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
8期
643-648
,共6页
陈怿%童华生%潘志国%陈玉兰%林幼萍%江东新%苏磊
陳懌%童華生%潘誌國%陳玉蘭%林幼萍%江東新%囌磊
진역%동화생%반지국%진옥란%림유평%강동신%소뢰
中暑%血必净注射液%内毒素血症%炎症反应%肠损伤
中暑%血必淨註射液%內毒素血癥%炎癥反應%腸損傷
중서%혈필정주사액%내독소혈증%염증반응%장손상
Heat-stroke%Xuebijing injection%Endotoxemia%Inflammation%Intestinal injury
目的：观察血必净注射液预处理对重症中暑大鼠炎症反应的影响，并从减轻小肠损伤方面探讨其可能机制。方法 SPF级健康成年雄性Wistar大鼠36只，按随机数字表法分为假手术组、重症中暑模型组和血必净预处理组（血必净组），每组12只。将大鼠置于人工气候舱内〔温度（40±2）℃，湿度（65±5）%〕制备经典中暑模型，热应激时间为60 min；假手术组大鼠置于25℃室温下观察。于实验开始时及热应激后取股动脉血，采用酶联免疫吸附试验（ELISA）检测血中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-1β、IL-6）和脂多糖（LPS）浓度。实验结束后开腹获取小肠组织，苏木素-伊红（HE）染色，光镜下观察组织病理学改变并计算小肠损伤病理评分；免疫组化法测定小肠组织诱导型一氧化氮合酶（iNOS）表达；原位末端缺刻标记法（TUNEL）检测小肠组织细胞凋亡；蛋白质免疫印迹试验（Western Blot）检测小肠组织紧密连接occludin蛋白表达。结果热应激后，模型组大鼠血中TNF-α、IL-1β、IL-6和LPS水平明显高于假手术组〔TNF-α（μg/L）：443.00±110.10比98.36±44.61，IL-1β（μg/L）：436.37±163.64比64.24±16.15，IL-6（μg/L）：342.70±92.42比54.40±13.22，LPS（μg/L）：0.68±0.22比0.09±0.02，均P＜0.01〕；而血必净组上述各指标均较模型组明显降低〔TNF-α（μg/L）：340.45±68.57比443.00±110.10，IL-1β（μg/L）：191.33±82.78比436.37±163.64， IL-6（μg/L）：192.21±37.89比342.70±92.42，LPS（μg/L）：0.43±0.17比0.68±0.22，均P＜0.01〕。模型组小肠可出现炎性细胞浸润、肠黏膜坏死、出血等病理改变；血必净组病理改变减轻，且损伤病理评分明显低于模型组（分：2.10±1.15比3.20±0.67，P＜0.01）。模型组小肠组织iNOS表达及细胞凋亡较假手术组明显增加；而血必净组各指标则均较模型组明显减轻〔iNOS（校正A值）：0.32±0.15比0.74±0.17，细胞凋亡指数：0.23±0.08比0.56±0.07，均P＜0.01〕。假手术组小肠occludin蛋白表达量最高，血必净组次之，模型组最低（A值分别为0.96±0.25、0.62±0.20、0.33±0.11），且模型组与血必净组和假手术组比较差异均有统计学意义（均P＜0.01）。结论血必净注射液具有减轻重症中暑大鼠炎症反应及内毒素血症的作用，其机制可能与改善小肠组织的氧化损伤、细胞凋亡和紧密连接ocdludin蛋白表达异常有关。
目的：觀察血必淨註射液預處理對重癥中暑大鼠炎癥反應的影響，併從減輕小腸損傷方麵探討其可能機製。方法 SPF級健康成年雄性Wistar大鼠36隻，按隨機數字錶法分為假手術組、重癥中暑模型組和血必淨預處理組（血必淨組），每組12隻。將大鼠置于人工氣候艙內〔溫度（40±2）℃，濕度（65±5）%〕製備經典中暑模型，熱應激時間為60 min；假手術組大鼠置于25℃室溫下觀察。于實驗開始時及熱應激後取股動脈血，採用酶聯免疫吸附試驗（ELISA）檢測血中腫瘤壞死因子-α（TNF-α）、白細胞介素（IL-1β、IL-6）和脂多糖（LPS）濃度。實驗結束後開腹穫取小腸組織，囌木素-伊紅（HE）染色，光鏡下觀察組織病理學改變併計算小腸損傷病理評分；免疫組化法測定小腸組織誘導型一氧化氮閤酶（iNOS）錶達；原位末耑缺刻標記法（TUNEL）檢測小腸組織細胞凋亡；蛋白質免疫印跡試驗（Western Blot）檢測小腸組織緊密連接occludin蛋白錶達。結果熱應激後，模型組大鼠血中TNF-α、IL-1β、IL-6和LPS水平明顯高于假手術組〔TNF-α（μg/L）：443.00±110.10比98.36±44.61，IL-1β（μg/L）：436.37±163.64比64.24±16.15，IL-6（μg/L）：342.70±92.42比54.40±13.22，LPS（μg/L）：0.68±0.22比0.09±0.02，均P＜0.01〕；而血必淨組上述各指標均較模型組明顯降低〔TNF-α（μg/L）：340.45±68.57比443.00±110.10，IL-1β（μg/L）：191.33±82.78比436.37±163.64， IL-6（μg/L）：192.21±37.89比342.70±92.42，LPS（μg/L）：0.43±0.17比0.68±0.22，均P＜0.01〕。模型組小腸可齣現炎性細胞浸潤、腸黏膜壞死、齣血等病理改變；血必淨組病理改變減輕，且損傷病理評分明顯低于模型組（分：2.10±1.15比3.20±0.67，P＜0.01）。模型組小腸組織iNOS錶達及細胞凋亡較假手術組明顯增加；而血必淨組各指標則均較模型組明顯減輕〔iNOS（校正A值）：0.32±0.15比0.74±0.17，細胞凋亡指數：0.23±0.08比0.56±0.07，均P＜0.01〕。假手術組小腸occludin蛋白錶達量最高，血必淨組次之，模型組最低（A值分彆為0.96±0.25、0.62±0.20、0.33±0.11），且模型組與血必淨組和假手術組比較差異均有統計學意義（均P＜0.01）。結論血必淨註射液具有減輕重癥中暑大鼠炎癥反應及內毒素血癥的作用，其機製可能與改善小腸組織的氧化損傷、細胞凋亡和緊密連接ocdludin蛋白錶達異常有關。
목적：관찰혈필정주사액예처리대중증중서대서염증반응적영향，병종감경소장손상방면탐토기가능궤제。방법 SPF급건강성년웅성Wistar대서36지，안수궤수자표법분위가수술조、중증중서모형조화혈필정예처리조（혈필정조），매조12지。장대서치우인공기후창내〔온도（40±2）℃，습도（65±5）%〕제비경전중서모형，열응격시간위60 min；가수술조대서치우25℃실온하관찰。우실험개시시급열응격후취고동맥혈，채용매련면역흡부시험（ELISA）검측혈중종류배사인자-α（TNF-α）、백세포개소（IL-1β、IL-6）화지다당（LPS）농도。실험결속후개복획취소장조직，소목소-이홍（HE）염색，광경하관찰조직병이학개변병계산소장손상병리평분；면역조화법측정소장조직유도형일양화담합매（iNOS）표체；원위말단결각표기법（TUNEL）검측소장조직세포조망；단백질면역인적시험（Western Blot）검측소장조직긴밀련접occludin단백표체。결과열응격후，모형조대서혈중TNF-α、IL-1β、IL-6화LPS수평명현고우가수술조〔TNF-α（μg/L）：443.00±110.10비98.36±44.61，IL-1β（μg/L）：436.37±163.64비64.24±16.15，IL-6（μg/L）：342.70±92.42비54.40±13.22，LPS（μg/L）：0.68±0.22비0.09±0.02，균P＜0.01〕；이혈필정조상술각지표균교모형조명현강저〔TNF-α（μg/L）：340.45±68.57비443.00±110.10，IL-1β（μg/L）：191.33±82.78비436.37±163.64， IL-6（μg/L）：192.21±37.89비342.70±92.42，LPS（μg/L）：0.43±0.17비0.68±0.22，균P＜0.01〕。모형조소장가출현염성세포침윤、장점막배사、출혈등병리개변；혈필정조병리개변감경，차손상병리평분명현저우모형조（분：2.10±1.15비3.20±0.67，P＜0.01）。모형조소장조직iNOS표체급세포조망교가수술조명현증가；이혈필정조각지표칙균교모형조명현감경〔iNOS（교정A치）：0.32±0.15비0.74±0.17，세포조망지수：0.23±0.08비0.56±0.07，균P＜0.01〕。가수술조소장occludin단백표체량최고，혈필정조차지，모형조최저（A치분별위0.96±0.25、0.62±0.20、0.33±0.11），차모형조여혈필정조화가수술조비교차이균유통계학의의（균P＜0.01）。결론혈필정주사액구유감경중증중서대서염증반응급내독소혈증적작용，기궤제가능여개선소장조직적양화손상、세포조망화긴밀련접ocdludin단백표체이상유관。
ObjectiveTo observe the effect of Xuebijing injection pretreatment on systemic inflammatory response induced by severe heat-stroke, and to investigate the mechanism of alleviation of intestinal injury in rats. Methods Thirty-six healthy adult male Wistar rats with grade SPF were randomly assigned into three groups with randomized number method, namely sham group, severe heat-stroke model group, and Xuebijing pretreatment group (XBJ group), with 12 rats in each group. The animals were placed in a pre-warm chamber [temperature (40±2)℃, humidity (65±5)%] in order to induce typical heat-stroke. The duration of heat-stress was 60 minutes, while the animals in sham group were exposed to ambient temperature of 25℃. Arterial blood samples were collected at the beginning and the end of heat-stress, the concentrations of tumor necrosis factor-α(TNF-α), interleukins (IL-1β, IL-6), and lipopolysaccharide (LPS) in peripheral blood were determined by enzyme linked immunosorbent assay (ELISA). The intestinal tissues were harvested after heat-stress, and the pathological changes in intestine tissues were observed after hematoxylin-eosin (HE) staining and under optical microscope. The pathological injury scores were calculated. Immunohistochemistry was performed to determine inducible nitric oxide synthase (iNOS) expression in intestinal tissue. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Western Blot was used to measure the tight junction protein occludin expression.Results The concentrations of TNF-α, IL-1β, IL-6 and LPS in blood of the rats after heat-stress in model group were significantly higher than those of sham group [TNF-α (μg/L): 443.00±110.10 vs. 98.36±44.61, IL-1β (μg/L): 436.37±163.64 vs. 64.24±16.15, IL-6 (μg/L): 342.70±92.42 vs. 54.40±13.22, LPS (μg/L): 0.68±0.22 vs. 0.09±0.02, allP< 0.01], but the levels of these parameters in XBJ group were significantly lower than those of model group [TNF-α (μg/L):340.45±68.57 vs. 443.00±110.10, IL-1β (μg/L): 191.33±82.78 vs. 436.37±163.64, IL-6 (μg/L): 192.21±37.89 vs. 342.70±92.42, LPS (μg/L): 0.43±0.17 vs. 0.68±0.22, allP< 0.01]. Infiltration of inflammatory cells, necrosis and hemorrhage in intestinal mucosa were found in the intestine of heat-stroke animals in model group. The pathological lesions in XBJ group were milder than those of model group, with a decreased pathological injury score compared with model group (2.10±1.15 vs. 3.20±0.67,P< 0.01). The expression of iNOS and apoptosis of cells in intestinal tissue in model group were increased compared with that of sham group, but they were significantly less marked in XBJ group compared with model group [iNOS (adjustedA value): 0.32±0.15 vs. 0.74±0.17, apoptotic index: 0.23±0.08 vs. 0.56±0.07, bothP< 0.01]. The order of expression for occludin protein from high to low was sham group, XBJ group and model group (A value was 0.96±0.25, 0.62±0.20, 0.33±0.11, respectively). Furthermore, there was significant difference in the expression of occludin protein between model group and both XBJ group and sham group (bothP<0.01).Conclusions Xuebijing injection alleviates inflammation and endotoxemia produced by severe heat-stroke in rats. The mechanism may be related to amelioration of oxidative injury, apoptosis, and dysfunction of tight junction protein occludin expression.
