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血必净注射液预处理通过减轻小肠损伤缓解重症中暑大鼠全身炎症反应

Pretreatment with Xuebijing injection alleviates systemic inflammatory response induced by severe heat-stroke via ameliorating intestinal injury in rats

摘要:

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。

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abstracts:

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.

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作者: 陈怿 [1] 童华生 [2] 潘志国 [2] 陈玉兰 [1] 林幼萍 [1] 江东新 [1] 苏磊 [2]
作者单位: 东莞市第五人民医院 暨南大学医学院附属东莞医院 重症医学科, 广东东莞,523900 [1] 515000 广东,广州军区广州总医院重症医学科,解放军热区创伤救治与组织修复重点实验室 [2]
期刊: 《中华危重病急救医学》2015年8期 643-648页 MEDLINEISTICPKUCSCD
栏目名称: 论著
DOI: 10.3760/cma.j.issn.2095-4352.2015.08.005
发布时间: 2015-09-14
基金项目:
国家自然科学基金(81471839) 广东省科技计划项目(2013B031800010) 广东省东莞市社会科技发展项目
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