BAPTA-AM通过抑制炎症和氧化损伤改善脂多糖诱导的小鼠急性肺损伤  

BAPTA-AM mitigates LPS-induced acute lung injury in mice by inhibiting inflammation and oxidative damage

作　　者：俞巧丽 莫泽君 宋必卫[3] YU Qiao-li;MO Ze-jun;SONG Bi-wei(Department of Pharmacy,Zhejiang Hospital,Hangzhou 310007,China.2.Department of Pharmacy,The Second Affiliated Hospital of Zhejiang University School of Medicine,Hangzhou 310009,China;Department of Pharmacology,College of Pharmaceutical Science,Zhejiang University of Technology,Hangzhou 310014,China)

机构地区：[1]浙江医院药剂科,浙江杭州310007 [2]浙江大学医学院附属第二医院药剂科,浙江杭州310009 [3]浙江工业大学药学院药理学科,浙江杭州310014

出　　处：《中国药理学与毒理学杂志》2019年第12期1042-1049,共8页Chinese Journal of Pharmacology and Toxicology

摘　　要：目的探讨1,2-双(2-氨基苯氧基)-乙烷-N,N,N’,N’-四乙酸(BAPTA-AM)对急性肺损伤(ALI)的保护作用及机制。方法体外实验:A549细胞分为正常对照组、缺糖缺氧复糖复氧(OGD/R)模型组(A549细胞首先于无糖RPMI-1640培养液培养,37℃培养箱中缺氧培养12 h;然后用含2 g·L-1葡萄糖的RPMI-1640培养液并在正常含氧量培养箱内继续培养6 h)、BAPTA-AM 0.005,0.05,0.5和5 nmol·L-1组(加相应浓度BAPTA-AM,培养30 min后,同模型组处理)。噻唑蓝(MTT)法检测细胞存活,用试剂盒检测乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)活性及一氧化氮(NO)和丙二醛(MDA)含量,罗丹明123荧光探针检测线粒体膜电位(Δψ)。体内实验:ICR小鼠分为正常对照组、ALI模型组〔iv脂多糖(LPS)10 mg·kg-1〕、地塞米松(Dex)5 mg·kg-1阳性对照和BAPTA-AM纳米脂质体(BAPTA-AML)25,50,100μg·kg-1组(iv LPS10 mg·kg-1后立即给予相应浓度BAPTA-AML或Dex 5 mg·kg-1),6 h后采集标本,测定肺系数和肺组织湿干比,用试剂盒检测血清LDH活性、肺组织MDA含量、SOD和髓过氧化物酶(MPO)活性,ELISA法测定血清TNF-α和IL-6含量,HE染色观察肺组织病理变化。结果体外实验结果表明,BAPTA-AM能提高损伤细胞存活率(P<0.05),降低LDH的释放和NO的合成(P<0.05),改善细胞氧化损伤,稳定Δψ。体内实验结果表明,BAPTA-AML能减轻ALI小鼠肺系数和肺组织湿干比(P<0.05),降低血清肿瘤坏死因子α(TNF-α)和白细胞介素(IL-6)含量及MPO活性(P<0.05),降低ALI模型小鼠血清LDH活性和肺组织MDA含量(P<0.05),提高肺组织SOD活性(P<0.05),降低肺组织MPO活性(P<0.05)及血清TNF-α和IL-6含量(P<0.05),改善肺组织病理改变,降低肺损伤评分(P<0.05)。结论 BAPTA-AM通过改善炎症和氧化损伤,减轻肺水肿,保护肺组织,具有良好的治疗ALI作用。OBJECTIVE To investigate the protective effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA-AM)on acute lung injury(ALI)and elucidate potential pharmaco⁃logical mechanisms.METHODS In vitro,A549 cells were divided into the normal group,oxygen-glucose deprivation/recovery(OGD/R)model(A549 cells were cultured in the absence of serum and glucose in RPMI-1640,exposed to 95%N2 and 5%CO2 for 12 h.Then,the culture medium was replaced with RPMI-1640 containing normal glucose and 10%(V/V)FBS under 5%CO2 and normoxic conditions),BAPTAAM 0.005,0.05,0.5 and 5 nmol·L^-1 groups(a given concentration of BAPTA-AM was added to the cell cultures 30 min prior to ODG/R).The cell vitality,lactate dehydrogenase(LDH)and superoxide dis⁃mutase(SOD)activity,nitric oxide(NO)and malondialdehyde(MDA)levels were tested after 6 h,while rhodamine 123 fluorescence probe was used to detect the mitochondrial membrane potential(Δψ).In vivo,ICR mice were divided into the normal,ALI model(lipopolysaccharide10 mg·kg^-1,iv),dexametha⁃sone(Dex)5 mg·kg^-1 and BAPTA-AM nanoliposome(BAPTA-AML)25,50,100μg·kg^-1 groups[a given concentration of BAPTA-AML(iv)and Dex(iv)5 mg·kg-1 injected immediately after lipopolysaccha⁃ride].Specimens were collected after 6 h,the lung coefficient and lung wet dry(W/D)ratio were tested and LDH,MDA,SOD and myeloperoxidase(MPO)were measured using colorimetry,ELISA method for determination of TNF-αand IL-6.HE stain was used to observe histopathological changes of lung tissue.RESULTS In vitro,BAPTA-AM could improve cell vitality(P<0.05),reverse the increase in LDH(P<0.05)and NO release(P<0.05),protect A549 cells from oxidative stress and keepΔψstable.In vivo,BAPTA-AML treated mice manifested a lower lung coefficient(P<0.05)and lung W/D ratio(P<0.05).The inhibition of LDH,MPO,TNF-αand IL-6(P<0.05)and improvement of oxidative damage were observed.BAPTA-AM could ameliorate histopathological changes and reduce score of lung injury pathology(P<0.05).CONCLUSION BAPTA-AM could ameliorate inflam

关 键 词：BAPTA-AM 急性肺损伤 炎症 氧化损伤 

分 类 号：R974[医药卫生—药品]