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
1期
17-22
,共6页
张立亚%崔尧丽%王兵%于金宝%王林林%王玉亮%王勇强
張立亞%崔堯麗%王兵%于金寶%王林林%王玉亮%王勇彊
장립아%최요려%왕병%우금보%왕림림%왕옥량%왕용강
创伤%休克,失血性%肝损伤%程序性坏死特异性抑制剂-1%缺血/再灌注损伤
創傷%休剋,失血性%肝損傷%程序性壞死特異性抑製劑-1%缺血/再灌註損傷
창상%휴극,실혈성%간손상%정서성배사특이성억제제-1%결혈/재관주손상
Trauma%Hemorrhagic shock%Liver injury%Necrostatin-1%Ischemia/reperfusion injury
目的 探讨程序性坏死特异性抑制剂-1(Nec-1)对创伤失血性休克大鼠的肝脏保护作用.方法 采用左下肢股骨、胫骨骨折及腹部软组织损伤并失血/再灌注的方法制备大鼠创伤失血性休克模型.选择雄性SD大鼠,22只按随机数字表法分为模型组和Nec-1组,每组11只,观察72 h死亡率.72只同法随机分为假手术组、模型组、Nec-1组,每组24只.假手术组仅麻醉和分离、结扎血管,不进行创伤、失血、再灌注;Nec-1组于再灌注前5 min经股静脉给予1 mg/kg Nec-1;模型组给予等体积溶剂.于再灌注后2、4、8h采集各组血清和肝组织,用全自动生化仪检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)的水平;光镜下观察肝组织病理学改变;采用逆转录-聚合酶链反应(RT-PCR)检测肝组织肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的mRNA表达;蛋白质免疫印迹试验(Western Blot)检测肝组织受体相互作用蛋白酶-1/3(RIP1/RIP3)的蛋白表达.结果 Nec-1组大鼠72 h死亡率较模型组明显降低[18.18％(2/11)比63.64％(7/11),P=0.040].模型组2h血清ALT、AST即较假手术组明显升高[ALT(U/L):110.21 ±22.32比80.98±19.94,AST (U/L):364.29±64.83比279.76±70.64,均P＜0.05],8h达高峰[ALT(U/L):387.41±47.11比82.76±22.44,AST(U/L):973.35±77.51比261.49±52.03,均P＜0.01].Nec-1组血清ALT、AST水平较模型组明显降低[ALT(U/L)4 h:144.64±33.79比213.96±36.21,8 h:159.48±43.57比387.41 ±47.11;AST(U/L)4 h:398.78±59.48比630.61±59.93,8 h:427.38±80.75比973.35±77.51,均P＜0.01].光镜下模型组大鼠肝窦扩张、淤血,肝细胞变性、坏死,大量炎性细胞浸润;Nec-1组肝组织损伤程度明显减轻.模型组肝组织TNF-α、IL-1β的mRNA表达和RIP1、RIP3的蛋白表达随时间延长呈逐渐升高趋势;给予Nec-1后各时间点TNF-α、IL-1β的mRNA表达均较模型组明显降低,以8h最为明显(TNF-α mRNA:1.457±0.081比2.317-0.062,IL-1β mRNA:0.690-0.087比1.812±0.112,均P＜0.01),而肝组织RIP1、RIP3的蛋白表达与模型组相比差异无统计学意义(RIP1蛋白8 h:0.561±0.033比0.587±0.036,RIP3蛋白8 h:0.976±0.040比1.044±0.115,均P＞0.05).结论 Nec-1对创伤失血性休克大鼠肝脏具有明显的保护作用,具体机制需进一步深入研究.
目的 探討程序性壞死特異性抑製劑-1(Nec-1)對創傷失血性休剋大鼠的肝髒保護作用.方法 採用左下肢股骨、脛骨骨摺及腹部軟組織損傷併失血/再灌註的方法製備大鼠創傷失血性休剋模型.選擇雄性SD大鼠,22隻按隨機數字錶法分為模型組和Nec-1組,每組11隻,觀察72 h死亡率.72隻同法隨機分為假手術組、模型組、Nec-1組,每組24隻.假手術組僅痳醉和分離、結扎血管,不進行創傷、失血、再灌註;Nec-1組于再灌註前5 min經股靜脈給予1 mg/kg Nec-1;模型組給予等體積溶劑.于再灌註後2、4、8h採集各組血清和肝組織,用全自動生化儀檢測血清丙氨痠轉氨酶(ALT)、天鼕氨痠轉氨酶(AST)的水平;光鏡下觀察肝組織病理學改變;採用逆轉錄-聚閤酶鏈反應(RT-PCR)檢測肝組織腫瘤壞死因子-α(TNF-α)和白細胞介素-1β(IL-1β)的mRNA錶達;蛋白質免疫印跡試驗(Western Blot)檢測肝組織受體相互作用蛋白酶-1/3(RIP1/RIP3)的蛋白錶達.結果 Nec-1組大鼠72 h死亡率較模型組明顯降低[18.18％(2/11)比63.64％(7/11),P=0.040].模型組2h血清ALT、AST即較假手術組明顯升高[ALT(U/L):110.21 ±22.32比80.98±19.94,AST (U/L):364.29±64.83比279.76±70.64,均P＜0.05],8h達高峰[ALT(U/L):387.41±47.11比82.76±22.44,AST(U/L):973.35±77.51比261.49±52.03,均P＜0.01].Nec-1組血清ALT、AST水平較模型組明顯降低[ALT(U/L)4 h:144.64±33.79比213.96±36.21,8 h:159.48±43.57比387.41 ±47.11;AST(U/L)4 h:398.78±59.48比630.61±59.93,8 h:427.38±80.75比973.35±77.51,均P＜0.01].光鏡下模型組大鼠肝竇擴張、淤血,肝細胞變性、壞死,大量炎性細胞浸潤;Nec-1組肝組織損傷程度明顯減輕.模型組肝組織TNF-α、IL-1β的mRNA錶達和RIP1、RIP3的蛋白錶達隨時間延長呈逐漸升高趨勢;給予Nec-1後各時間點TNF-α、IL-1β的mRNA錶達均較模型組明顯降低,以8h最為明顯(TNF-α mRNA:1.457±0.081比2.317-0.062,IL-1β mRNA:0.690-0.087比1.812±0.112,均P＜0.01),而肝組織RIP1、RIP3的蛋白錶達與模型組相比差異無統計學意義(RIP1蛋白8 h:0.561±0.033比0.587±0.036,RIP3蛋白8 h:0.976±0.040比1.044±0.115,均P＞0.05).結論 Nec-1對創傷失血性休剋大鼠肝髒具有明顯的保護作用,具體機製需進一步深入研究.
목적 탐토정서성배사특이성억제제-1(Nec-1)대창상실혈성휴극대서적간장보호작용.방법 채용좌하지고골、경골골절급복부연조직손상병실혈/재관주적방법제비대서창상실혈성휴극모형.선택웅성SD대서,22지안수궤수자표법분위모형조화Nec-1조,매조11지,관찰72 h사망솔.72지동법수궤분위가수술조、모형조、Nec-1조,매조24지.가수술조부마취화분리、결찰혈관,불진행창상、실혈、재관주;Nec-1조우재관주전5 min경고정맥급여1 mg/kg Nec-1;모형조급여등체적용제.우재관주후2、4、8h채집각조혈청화간조직,용전자동생화의검측혈청병안산전안매(ALT)、천동안산전안매(AST)적수평;광경하관찰간조직병이학개변;채용역전록-취합매련반응(RT-PCR)검측간조직종류배사인자-α(TNF-α)화백세포개소-1β(IL-1β)적mRNA표체;단백질면역인적시험(Western Blot)검측간조직수체상호작용단백매-1/3(RIP1/RIP3)적단백표체.결과 Nec-1조대서72 h사망솔교모형조명현강저[18.18％(2/11)비63.64％(7/11),P=0.040].모형조2h혈청ALT、AST즉교가수술조명현승고[ALT(U/L):110.21 ±22.32비80.98±19.94,AST (U/L):364.29±64.83비279.76±70.64,균P＜0.05],8h체고봉[ALT(U/L):387.41±47.11비82.76±22.44,AST(U/L):973.35±77.51비261.49±52.03,균P＜0.01].Nec-1조혈청ALT、AST수평교모형조명현강저[ALT(U/L)4 h:144.64±33.79비213.96±36.21,8 h:159.48±43.57비387.41 ±47.11;AST(U/L)4 h:398.78±59.48비630.61±59.93,8 h:427.38±80.75비973.35±77.51,균P＜0.01].광경하모형조대서간두확장、어혈,간세포변성、배사,대량염성세포침윤;Nec-1조간조직손상정도명현감경.모형조간조직TNF-α、IL-1β적mRNA표체화RIP1、RIP3적단백표체수시간연장정축점승고추세;급여Nec-1후각시간점TNF-α、IL-1β적mRNA표체균교모형조명현강저,이8h최위명현(TNF-α mRNA:1.457±0.081비2.317-0.062,IL-1β mRNA:0.690-0.087비1.812±0.112,균P＜0.01),이간조직RIP1、RIP3적단백표체여모형조상비차이무통계학의의(RIP1단백8 h:0.561±0.033비0.587±0.036,RIP3단백8 h:0.976±0.040비1.044±0.115,균P＞0.05).결론 Nec-1대창상실혈성휴극대서간장구유명현적보호작용,구체궤제수진일보심입연구.
Objective To investigate the effects of necrostatin-1 (Nec-1) on the liver of rats with trauma induced hemorrhagic shock.Methods Trauma induced hemorrhagic shock model was produced by adopting the left femur,tibia fracture and soft tissue injury,bleeding and reperfusion in male Sprague-Dawley (SD) rats.A total of 22 rats were divided into model group and Nec-1 group with 11 rats in each group by randomized digital number method and the 72-hour mortality was observed.In addition,72 rats were randomly divided into sham group,model group,Nec-1 group with 24 rats in each group.Rats in sham group were only received anesthesia,separating and ligating blood vessels,without trauma induced hemorrhagic and reperfusion,and the rats in Nec-1 group were received 1 mg/kg Nec-1 through femoral vein 5 minutes before reperfusion,while the rats in model group were received the same amount of solvent.The serum and liver tissues of each group were collected at 2,4,8 hours after reperfusion.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by automatic biochemistry analyzer.The pathology changes in liver were observed by hematoxylin-eosin (HE) staining.The mRNA expressions of tumor necrosis factor-oα (TNF-α) and interleukin-1β (IL-1β) in the liver were detcrmined by reverse transcription-polymerase chain reaction (RT-PCR).The protein expressions of receptor interaction of protease 1/3 (RIP1/RIP3) were also assessed by Western Blot analysis.Results Compared with model group,Nec-1 significantly reduced the 72-hour mortality [18.18％ (2/11) vs.63.64％ (7/11),P=0.040].Two hours after trauma induced hemorrhagic shock and reperfusion,the expressions of ALT and AST in model group were significantly increased compared with those in sham group [ALT (U/L):110.21 ±22.32 vs.80.98 ± 19.94,AST (U/L):364.29 ±64.83 vs.279.76 ±70.64,both P＜0.05],and reached the peak at 8 hours [ALT (U/L):387.41 ± 47.11 vs.82.76 ± 22.44,AST (U/L):973.35 ± 77.51 vs.261.49 ±52.03,both P＜0.01].Levels of serum ALT and AST in Nec-1 group were significantly decreased compared with model group [ALT (U/L) 4 hours:144.64± 33.79 vs.213.96± 36.21,8 hours:159.48 ± 43.57 vs.387.41 ± 47.11; AST (U/L) 4 hours:398.78 ± 59.48 vs.630.61 ± 59.93,8 hours:427.38 ± 80.75 vs.973.35 ± 77.51,all P＜0.01].Under light microscopy,it was noted that the hepatic sinus expansion,liver cells degeneration,necrosis,as well as infiltration of abundant inflammatory cells were observed.But the pathology changes in hepatic tissues were significantly mitigated in Nec-1 group.Along with the time extension,the mRNA expressions of TNF-α and IL-1β and the protein expressions of RIP1 and RIP3 were markedly up-regulated.Compared with model group,difference in the mRNA expressions of TNF-α and IL-1β in hepatic tissues in Nec-1 group were statistically significant,and the most obvious difference was at 8 hours [TNF-α mRNA:1.457 ± 0.081 vs.2.317 ± 0.062,IL-1β mRNA:0.690 ± 0.087 vs.1.812 ± 0.112,both P＜0.01].But there was no statistically significant difference in RIP1 and RIP3 between Nec-1 group and model group [RIP1 protein 8 hours:0.561 ± 0.033 vs.0.587 ± 0.036,RIP3 protein 8 hours:0.976 ± 0.040 vs.1.044 ± 0.115,both P＞0.05].Conclusion Nec-1 may be remarkable protect effect on the liver of rats with trauma induced hemorrhage shock and reperfusion,and the intrinsic mechanisms need further investigation.
