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
11期
810-814
,共5页
穆恩%丁仁彧%安欣%李鑫%陈松%马晓春
穆恩%丁仁彧%安訢%李鑫%陳鬆%馬曉春
목은%정인욱%안흔%리흠%진송%마효춘
肝素%一氧化氮合酶%转化生长因子-β/Smad信号转导途径%急性肺损伤%急性呼吸窘迫综合征
肝素%一氧化氮閤酶%轉化生長因子-β/Smad信號轉導途徑%急性肺損傷%急性呼吸窘迫綜閤徵
간소%일양화담합매%전화생장인자-β/Smad신호전도도경%급성폐손상%급성호흡군박종합정
Heparin%Nitric oxide synthase%Transforming growth factor-β/Smad signaling pathway%Acute lung injury%Acute respiratory distress syndrome
目的：探讨肝素对脂多糖（LPS）致急性肺损伤（ALI）的保护作用及可能机制。方法32只SD大鼠按随机数字表法分为对照组、肝素对照组、模型组和肝素治疗组，每组8只。采用气管内滴入LPS 1 mg/kg的方法制备大鼠ALI模型，对照组和肝素对照组滴入等量生理盐水；肝素对照组和肝素治疗组于制模后每小时静脉注射肝素50 U/kg。24 h后各组大鼠进行肺泡灌洗，采用酶联免疫吸附试验（ELISA）检测支气管肺泡灌洗液（BALF）中炎症介质表达；取肺组织，测定肺湿/干质量（W/D）比值，光镜下观察肺组织病理改变，并检测其丙二醛（MDA）、一氧化氮（NO）和髓过氧化物酶（MPO）水平；反转录-聚合酶链反应（RT-PCR）检测肺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）检测肺组织iNOS、转化生长因子-β1（TGF-β1）和磷酸化Smad表达；免疫组化法检测肺组织iNOS表达。结果光镜下观察对照组和肝素对照组肺组织结构完整、肺泡腔清晰；模型组肺泡壁增厚，有明显的炎性细胞浸润、肺泡出血和结构破坏；肝素治疗组病理改变较模型组明显减轻。与对照组和肝素对照组比较，模型组肺W/D比值，肺组织MDA、NO、MPO，BALF中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平均明显升高。与模型组比较，肝素治疗组肺W/D比值，肺组织MDA、NO、MPO，BALF中TNF-α和IL-6水平均明显降低〔W/D比值：7.54±0.17比10.69±0.15，MDA（mmol/mg）：2.01±0.30比2.51±0.25，NO（μmol/L）：3.07±0.21比3.89±0.14，MPO （U/g）：1.94±0.09比2.74±0.20，TNF-α（μg/L）：201.80±0.27比297.53±0.34，IL-6（μg/L）：38.41±0.25比46.31±0.31，均P＜0.05〕。RT-PCR结果显示，肝素治疗组iNOS mRNA表达明显低于模型组（2-ΔΔCt：3.04±0.18比4.37±0.15，P＜0.05）。Western Blot检测结果显示，与对照组比较，模型组肺组织iNOS、TGF-β1蛋白表达及Smad2、Smad3磷酸化水平明显增加；而肝素治疗组蛋白表达较模型组明显受到抑制。免疫组化结果显示，肝素治疗组肺泡上皮细胞和微血管内皮细胞iNOS阳性细胞表达较模型组明显减少。结论肝素能通过抑制一氧化氮合酶的表达和TGF-β/Smad信号转导途径来发挥对LPS致大鼠ALI的保护作用。
目的：探討肝素對脂多糖（LPS）緻急性肺損傷（ALI）的保護作用及可能機製。方法32隻SD大鼠按隨機數字錶法分為對照組、肝素對照組、模型組和肝素治療組，每組8隻。採用氣管內滴入LPS 1 mg/kg的方法製備大鼠ALI模型，對照組和肝素對照組滴入等量生理鹽水；肝素對照組和肝素治療組于製模後每小時靜脈註射肝素50 U/kg。24 h後各組大鼠進行肺泡灌洗，採用酶聯免疫吸附試驗（ELISA）檢測支氣管肺泡灌洗液（BALF）中炎癥介質錶達；取肺組織，測定肺濕/榦質量（W/D）比值，光鏡下觀察肺組織病理改變，併檢測其丙二醛（MDA）、一氧化氮（NO）和髓過氧化物酶（MPO）水平；反轉錄-聚閤酶鏈反應（RT-PCR）檢測肺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）檢測肺組織iNOS、轉化生長因子-β1（TGF-β1）和燐痠化Smad錶達；免疫組化法檢測肺組織iNOS錶達。結果光鏡下觀察對照組和肝素對照組肺組織結構完整、肺泡腔清晰；模型組肺泡壁增厚，有明顯的炎性細胞浸潤、肺泡齣血和結構破壞；肝素治療組病理改變較模型組明顯減輕。與對照組和肝素對照組比較，模型組肺W/D比值，肺組織MDA、NO、MPO，BALF中腫瘤壞死因子-α（TNF-α）和白細胞介素-6（IL-6）水平均明顯升高。與模型組比較，肝素治療組肺W/D比值，肺組織MDA、NO、MPO，BALF中TNF-α和IL-6水平均明顯降低〔W/D比值：7.54±0.17比10.69±0.15，MDA（mmol/mg）：2.01±0.30比2.51±0.25，NO（μmol/L）：3.07±0.21比3.89±0.14，MPO （U/g）：1.94±0.09比2.74±0.20，TNF-α（μg/L）：201.80±0.27比297.53±0.34，IL-6（μg/L）：38.41±0.25比46.31±0.31，均P＜0.05〕。RT-PCR結果顯示，肝素治療組iNOS mRNA錶達明顯低于模型組（2-ΔΔCt：3.04±0.18比4.37±0.15，P＜0.05）。Western Blot檢測結果顯示，與對照組比較，模型組肺組織iNOS、TGF-β1蛋白錶達及Smad2、Smad3燐痠化水平明顯增加；而肝素治療組蛋白錶達較模型組明顯受到抑製。免疫組化結果顯示，肝素治療組肺泡上皮細胞和微血管內皮細胞iNOS暘性細胞錶達較模型組明顯減少。結論肝素能通過抑製一氧化氮閤酶的錶達和TGF-β/Smad信號轉導途徑來髮揮對LPS緻大鼠ALI的保護作用。
목적：탐토간소대지다당（LPS）치급성폐손상（ALI）적보호작용급가능궤제。방법32지SD대서안수궤수자표법분위대조조、간소대조조、모형조화간소치료조，매조8지。채용기관내적입LPS 1 mg/kg적방법제비대서ALI모형，대조조화간소대조조적입등량생리염수；간소대조조화간소치료조우제모후매소시정맥주사간소50 U/kg。24 h후각조대서진행폐포관세，채용매련면역흡부시험（ELISA）검측지기관폐포관세액（BALF）중염증개질표체；취폐조직，측정폐습/간질량（W/D）비치，광경하관찰폐조직병리개변，병검측기병이철（MDA）、일양화담（NO）화수과양화물매（MPO）수평；반전록-취합매련반응（RT-PCR）검측폐조직유도형일양화담합매（iNOS）mRNA표체；단백질면역인적시험（Western Blot）검측폐조직iNOS、전화생장인자-β1（TGF-β1）화린산화Smad표체；면역조화법검측폐조직iNOS표체。결과광경하관찰대조조화간소대조조폐조직결구완정、폐포강청석；모형조폐포벽증후，유명현적염성세포침윤、폐포출혈화결구파배；간소치료조병리개변교모형조명현감경。여대조조화간소대조조비교，모형조폐W/D비치，폐조직MDA、NO、MPO，BALF중종류배사인자-α（TNF-α）화백세포개소-6（IL-6）수평균명현승고。여모형조비교，간소치료조폐W/D비치，폐조직MDA、NO、MPO，BALF중TNF-α화IL-6수평균명현강저〔W/D비치：7.54±0.17비10.69±0.15，MDA（mmol/mg）：2.01±0.30비2.51±0.25，NO（μmol/L）：3.07±0.21비3.89±0.14，MPO （U/g）：1.94±0.09비2.74±0.20，TNF-α（μg/L）：201.80±0.27비297.53±0.34，IL-6（μg/L）：38.41±0.25비46.31±0.31，균P＜0.05〕。RT-PCR결과현시，간소치료조iNOS mRNA표체명현저우모형조（2-ΔΔCt：3.04±0.18비4.37±0.15，P＜0.05）。Western Blot검측결과현시，여대조조비교，모형조폐조직iNOS、TGF-β1단백표체급Smad2、Smad3린산화수평명현증가；이간소치료조단백표체교모형조명현수도억제。면역조화결과현시，간소치료조폐포상피세포화미혈관내피세포iNOS양성세포표체교모형조명현감소。결론간소능통과억제일양화담합매적표체화TGF-β/Smad신호전도도경래발휘대LPS치대서ALI적보호작용。
Objective To investigate whether heparin has a beneficial effect on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in rats,and to explore the possible underlying mechanisms. Methods Thirty-two adult Sprague-Dawley(SD)rats were randomly assigned into the control,heparin control,model,and heparin treatment groups,with 8 in each group. ALI rat model was reproduced by intratracheal instillation of LPS at a dose of 1 mg/kg. The rats in the control and heparin control groups received an equal volume of normal saline at the same times. The rats in the heparin control and heparin treatment groups were intravenously received 50 U/kg heparin every 1 hour after the induction of ALI. Animals were sacrificed 24 hours after LPS challenge. Bronchoalveolar lavage fluid(BALF) and lung tissue samples were collected. Histopathological evaluation,lung wet/dry(W/D)ratio,malondialdehyde (MDA),nitric oxide(NO)and myeloperoxidase(MPO)were analyzed. Enzyme-linked immunosorbent assay(ELISA) was used to measure the concentration of inflammatory factor in BALF. Expression of inducible nitric oxide synthase (iNOS)mRNA in the lung of rats was measured by reverse transcription-polymerase chain reaction(RT-PCR). Western Blot was used to determine the expression of transforming growth factor-β1(TGF-β1)and phosphorylation of Smad in the lung tissues. The expression of iNOS in lung was determined by immunohistochemistry. Results In the control and heparin control groups,lung tissue showed a normal structure and clear pulmonary alveoli under a light microscope. In the model group,ALI characters such as extensive thickening of the alveolar wall,significant infiltration of inflammatory cells,demolished structure of pulmonary alveoli,and hemorrhage were found. In the heparin treatment group,heparin treatment markedly alleviated LPS-induced these pathological changes in lung. Compared with control and heparin control groups,lung W/D ratio,lung MDA,NO and MPO levels,and tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6)in BALF in the model group were increased significantly. Compared with the model group, lung W/D ratio,lung MDA,NO and MPO levels,and TNF-αand IL-6 in BALF in the heparin treatment group were significantly decreased〔W/D ratio:7.54±0.17 vs. 10.69±0.15,MDA(mmol/mg):2.01±0.30 vs. 2.51±0.25,NO (μmol/L):3.07±0.21 vs. 3.89±0.14,MPO(U/g):1.94±0.09 vs. 2.74±0.20,TNF-α(μg/L):201.80±0.27 vs. 297.53±0.34,IL-6(μg/L):38.41±0.25 vs. 46.31±0.31,all P<0.05〕. RT-PCR showed that the expression of iNOS mRNA in the heparin treatment group was significantly lower than that in the model group(2-ΔΔCt:3.04±0.18 vs. 4.37±0.15,P<0.05). Western Blot showed that compared with control group,the protein expressions of iNOS and TGF-β1,and phosphorylation of Smad2 and Smad3 were significantly increased,and the heparin could inhibit the protein expressions compared with model group. Immunohistochemistry showed that positive expressions of iNOS in alveolar epithelial cell and capillary endothelial cell in the heparin treatment group were significantly lower than those in the model group. Conclusion Heparin significantly ameliorated the lung injury induced by LPS in rats via the inhibition of nitric oxide synthase expression and the TGF-β/Smad pathway.
