摘要目的 观察半胱氨酸天冬氨酸蛋白酶1抑制剂VX765对冷束缚诱导小鼠急性胃溃疡胃黏膜的保护效应. 方法 将24只无特殊病原体级雄性C57BL/6J小鼠按照随机数字表法分为正常对照组、冷束缚组、VX765预处理+冷束缚组、雷贝拉唑预处理+冷束缚组,每组6只.正常对照组小鼠仅在腹腔内注射10 mL/kg二甲基亚砜(DMSO)和PBS混合液.冷束缚组小鼠在腹腔内注射DMSO和PBS混合液,30 min后行冷束缚实验诱导急性胃溃疡.VX765预处理+冷束缚组、雷贝拉唑预处理+冷束缚组小鼠同前造成急性胃溃疡,实验前30 min分别腹腔注射含10 mg VX765的DMSO、PBS混合液(12.5 μmol/kg)及含20 mg雷贝拉唑的PBS、DMSO 混合液(40 mg/kg).冷束缚实验结束后4h,采用ELISA法测定血清TNF-α、IL-6含量,观察胃组织的大体情况、评估溃疡指数、行HE染色观察胃组织的病理学变化,采用蛋白质印迹法检测胃组织中IL-1β、IL-18及剪切型半胱氨酸天冬氨酸蛋白酶1的相对表达量.正常对照组小鼠于相同时相点同前检测.对数据行单因素方差分析、Bonferroni检验. 结果 正常对照组小鼠血清中TNF-α和IL-6含量和胃组织中剪切型半胱氨酸天冬氨酸蛋白酶1、IL-1β、IL-18含量均明显低于冷束缚组(P值均小于0.01).VX765预处理+冷束缚组小鼠血清和胃组织中的前述炎性指标含量均明显低于冷束缚组(P值均小于0.01).雷贝拉唑预处理+冷束缚组与冷束缚组小鼠血清和胃组织中的前述炎性指标含量比较,差异均无统计学意义(P值均大于0.05).VX765预处理+冷束缚组小鼠血清和胃组织中的前述炎性指标含量均明显低于雷贝拉唑预处理+冷束缚组(P值均小于0.01).正常对照组小鼠胃黏膜表面光滑平整,黏膜细胞形态正常、结构清晰.冷束缚组小鼠多发性胃黏膜出血,黏膜细胞排列混乱,坏死组织周围有大量炎性细胞浸润.VX765预处理+冷束缚组、雷贝拉唑预处理+冷束缚组小鼠胃黏膜出血数目显著减少、黏膜结构尚完整,坏死组织周围有少量炎性细胞浸润.正常对照组、冷束缚组、VX765预处理+冷束缚组、雷贝拉唑预处理+冷束缚组小鼠的溃疡指数分别为0、1 8.7±1.1、6.3±1.5、8.2±1.3;其中正常对照组明显低于其余3组(P值均小于0.05),VX765预处理+冷束缚组与雷贝拉唑预处理+冷束缚组相近(P＞0.05),该2组均明显低于冷束缚组(P值均小于0.05). 结论 VX765可以有效阻断半胱氨酸天冬氨酸蛋白酶1的活化、减少炎症因子的生成、减轻炎症反应,对冷束缚诱导小鼠急性胃溃疡胃黏膜有一定的保护作用.

abstractsObjective To investigate the protective effects of caspase-1 inhibitor VX765 on gastric mucosa of mice with cold-restraint stress-induced acute gastric ulcer.Methods Twenty-four specific pathogen free male C57BL/6J mice were divided into normal control group (NC),cold restrain group (CR),VX765 pre-treatment + cold restrain group (VCR),and rabeprazole pre-treatment + cold restrain group (RCR) according to the random number table,with 6 mice in each group.Mice in group NC were injected intraperitoneally with solution of 10 mL/kg dimethylsulfoxide (DMSO) and phosphate buffer solution (PBS).Mice in group CR were inflicted with acute gastric ulcer induced by cold-restraint stress 30 minutes after intraperitoneal injection of solution of DMSO and PBS.Mice in groups VCR and RCR were inflicted with acute gastric ulcer as above 30 minutes after intraperitoneal injection of solution of DMSO and PBS with dose of 12.5 μmol/kg containing 10 mg VX765 and 40 mg/kg containing 20 mg rabeprazole,respectively.Four hour after cold-restraint stress,serum content of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6)was determined by enzyme-linked immunosorbent assay.Gross condition of gastric tissue was observed.Ulcer index was evaluated.Pathological change of gastric tissue was observed with HE staining.The relative expression of IL-1β,IL-18,and cleaved-caspase-1 in gastric tissue were detected by Western blotting.Mice in group NC were detected as above at the same time point.Data were processed with one-way analysis of variance and Bonferroni test.Results The serum content of TNF-α and IL-6 and the relative expression of cleaved-caspase-1,IL-1β,and IL-18 in gastric tissue of mice in group NC were significantly lower than those in group CR (with P values below 0.01).The content of the above-mentioned inflammatory indexes in serum and gastric tissue of mice in group VCR was significantly lower than that in group CR (with P values below 0.01).There were no statistically significant differences in content of the above-mentioned inflammatory indexes in serum and gastric tissue of mice between groups RCR and CR (with P values above 0.05).The content of the above-mentioned inflammatory indexes in serum and gastric tissue of mice in group VCR was significantly lower than that in group RCR (with P values below 0.01).Surface of gastric mucosa was smooth and morphology of mueosal cells was normal with clear structure of mice in group NC.Multiple hemorrhage of gastric mucosa,disorderly arrangement of mucosal cells,and large number of inflammatory cell infiltration around necrotic tissue were observed in mice of group CR.Decreased number of gastric mucosa bleeding,intact mucosal structure,and small amount of inflammatory cell infiltration around necrotic tissue were observed in mice of groups VCR and RCR.The ulcer indexes of mice in groups NC,CR,VCR,and RCR were 0,18.7 ± 1.1,6.3 ± 1.5,and 8.2 ± 1.3,respectively.The ulcer index of mice in group NC was significantly lower than that in the other 3 groups (with P values below 0.05).The ulcer indexes of mice in groups VCR and RCR were close (P ＞ 0.05),which were significantly lower than ulcer index of mice in group CR (with P values below 0.05).Conclusions VX765 can effectively inhibit the activation of caspase-1,reduce production of inflammatory factor,and alleviate inflammatory response,which have protective effects on gastric mucosa of mice with cold-restraint stress-induced acute gastric ulcer.