中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2014年
7期
493-497
,共5页
张锦丽%秦魏婷%吕汪洄%沈唯长%王旭%孙炳伟
張錦麗%秦魏婷%呂汪洄%瀋唯長%王旭%孫炳偉
장금려%진위정%려왕회%침유장%왕욱%손병위
脓毒症%苦柯胺B%炎症反应%肺
膿毒癥%苦柯胺B%炎癥反應%肺
농독증%고가알B%염증반응%폐
Sepsis%Kukoamine B%Inflammatory response%Lung
目的 探讨苦柯胺B(KB)对脓毒症小鼠肺部炎症反应的抑制作用及分子机制.方法 将28只雄性ICR小鼠按随机数字表法分为对照组(8只)、脂多糖(LPS)组(10只)、LPS +KB组(10只).腹腔注射LPS 20 mg/kg制备脓毒症模型(LPS组);对照组给予等体积生理盐水;LPS +KB组于LPS刺激4h后经尾静脉注射KB 20 μg/kg.于LPS刺激后8h检测动物血浆LPS浓度及肺组织髓过氧化物酶(MPO)活性;采用酶联免疫吸附试验(ELISA)检测血浆、肺泡灌洗液及肺组织匀浆中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的含量;用蛋白质免疫印迹试验(Western Blot)检测肺组织核转录因子-κB(NF-κB)活性和诱导型一氧化氮合酶(iNOS)蛋白表达;用苏木素-伊红(HE)染色观察肺组织病理学改变;免疫组化法观察肺组织中细胞间黏附分子-1 (ICAM-1)蛋白表达.结果 与对照组比较,LPS组血浆LPS浓度(kEU/L:1 155.650±147.149比31.390±18.859)、MPO活性(U/g:1.177 ±0.093比0.775±0.166)、NF-κB活性(灰度值:1.557±0.105比0.824±0.032)、iNOS蛋白表达(灰度值:0.650±0.129比0.392±0.097)均显著升高(均P＜0.05);KB干预后LPS浓度(624.461±149.012)、MPO活性(0.919±0.023)、NF-κB活性(1.127±0.074)、iNOS蛋白表达(0.425±0.066)均被明显抑制(均P＜0.05).与对照组比较,LPS组血浆、肺泡灌洗液、肺组织匀浆中TNF-α(ng/L:47.325±13.864比6.534±0.544,13.382±2.231比3.748±0.692,31.127±7.399比14.948±4.673)、IL-1β(ng/L:74.329±11.890比29.921±6.487,9.422±2.674比1.105±0.364,528.509±32.073比109.945±13.561)浓度均显著增加(均P＜0.05);而应用KB干预后TNF-α(20.331±7.789、7.145±1.202、15.966±2.946)、IL-1β(57.707±8.098、2.212±0.878、426.154±11.270)浓度均明显降低(血浆TNF-α:F=16.052、P=0.002,IL-1β:F=20.649、P=0.000;肺泡灌洗液TNF-α:F=31.134、P=0.001,IL-1β:F=22.792、P=0.002;肺组织匀浆TNF-α:F=10.013、P=0.009,IL-1β:F=319.857、P=0.000).光镜下观察显示,与LPS组比较,KB干预后肺组织病理改变明显减轻,ICAM-1表达明显减少.结论 KB作为新型的LPS中和剂,能够拮抗LPS对脓毒症小鼠的炎症刺激作用,减轻肺部炎症反应,保护脓毒症小鼠的肺脏功能.
目的 探討苦柯胺B(KB)對膿毒癥小鼠肺部炎癥反應的抑製作用及分子機製.方法 將28隻雄性ICR小鼠按隨機數字錶法分為對照組(8隻)、脂多糖(LPS)組(10隻)、LPS +KB組(10隻).腹腔註射LPS 20 mg/kg製備膿毒癥模型(LPS組);對照組給予等體積生理鹽水;LPS +KB組于LPS刺激4h後經尾靜脈註射KB 20 μg/kg.于LPS刺激後8h檢測動物血漿LPS濃度及肺組織髓過氧化物酶(MPO)活性;採用酶聯免疫吸附試驗(ELISA)檢測血漿、肺泡灌洗液及肺組織勻漿中腫瘤壞死因子-α(TNF-α)和白細胞介素-1β(IL-1β)的含量;用蛋白質免疫印跡試驗(Western Blot)檢測肺組織覈轉錄因子-κB(NF-κB)活性和誘導型一氧化氮閤酶(iNOS)蛋白錶達;用囌木素-伊紅(HE)染色觀察肺組織病理學改變;免疫組化法觀察肺組織中細胞間黏附分子-1 (ICAM-1)蛋白錶達.結果 與對照組比較,LPS組血漿LPS濃度(kEU/L:1 155.650±147.149比31.390±18.859)、MPO活性(U/g:1.177 ±0.093比0.775±0.166)、NF-κB活性(灰度值:1.557±0.105比0.824±0.032)、iNOS蛋白錶達(灰度值:0.650±0.129比0.392±0.097)均顯著升高(均P＜0.05);KB榦預後LPS濃度(624.461±149.012)、MPO活性(0.919±0.023)、NF-κB活性(1.127±0.074)、iNOS蛋白錶達(0.425±0.066)均被明顯抑製(均P＜0.05).與對照組比較,LPS組血漿、肺泡灌洗液、肺組織勻漿中TNF-α(ng/L:47.325±13.864比6.534±0.544,13.382±2.231比3.748±0.692,31.127±7.399比14.948±4.673)、IL-1β(ng/L:74.329±11.890比29.921±6.487,9.422±2.674比1.105±0.364,528.509±32.073比109.945±13.561)濃度均顯著增加(均P＜0.05);而應用KB榦預後TNF-α(20.331±7.789、7.145±1.202、15.966±2.946)、IL-1β(57.707±8.098、2.212±0.878、426.154±11.270)濃度均明顯降低(血漿TNF-α:F=16.052、P=0.002,IL-1β:F=20.649、P=0.000;肺泡灌洗液TNF-α:F=31.134、P=0.001,IL-1β:F=22.792、P=0.002;肺組織勻漿TNF-α:F=10.013、P=0.009,IL-1β:F=319.857、P=0.000).光鏡下觀察顯示,與LPS組比較,KB榦預後肺組織病理改變明顯減輕,ICAM-1錶達明顯減少.結論 KB作為新型的LPS中和劑,能夠拮抗LPS對膿毒癥小鼠的炎癥刺激作用,減輕肺部炎癥反應,保護膿毒癥小鼠的肺髒功能.
목적 탐토고가알B(KB)대농독증소서폐부염증반응적억제작용급분자궤제.방법 장28지웅성ICR소서안수궤수자표법분위대조조(8지)、지다당(LPS)조(10지)、LPS +KB조(10지).복강주사LPS 20 mg/kg제비농독증모형(LPS조);대조조급여등체적생리염수;LPS +KB조우LPS자격4h후경미정맥주사KB 20 μg/kg.우LPS자격후8h검측동물혈장LPS농도급폐조직수과양화물매(MPO)활성;채용매련면역흡부시험(ELISA)검측혈장、폐포관세액급폐조직균장중종류배사인자-α(TNF-α)화백세포개소-1β(IL-1β)적함량;용단백질면역인적시험(Western Blot)검측폐조직핵전록인자-κB(NF-κB)활성화유도형일양화담합매(iNOS)단백표체;용소목소-이홍(HE)염색관찰폐조직병이학개변;면역조화법관찰폐조직중세포간점부분자-1 (ICAM-1)단백표체.결과 여대조조비교,LPS조혈장LPS농도(kEU/L:1 155.650±147.149비31.390±18.859)、MPO활성(U/g:1.177 ±0.093비0.775±0.166)、NF-κB활성(회도치:1.557±0.105비0.824±0.032)、iNOS단백표체(회도치:0.650±0.129비0.392±0.097)균현저승고(균P＜0.05);KB간예후LPS농도(624.461±149.012)、MPO활성(0.919±0.023)、NF-κB활성(1.127±0.074)、iNOS단백표체(0.425±0.066)균피명현억제(균P＜0.05).여대조조비교,LPS조혈장、폐포관세액、폐조직균장중TNF-α(ng/L:47.325±13.864비6.534±0.544,13.382±2.231비3.748±0.692,31.127±7.399비14.948±4.673)、IL-1β(ng/L:74.329±11.890비29.921±6.487,9.422±2.674비1.105±0.364,528.509±32.073비109.945±13.561)농도균현저증가(균P＜0.05);이응용KB간예후TNF-α(20.331±7.789、7.145±1.202、15.966±2.946)、IL-1β(57.707±8.098、2.212±0.878、426.154±11.270)농도균명현강저(혈장TNF-α:F=16.052、P=0.002,IL-1β:F=20.649、P=0.000;폐포관세액TNF-α:F=31.134、P=0.001,IL-1β:F=22.792、P=0.002;폐조직균장TNF-α:F=10.013、P=0.009,IL-1β:F=319.857、P=0.000).광경하관찰현시,여LPS조비교,KB간예후폐조직병리개변명현감경,ICAM-1표체명현감소.결론 KB작위신형적LPS중화제,능구길항LPS대농독증소서적염증자격작용,감경폐부염증반응,보호농독증소서적폐장공능.
Objective To investigate the inhibitory effect of kukoamine B (KB) on lung inflammatory responses in mice with sepsis and its possible molecular mechanism.Methods Twenty-eight male mice were randomly divided into control group (n=8),lipopolysaccharide (LPS) group (n=10),and LPS + KB group (n=10).Sepsis model was reproduced by intra-peritoneal injection of 20 mg/kg LPS,while equivalent normal saline was given in control group,and 20 μg/kg KB was injected through caudal vein 4 hours after LPS challenge in LPS + KB group.After 8 hours of LPS challenge,the concentration of LPS in plasma and the activity of myeloperoxidase (MPO) in the lung tissue were determined.The contents of tumor necrosis factor-α (TNF-α) and interleukin-lβ (IL-1β) in plasma,alveolar lavage fluid and lung tissue homogenates were assessed by enzyme linked immunosorbent assay (ELISA).The activation of nuclear factor-κB (NF-κB) and the expression of inducible nitric oxide synthase (iNOS) in lung tissue were determined by Western Blot.The pathological changes in lung tissues were observed with hematoxylin-eosin (HE) staining.The expression of intercellular adhesion molecule-1 (ICAM-1) in lung tissue was determined by immunohistochemistry.Results Compared with control group,the concentration of LPS in plasma (kEU/L:1 155.650 ± 147.149 vs.31.390 ± 18.859),MPO activity (U/g:1.177 ±0.093 vs.0.775 ±0.166),NF-κB activity (gray value:1.557 ±0.105 vs.0.824 ±0.032) and the expression of iNOS (gray value:0.650 ±0.129 vs.0.392 ±0.097) were significantly increased in LPS group (all P＜0.05).After KB intervention,the concentration of LPS (624.461 ± 149.012),MPO activity (0.919 ±0.023),NF-κB activity (1.127 ±0.074) and the expression ofiNOS (0.425 ± 0.066) were significantly lowered (all P＜0.05).Compared with control group,the contents of TNF-α (ng/L:47.325 ± 13.864 vs.6.534 ± 0.544,13.382 ± 2.231 vs.3.748 ± 0.692,31.127 ± 7.399 vs.14.948 ± 4.673) and IL-1β (ng/L:74.329 ± 11.890 vs.29.921 ± 6.487,9.422 ± 2.674 vs.1.105 ± 0.364,528.509 ± 32.073 vs.109.945 ± 13.561) in plasma,alveolar lavage fluid and lung tissue homogenates were obviously enhanced in LPS group (all P＜0.05).With KB intervention,the contents of TNF-α (20.331 ± 7.789,7.145 ± 1.202,15.966 ± 2.946) and IL-1β (57.707 ±8.098,2.212 ± 0.878,426.154 ± 11.270) were markedly reduced (plasma TNF-α:F=16.052,P=0.002; IL-1β:F=20.649,P=0.000; lung tissue homogenates TNF-α:F=31.134,P=0.001; IL-1β:F=22.792,P=0.002;alveolar lavage fluid TNF-α:F=10.013,P=0.009; IL-1β:F=319.857,P=0.000).In addition,leukocyte infiltration to the lung tissue was attenuated,and the expression of ICAM-1 was reduced by KB in histological examination.Conclusion KB,as a neutralizer of LPS,can inhibit the release of inflammatory mediators,reduce the pulmonary inflammatory response and protect the function of lung in septic mice.
