高氧对幽门螺杆菌的清除作用及基于脂质组学的机制
Chinese Journal of Pharmacology and Toxicology（2023）
西安交通大学第二附属医院
Abstract
目的 研究高氧对幽门螺杆菌(Hp)的清除作用,并基于脂质组学探讨其可能机制.方法 ①高氧对Hp的清除作用.将过氧化氢300 g·L-1加入生理盐水配制成含氧量为13～416 mg·L-1高氧水溶液.将Hp分为Hp对照组(等体积生理盐水)、枸橼酸铋钾组(7.3 g·L-1)及高氧组(高氧溶液13,26,52,104,208和416 mg·L-1).各组Hp置微需氧环境培养5 d后,紫外分光光度法检测菌落洗脱液在600 nm吸光度(A600 nm)值.蒙古沙鼠ig给予Hp 1×1011 CFU·L-1悬液,每天1次,连续20 d,建立蒙古沙鼠Hp感染模型;选取60只感染模型沙鼠,分为Hp模型组(等体积生理盐水)、Hp模型+高氧(高氧溶液26 mg·L-1)组、Hp模型+三联药物组(克拉霉素8.3 g·L-1、枸橼酸铋钾7.3 g·L-1和替硝唑16.6 g·L-1),选取20只健康沙鼠作为正常对照组(等体积生理盐水),各组分别于给药14 d后及停药14 d后处死半数沙鼠,吉姆萨染色计数胃黏膜Hp数目.②高氧对Hp细胞膜的损伤作用.将Hp分为Hp对照组(等体积生理盐水)、高氧(7,13和26 mg·L-1高氧溶液)组、阿莫西林40 mg·L-1组,SYTO 9/PI染色后使用流式细胞术检测Hp的存活率;BCA与ELISA法分别检测Hp对照组、高氧组胞质蛋白与DNA泄漏量;透射电镜观察高氧26 mg·L-1组、阿莫西林40 mg·L-1组和Hp对照组菌膜完整性.③高氧处理前后Hp脂质种类和含量变化.将Hp分为Hp对照组、高氧40 min和高氧12 h组,高效液相色谱-串联质谱法检测高氧26 mg·L-1处理40 min和12 h后脂质种类及丰度,数据挖掘算法分析筛选差异脂质代谢物.结果 ①高氧浓度依赖性降低菌落洗脱液A600 nm值(P<0.05,P<0.01,r=-0.99).蒙古沙鼠胃黏膜存在Hp定植,表明感染模型建立成功;Hp模型+高氧组沙鼠胃黏膜Hp数目显著低于Hp模型组(P<0.01);②高氧组Hp的存活率显著低于阿莫西林组(P<0.01);透射电镜结果显示,Hp对照组细胞膜完整、透明,胞质均匀分布.高氧组菌膜损伤、破裂、模糊不清,胞质溢出;高氧各组菌体胞质蛋白与DNA泄漏量均显著高于正常对照组(P<0.01).③与Hp对照组相比,高氧12 h组脂质分布具有显著差异(P<0.01).脂质组学分析筛选出2557个差异脂质,其中正、负离子模式下分别有1752和805个;正、负离子模式的上调差异脂质分别为200和85个,下调分别为73和55个,正离子模式差异脂质主要集中于脂肪酰类、甘油磷脂类和多聚乙烯类;负离子模式主要集中于脂肪酰类、固醇脂类和甘油脂类.结论 高氧可清除Hp,其除菌机制与破坏Hp细胞膜的脂质结构相关.
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Key words
Helicobacter pylori,Mongolian gerbils,enriched oxygen,cell membrane,lipidomics
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