂在内毒素(LPS)腹腔注射诱导的幼年大鼠急性胃黏膜损伤中的作用。方法 18日龄Wistar大鼠192只,随机分为对照组、LPS组、PAF受体拮抗剂预防组和治疗组。采用LPS(O55:B5脂多糖)5mg/kg腹腔注射制备幼年大鼠LPS血症模型,预防组和治疗组分别于LPS腹腔注射前后0.5h应用5mg/kg PAF受体拮抗剂BN52021(Ginkgolide B),对照组腹腔注射等量生理盐水。于LPS注射后1.5、3、6、24、48、72h处死动物,每组每时间点8只,肉眼及光学显微镜下观察胃黏膜损伤情况,采用放射免疫法测定胃黏膜PGE2含量,免疫组织化学SP法测定胃黏膜COX-2蛋白的表达,半定量RT-PCR法测定胃黏膜COX-2 m RNA的表达。结果 LPS组腹腔注射LPS后1.5h黏膜上皮细胞水肿,3h组织充血、水肿,6h胃黏膜损伤最重,黏膜内有出血,细胞核碎裂、固缩,凋亡小体出现;24h上皮脱落、中性粒细胞浸润,48h黏膜层变薄、腺体减少,72h未见明显异常。预防组和治疗组改变轻微。与对照组比较,LPS组胃黏膜PGE2含量在3h时明显降低(P<0.05),6h时达最低(P<0.01),预防组胃黏膜PGE2含量在3、6h明显增高(P<0.05),治疗组PGE2含量在6h时明显增高(P<0.05)。与LPS组比较,预防组、治疗组胃黏膜PGE2含量在6h时均明显增高(P<0.01)。对照组胃黏膜组织未见明显COX-2蛋白及m RNA表达;与对照组比较,LPS组腹腔注射LPS后6h胃黏膜组织胞质即有COX-2蛋白表达,24、48、72h时明显增高(P<0.01),其m RNA水平亦上调;预防组和治疗组6h COX-2蛋白、m RNA水平明显增高(P<0.01);预防组和治疗组6h COX-2蛋白、24h COX-2 m RNA与LPS组比较亦明显增高(P<0.01)。结论 PAF受体拮抗剂可上调COX-2 m RNA及蛋白表达,使PGE2含量增加,对胃黏膜有保护作用。

Objective To investigate the protective effect ofprostaglandin E2 （PGE2）, cyclooxygenase-2 （COX-2） and platelet activating factor （PAF） receptor antagonist on endotoxin-induced acute gastric mucosal injury in young rats. Methods Eighteen-day old Wistar rats were randomly divided into 4 groups： normal control group, model group （LPS group）, PAF antagonist prevention group, and PAF antagonist treatment group. The model of endotoxemia in young rats was reproduced by intraperitoneal injection of endotoxin （5mg/kg of O55：B5 lipopolysaccharide）. The rats in PAF prevention and treatment group received PAF antagonist BN52021 （Ginkgolide B, 5mg/kg） 0.Sh before or after modeling. The rats in control group were given intraperitoneal injection of same amount of normal saline （lml/kg）. The animals were sacrificed 1.5, 3, 6, 24, 48 and 72h after intraperitoneal injection of endotoxin （8 in each group）. The pathologic changes in gastric mucosa were observed after HE staining. The content of PGE2 was measured by radioimmunoassay, the expression of COX-2 protein was determined by immunohistochemistry SP method, and the expression of COX-2 mRNA was assessed with RT-PCR method. Results Pathological changes in gastric mucosa were found to be edema of epithelial cells at 1.5h, and hyperemia and edema 3h after intraperitoneal injection of endotoxin in LPS group. The changes were most marked at 6h, including bleeding, karyorrhexis, pyknosis and apoptosis of epithelial cells of gastric mucosa. Exfoliation of the epithelium and neutrophil infiltration were observed at 24h, thinning of mucosa and a decrease in glands were observed at 48h, but no further changes were observed at 72h. However, all the above changes were significantly alleviated in prevention and treatment groups. The PGE2 content of gastric mucosa was lowered at 3h （P〈0.0S）, and it was lowest at 6h （P〈0.01） after endotoxin injection in LPS groupp and significant difference was found between LPS group and control group. The PGEz content of gastric mucosa was obviously increased at 3h and 6h in prevention group （P〈0.05）, and at 6h in treatment group （P〈0.05）. The differences at 6h were significant （P〈0.01） among prevention group, treatment group and LPS group. No expression of COX- 2 protein or mRNA was seen in gastric mucosal tissue of control group. In contrast with control group, cytoplasm COX-2 protein of gastric mucosal tissue was seen to express at 6h after endotoxin injection in LPS group, and it was obviously enhanced at 24, 48 and 72h （P〈0.01）, and the COX-2 mKNA level was also elevated. The expressions of COX-2 protein and mRNA were increased obviously at 6h in PAF antagonist prevention group and treatment group （P〈0.01）. The expressions of COX-2 protein at 6h and COX-2 mRNA at 24h were obviously elevated in prevention group and treatment group compared with those of LPS group （P〈 0.01）. Conclusion PAF receptor antagonist may up-regulate the expression level of COX-2 protein and mRNA, increase PGE2 content, alleviate acute gastric mucosal injury, and promote the healing of gastric mucosal injury.