中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
2期
121-126
,共6页
吕汪洄%秦魏婷%张锦丽%沈唯长%王旭%孙炳伟
呂汪洄%秦魏婷%張錦麗%瀋唯長%王旭%孫炳偉
려왕회%진위정%장금려%침유장%왕욱%손병위
脂多糖%苦柯胺B%小肠%炎症反应
脂多糖%苦柯胺B%小腸%炎癥反應
지다당%고가알B%소장%염증반응
Lipopolysaccharide%Kukoamine B%Small intestine%Inflammatory response
目的：探讨苦柯胺B（KB）对脓毒症小鼠小肠炎症反应的抑制作用及其分子机制。方法按照随机数字表法将24只雄性ICR小鼠分为对照组、模型组、KB干预组，每组8只。腹腔注射脂多糖（LPS）20 mg/kg制备脓毒症动物模型，对照组注射等量生理盐水；KB干预组于制模后4 h经尾静脉注射KB 20μg/kg进行干预。各组于注射LPS后8 h取心脏血和空肠、回肠组织，检测血浆LPS含量；采用酶联免疫吸附试验（ELISA）检测血浆及小肠组织肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）水平；光镜下观察小肠组织病理学改变；比色法检测小肠组织髓过氧化物酶（MPO）活性；免疫组化法观察小肠组织细胞间黏附分子-1（ICAM-1）表达；反转录-聚合酶链反应（RT-PCR）检测小肠组织诱导型一氧化氮合酶（iNOS） mRNA表达；蛋白质免疫印迹试验（Western Blot）检测小肠组织核转录因子-κB（NF-κB）蛋白表达。结果模型小鼠表现为肠组织微血管通透性增加，间质水肿，白细胞浸润；血浆LPS、TNF-α、IL-1β水平及小肠TNF-α、IL-1β、 MPO活性、 ICAM-1阳性表达、 iNOS mRNA、NF-κB蛋白表达均明显升高；而经KB干预后，小肠组织微血管通透性降低，水肿程度减轻，白细胞浸润显著减少，血浆LPS、TNF-α、IL-1β及小肠TNF-α、IL-1β、MPO活性、ICAM-1阳性表达、 iNOS mRNA和NF-κB蛋白表达均较模型组均明显下降〔血浆LPS（kEU/L）：654.09±28.13比1155.65±47.15，TNF-α（ng/L）：12.75±0.47比30.61±0.71， IL-1β（ng/L）：53.06±5.32比64.47±2.61；空肠TNF-α（ng/L）：43.27±1.20比64.82±2.09，IL-1β（ng/L）：326.38±14.47比535.22±13.48， MPO（U/g）：0.14±0.01比0.32±0.02，iNOS mRNA（2-ΔΔCt）：2.39±0.13比10.80±0.22，NF-κB蛋白（灰度值）：0.687±0.062比1.404±0.046；回肠TNF-α（ng/L）：62.75±3.92比104.24±2.82， IL-1β（ng/L）：408.06±1.70比521.97±1.16， MPO（U/g）：0.36±0.08比0.66±0.05，iNOS mRNA（2-ΔΔCt）：1.65±0.11比3.59±0.29， NF-κB蛋白（灰度值）：0.830±0.114比1.609±0.051，均P＜0.05〕。结论 KB可以通过结合LPS，抑制LPS/Toll样受体4（TLR4）信号通路活化，从而显著抑制LPS诱导的脓毒症小鼠小肠炎症反应，保护小肠功能。
目的：探討苦柯胺B（KB）對膿毒癥小鼠小腸炎癥反應的抑製作用及其分子機製。方法按照隨機數字錶法將24隻雄性ICR小鼠分為對照組、模型組、KB榦預組，每組8隻。腹腔註射脂多糖（LPS）20 mg/kg製備膿毒癥動物模型，對照組註射等量生理鹽水；KB榦預組于製模後4 h經尾靜脈註射KB 20μg/kg進行榦預。各組于註射LPS後8 h取心髒血和空腸、迴腸組織，檢測血漿LPS含量；採用酶聯免疫吸附試驗（ELISA）檢測血漿及小腸組織腫瘤壞死因子-α（TNF-α）和白細胞介素-1β（IL-1β）水平；光鏡下觀察小腸組織病理學改變；比色法檢測小腸組織髓過氧化物酶（MPO）活性；免疫組化法觀察小腸組織細胞間黏附分子-1（ICAM-1）錶達；反轉錄-聚閤酶鏈反應（RT-PCR）檢測小腸組織誘導型一氧化氮閤酶（iNOS） mRNA錶達；蛋白質免疫印跡試驗（Western Blot）檢測小腸組織覈轉錄因子-κB（NF-κB）蛋白錶達。結果模型小鼠錶現為腸組織微血管通透性增加，間質水腫，白細胞浸潤；血漿LPS、TNF-α、IL-1β水平及小腸TNF-α、IL-1β、 MPO活性、 ICAM-1暘性錶達、 iNOS mRNA、NF-κB蛋白錶達均明顯升高；而經KB榦預後，小腸組織微血管通透性降低，水腫程度減輕，白細胞浸潤顯著減少，血漿LPS、TNF-α、IL-1β及小腸TNF-α、IL-1β、MPO活性、ICAM-1暘性錶達、 iNOS mRNA和NF-κB蛋白錶達均較模型組均明顯下降〔血漿LPS（kEU/L）：654.09±28.13比1155.65±47.15，TNF-α（ng/L）：12.75±0.47比30.61±0.71， IL-1β（ng/L）：53.06±5.32比64.47±2.61；空腸TNF-α（ng/L）：43.27±1.20比64.82±2.09，IL-1β（ng/L）：326.38±14.47比535.22±13.48， MPO（U/g）：0.14±0.01比0.32±0.02，iNOS mRNA（2-ΔΔCt）：2.39±0.13比10.80±0.22，NF-κB蛋白（灰度值）：0.687±0.062比1.404±0.046；迴腸TNF-α（ng/L）：62.75±3.92比104.24±2.82， IL-1β（ng/L）：408.06±1.70比521.97±1.16， MPO（U/g）：0.36±0.08比0.66±0.05，iNOS mRNA（2-ΔΔCt）：1.65±0.11比3.59±0.29， NF-κB蛋白（灰度值）：0.830±0.114比1.609±0.051，均P＜0.05〕。結論 KB可以通過結閤LPS，抑製LPS/Toll樣受體4（TLR4）信號通路活化，從而顯著抑製LPS誘導的膿毒癥小鼠小腸炎癥反應，保護小腸功能。
목적：탐토고가알B（KB）대농독증소서소장염증반응적억제작용급기분자궤제。방법안조수궤수자표법장24지웅성ICR소서분위대조조、모형조、KB간예조，매조8지。복강주사지다당（LPS）20 mg/kg제비농독증동물모형，대조조주사등량생리염수；KB간예조우제모후4 h경미정맥주사KB 20μg/kg진행간예。각조우주사LPS후8 h취심장혈화공장、회장조직，검측혈장LPS함량；채용매련면역흡부시험（ELISA）검측혈장급소장조직종류배사인자-α（TNF-α）화백세포개소-1β（IL-1β）수평；광경하관찰소장조직병이학개변；비색법검측소장조직수과양화물매（MPO）활성；면역조화법관찰소장조직세포간점부분자-1（ICAM-1）표체；반전록-취합매련반응（RT-PCR）검측소장조직유도형일양화담합매（iNOS） mRNA표체；단백질면역인적시험（Western Blot）검측소장조직핵전록인자-κB（NF-κB）단백표체。결과모형소서표현위장조직미혈관통투성증가，간질수종，백세포침윤；혈장LPS、TNF-α、IL-1β수평급소장TNF-α、IL-1β、 MPO활성、 ICAM-1양성표체、 iNOS mRNA、NF-κB단백표체균명현승고；이경KB간예후，소장조직미혈관통투성강저，수종정도감경，백세포침윤현저감소，혈장LPS、TNF-α、IL-1β급소장TNF-α、IL-1β、MPO활성、ICAM-1양성표체、 iNOS mRNA화NF-κB단백표체균교모형조균명현하강〔혈장LPS（kEU/L）：654.09±28.13비1155.65±47.15，TNF-α（ng/L）：12.75±0.47비30.61±0.71， IL-1β（ng/L）：53.06±5.32비64.47±2.61；공장TNF-α（ng/L）：43.27±1.20비64.82±2.09，IL-1β（ng/L）：326.38±14.47비535.22±13.48， MPO（U/g）：0.14±0.01비0.32±0.02，iNOS mRNA（2-ΔΔCt）：2.39±0.13비10.80±0.22，NF-κB단백（회도치）：0.687±0.062비1.404±0.046；회장TNF-α（ng/L）：62.75±3.92비104.24±2.82， IL-1β（ng/L）：408.06±1.70비521.97±1.16， MPO（U/g）：0.36±0.08비0.66±0.05，iNOS mRNA（2-ΔΔCt）：1.65±0.11비3.59±0.29， NF-κB단백（회도치）：0.830±0.114비1.609±0.051，균P＜0.05〕。결론 KB가이통과결합LPS，억제LPS/Toll양수체4（TLR4）신호통로활화，종이현저억제LPS유도적농독증소서소장염증반응，보호소장공능。
ObjectiveTo study the role of Kukoamine B (KB) in inhibiting the inflammatory response of small intestine in septic mice and its molecular mechanisms.Methods Twenty-four male ICR mice were randomly divided into control group, model group, and KB intervention group (each,n= 8). Sepsis model was reproduced by intra-peritoneal injection of 20 mg/kg lipopolysaccharide (LPS), while equivalent normal saline was given in control group, and 20μg/kg KB was injected through caudal vein 4 hours after LPS challenge in KB intervention group. The blood/tissue samples (jejunum and ileum) were harvested 8 hours after LPS injection. The levels of plasma LPS, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured. The pathological changes in small intestine tissues were observed under light microscope, while the levels of inflammatory cytokines TNF-α and IL-1β in the tissue homogenates (jejunum and ileum) were assessed by enzyme linked immunosorbent assay (ELISA). The activity of myeloperoxidase (MPO) was measured by colorimetry. The expression of intercellular adhesion molecule-1 (ICAM-1) was determined by immunohistochemistry. The expressions of inducible nitric oxide synthase (iNOS) mRNA was assayed by reverse transcription-polymerase chain reaction (RT-PCR). The activation of nuclear factor-κΒ (NF-κΒ) was determined by Western Blot.Results The mice in model group were found to have an increase in microvascular permeability, interstitial edema, and infiltration of white blood cells, and the levels of LPS, TNF-α and IL-1β in their plasma, with an increase in concentrations of TNF-α and IL-1β, activity of MPO, positive expression of ICAM-1, expression of iNOS mRNA and NF-κB protein in small intestine (jejunum and ileum). Compared with model group, in mice with KB intervention, microvascular permeability, interstitial edema, and infiltration of white blood cells were reduced significantly, while the levels of LPS, TNF-α and IL-1β in plasma, the concentration of TNF-α and IL-1β, the activity of MPO, the positive expression of ICAM-1, the expression of iNOS mRNA and NF-κB protein in small intestine (jejunum and ileum) were significantly decreased [plasma LPS (kEU/L): 654.09±28.13 vs. 1 155.65±47.15, TNF-α (ng/L): 12.75±0.47 vs. 30.61±0.71, IL-1β (ng/L): 53.06±5.32 vs. 64.47±2.61; jejunum TNF-α(ng/L): 43.27±1.20 vs. 64.82±2.09, IL-1β (ng/L): 326.38±14.47 vs. 535.22±13.48, MPO (U/g): 0.14±0.01 vs. 0.32±0.02, iNOS mRNA (2-ΔΔCt): 2.39±0.13 vs. 10.80±0.22, NF-κB protein (gray value): 0.687±0.062 vs. 1.404±0.046; ileum TNF-α (ng/L): 62.75±3.92 vs. 104.24±2.82, IL-1β(ng/L): 408.06±1.70 vs. 521.97±1.16, MPO (U/g): 0.36±0.08 vs. 0.66±0.05, iNOS mRNA (2-ΔΔCt): 1.65±0.11 vs. 3.59±0.29, NF-κB protein (gray value):0.830±0.114 vs. 1.609±0.051, allP< 0.05].Conclusion KB can combine with LPS and inhibit LPS/Toll-like receptor 4 (TLR4) signaling pathway, thereby significantly inhibit the inflammatory response and protect the function of the small intestine in LPS-induced septic mice.
