外源性一氧化碳释放分子2对脓毒症大鼠T淋巴细胞凋亡的影响

Effect of Exogenous Carbon Monoxide Release Molecule 2 on Apoptosis of T Lymphocytes in Septic Rats

目的探讨外源性一氧化碳释放分子2对脓毒症大鼠T淋巴细胞的影响。方法采用腹腔注射LPS方法制备脓毒症模型。采用数字表法将28只雄性SD大鼠随机分为阴性对照组(n=7)、LPS组(n=7)、外源性一氧化碳释放分子2(CORM-2)(10mg/L LPS^+10μmol/L CORM-2干预)、无活性外源性一氧化碳释放分子2(iCORM-2)组(10mg/L LPS^+10μmol/L iCORM-2干预)。给予相应试剂预处理24h后处死大鼠,收集血液、脾脏、肠系膜淋巴结等标本,进行模型验证,应用流式细胞仪观察和计数CD4与CD8标记阳性T细胞数,计算CD4/CD8阳性细胞比值(CD4^+/CD8^+),苏木精-伊红(HE)染色观察脾脏组织病理变化,观察T淋巴细胞含量计数,用Western blot法检测Fas、caspase-9、caspase-3的蛋白表达。结果观察大鼠小肠光镜下形态,与阴性对照组比较,大鼠脓毒症模型造模成功。流式细胞仪观察,与阴性对照组比较,LPS组24h后CD4^+T细胞百分比显著降低(41.05%±5.26%vs 53.06%±6.28%),CD8^+T细胞百分比显著降低(33.98%±7.47%vs 40.33%±7.23%),CD4^+/CD8^+比值显著降低(1.21±0.70 vs 1.31±0.87),差异有统计学意义(P<0.05)。与LPS组比较,LPS^+CORM-2组24h后CD4^+T细胞百分比显著增加(50.02±5.58 vs 41.05±5.26),CD8^+T细胞百分比显著增加(38.12±7.41 vs 33.98±7.47),CD4^+/CD8^+比值显著增加(1.31±0.75 vs 1.21±0.70),差异有统计学意义(P<0.05)。LPS^+iCORM-2组与LPS组上述各指标比较,差异无统计学意义。取脾脏病理学观察,与阴性对照组比较,LPS组T淋巴细胞含量计数在光镜下观察明显减少,与LPS组比较,LPS^+CORM-2组T淋巴细胞含量计数在光镜下观察明显增加。用Western blot法检测Fas、caspase-9、caspase-3的蛋白表达,与阴性对照组比较,LPS组处理24h后,Fas、caspase-9、caspase-3蛋白表达明显上调(P<0.05),在同时间点,与LPS组比较,经过LPS^+CORM-2处理24h后,Fas、caspase-9、caspase-3蛋白表达显著降低(P<0.05),LPS^+iCORM-2组与LPS组上述各指标比较,差异无统计学意义(P>0.05)。结论外源性一氧化碳释放分子2能够在脓毒症模型中通过抑制凋亡蛋白的表达减少T淋巴细胞凋亡。

Objective To investigate the effect of exogenous carbon monoxide release molecule 2 on T lymphocytes in septic rats.Methods A sepsis model was prepared by intraperitoneal injection of LPS.Twenty-eight male Sprague-Dawley rats were divided into negative control group(n=7),LPS group(n=7)and exogenous carbon monoxide release molecule 2(CORM-2)(10mg/L LPS^+10μmol/L CORM-2 intervention),inactive exogenous carbon monoxide release molecule 2(iCORM-2)group(10mg/L LPS^+10μmol/L iCORM-2 intervention)according to random number table method.The rats were sacrificed 24h after the corresponding reagents were pretreated,and blood,spleen,mesenteric lymph nodes and other specimens were collected for model validation.The number of CD4^+and CD8^+T cells was observed and counted by flow cytometry,and the ratio of CD4^+/CD8^+cells was calculated.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of spleen tissue,and the T lymphocyte content was observed.The protein expression of Fas/Fasl,caspase-9 and caspase-3 was detected by Western blot.Results Flow cytometry showed that the percentage of CD4^+T cells were significantly increased(41.05%±5.26%vs 53.06%±6.28%)and the percentage of CD8^+T cells were significantly increased(33.98%±7.47%vs 40.33%±7.23%)after 24 hours in the LPS group compared with the negative control group.The ratio of CD4^+/CD8^+was significantly lower(1.21±0.70)than that of(1.31±0.87).The difference was statistically significant(P<0.05).Compared with the LPS group,the percentage of CD4^+T cells were increased significantly(50.02±5.58)after 24h in the LPS^+CORM-2 group(41.05±5.26),and the percentage of CD8^+T cells were increased significantly(38.12±7.41 vs 33.98±7.47).The ratio of CD4^+/CD8^+was significantly increased(1.31±0.75 vs 1.21±0.70),and the difference was statistically significant(P<0.05).There was no significant difference in the above indexes between the LPS^+iCORM-2 group and the LPS group.According to the pathological observation of spleen,compared with the negative control group,the T lymphocyte count in the LPS group was significantly reduced under light microscope.Compared with the LPS group,the T lymphocyte count in the LPS^+CORM-2 group was significantly increased under light microscope.The protein expression of Fas/Fasl,caspase-9 and caspase-3 was detected by Western blot.The protein expressions of Fas、caspase-9 and caspase-3 were up-regulated significantly 24 hours after LPS stimulation compared with control group(P<0.05),while the protein expressions of Fas、caspase-9 and caspase-3 were down-regulated significantly 24 hours after LPS^+CORM-2 stimulation compared with LPS group(P<0.05).There was no significant difference between the LPS^+iCORM-2 group and the LPS group.Conclusion Exogenous carbon monoxide 2 can reduce T lymphocyte apoptosis by inhibiting the expression of apoptotic proteins in a sepsis model.