摘要
目的探讨一氧化碳释放分子2(carbon monoxide-releasing molecule-2,CORM-2)调控T淋巴细胞的分化介导抗炎保护失血性休克大鼠肠屏障。方法56只健康雄性SD大鼠按照随机数字表法分为假手术组、休克组、二甲基亚砜组(dimethyl sulfoxide,DMSO)、灭活型一氧化碳释放分子2组(inactive carbon monoxide-releasing molecule-2,iCORM-2)、CORM-22 mg/kg组、CORM-24 mg/kg组及CORM-26 mg/kg共7组,每组8只。采用Wiggers改良法制备失血性休克大鼠模型,CORM-2各剂量组和iCORM-2组于制备休克模型前即刻腹腔注射不同剂量CORM-2和6 mg/kg iCORM-2,DMSO组腹腔注射与iCORM-2等量的2%DMSO,休克组和假手术组不给予药物干预。各组大鼠记录置管后或休克后不同时相平均动脉压变化。各组大鼠造模成功后23 h采用荧光素异硫氰酸酯(fliorescein isothiocyanate,FITC)-葡聚糖作为渗透性标记物测试肠壁通透性,并留取回肠组织观察肠道病理形态。免疫组化观察大鼠肠黏膜淋巴细胞转录因子T-bet、Foxp3的表达,Western Blot检测大鼠肠黏膜组织干扰素-γ(interferon-γ,IFN-γ)、白介素10(interleukin-10,IL-10)和转化生长因子-β(transforming growth factor-β,TGF-β)的表达。正态分布计量资料多组间均数比较采用单因素方差分析,非正态分布数据采用Kruskal Wallis秩和检验。结果与假手术组相比,休克组、DMSO组和iCORM-2组血清中FITC-葡聚糖浓度明显增加(均P<0.05);与休克其余各组比较,CORM-2各剂量组血清中FITC-葡聚糖浓度均减低(均P<0.05)。病理学改变显示休克组、DMSO组和iCORM-2组大鼠回肠组织损伤明显;CORM-2干预可减轻休克大鼠回肠黏膜损伤,且CORM-24 mg/kg组和CORM-26 mg/kg组回肠结构更完整。休克组和DMSO组肠黏膜淋巴细胞T-bet抗原表达较假手术组升高(均P<0.05);CORM-2各剂量组T-bet抗原表达较休克组降低(均P<0.05)。CORM-22 mg/kg组、CORM-24 mg/kg组及iCORM-2组Foxp3抗原表达较休克组和DMSO组均减低(均P<0.05),但CORM-26 mg/kg组与休克组或DMSO组比较差异无统计学意义(均P>0.05)。与假手术组相比,休克组IFN-γ表达升高(P<0.05),IL-10和TGF-β表达未见差异(均P>0.05);与休克组相比,CORM-2各剂量组IL-10蛋白表达升高(均P<0.05),其中CORM-24 mg/kg组和CORM-26 mg/kg组TGF-β表达上调(均P<0.05),但仅CORM-26 mg/kg组较休克组IFN-γ表达下调(P<0.05)。结论CORM-2可抑制1型辅助性T细胞的活化,降低炎症因子,增加抗炎因子,减轻休克缺血肠壁炎症,保护肠屏障。
Objective To investigate the protective effect of carbon monoxide releasing molecule 2(CORM-2)on intestinal barrier by regulating the differentiation of T lymphocytes in rats with hemorrhagic shock.Methods Healthy male Sprague-Dawley rats(n=56)were randomly(random number)divided into the sham operation group,shock group,dimethyl sulfoxide(DMSO)control group,inactivated carbon monoxide release molecule-2(iCORM-2)pretreatment group and three pretreatment CORM-2 with the doses of 2,4 and 6 mg/kg separately.The hemorrhagic shock was induced with the use of a modified Wiggers model.Rats in the CORM-2 group and iCORM-2 group were intraperitoneally injected with CORM-2 with the doses of 2,4 and 6 mg/kg and 6 mg/kg iCORM-2 instantly before shock induction.Rats in the DMSO group received intraperitoneal administration of 2%DMSO with the same volume of iCORM-2.Rats in the shock group and sham operation group were not pretreated before inducing shock.Mean arterial pressure of each rat was recorded at different phases after catheterization or shock.Twenty-three hours after shock induction,the permeability of intestinal barrier was measured by FITC-dextran flux,and then ileum tissues were harvested to observe histopathologic changes.Immunohistochemistry was used to detect the expression of transcription factors T-bet and Foxp3 of intestinal mucosa in rats,and the expression of interferon-γ(IFN-γ),interleukin-10(IL-10)and transforming growth factor-β(TGF-β)in intestinal mucosa was measured by Western blot.One-way analysis of variance or Kruskal Wallis rank sum test was used to compare the difference among groups for normal or non-normal distributed data.Results Compared with the sham operation group,serum concentrations of FITC-dextran were significantly increased in the shock group,DMSO group and iCORM-2 group(all P<0.05).The concentrations of FITC-dextran in serum of three CORM-2 pretreatment groups pretreatment were significantly decreased compared with other groups undergoing shock(all P<0.05).Rats in the shock group,DMSO group and iCORM-2 group showed severe ileum injury.CORM-2 intervention resulted in alleviation of intestinal mucosal injury in rats with shock,and rats in groups pretreatment CORM-2 at the doses of 4 and 6 mg/kg exhibited integrity of anatomic ileac structure.Compared with the sham operation group,T-bet levels of intestinal intraepithelial lymphocytes were increased in shock group and DMSO group(all P<0.05).Compared with the shock group,levels of T-bet were decreased in intestinal epithelium of three groups pretreatment with CORM-2 at the doses of 2,4 and 6 mg/kg(all P<0.05).Foxp3 levels in intestinal intraepithelial lymphocytes of the iCORM-2 group and two groups pretreatment with CORM-2 at the doses of 4 and 6 mg/kg were increased compared with the shock group and DMSO group(all P<0.05),but there was no significant difference among the shock group,DMSO group and group pretreatment with CORM-2 at 6 mg/kg(all P>0.05).The shock group showed increased expression of IFN-γ(all P<0.05),but unchangeable expression of IL-10 and TGF-β(allP>0.05)compared with the sham operation group.Compared with the shock group,the expression levels of IL-10 in three groups pretreatment with CORM-2 at the doses of 2,4 and 6 mg/kg were significantly increased(all P<0.05),and the expression levels of TGF-βwere increased in two groups pretreatment with CORM-2 at the doses of 4 and 6 mg/kg(all P<0.05).The expression of IFN-γin group pretreatment with CORM-2 at 6 mg/kg was significantly decreased compared with the shock group(P<0.05).Conclusions CORM-2 inhibited the activation of type 1 helper T cells to decrease the expression of proinflammatory factors and upregulated the expression of anti-inflammatory factors.Thus,CORM-2 reduced gut inflammation and protected the intestinal barrier.
作者
牛庆晟
张瑞
陈磊
王晓红
Niu Qingsheng;Zhang Rui;Chen Lei;Wang Xiaohong(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Ningxia Key Laboratory of Craniocerebral Diseases,Yinchuan 750004,China;Department of Intensive Care Unit,General Hospital of Ningxia Medical University,Yinchuan 750004,China)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2022年第5期628-635,共8页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金(81960348)
宁夏回族自治区自然科学基金(2021AAC03393)。
关键词
一氧化碳释放分子2
休克
肠
淋巴细胞
炎症
Carbon monoxide releasing molecule-2
Shock
Intestine
Lymphocyte
Inflammation
