高压氧调控HMGB1防治急性一氧化碳中毒后脑病的作用机制

The mechanism of hyperbaric oxygen regulating HMGB1 in the prevention and treatment of encephalopathy after acute CO poisoning

目的分析高压氧(HBO)治疗急性一氧化碳中毒后脑病(DEACMP)高迁移率族蛋白1(HMGB1)在大鼠脑组织的表达情况,探讨HBO通过调控HMGB1防治DEACMP病理过程的作用机制。方法选取SD大鼠108只,按照随机数字表法分为空白对照组(NC组)、CO染毒组(CO组)、染毒后HBO治疗组(HBO组),各组36只。各组分为染毒前、1、3、7、14、21 d六个亚组,各亚组6只。CO组和HBO组应用静态吸入染毒法建立CO中毒模型,HBO组大鼠进行HBO治疗,1次/d。用水迷宫实验检测各组大鼠3、7、14、21 d的平均逃避潜伏期和空间位置记忆保持能力。用酶联免疫吸附(ELISA)检测各组大鼠1、3、7、14、21 d血清HMGB1、白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)浓度。用免疫印迹法(Western blotting)检测各组大鼠脑组织3、7、14、21 d HMGB1、半胱氨酸蛋白酶-3(Caspase-3)表达改变情况。用海马组织原位末端标记法(TUNEL)检测各组大鼠3、7、14、21 d海马组织神经细胞凋亡情况。结果与NC组比较,CO组和HBO组大鼠7、14、21 d平均逃避潜伏期均明显增加,差异有统计学意义(P<0.05);与CO组比较,HBO组大鼠平均逃避潜伏期在14、21 d均明显缩短、平台象限活动时间在14、21 d明显增加,差异有统计学意义(P<0.05),与NC组比较,CO组大鼠血清HMGB1浓度在1、3、7、14、21 d均较高、血清TNF-α浓度在1、3、7 d均较高、血清IL-6浓度1、3 d均较高,差异均有统计学意义(P<0.05)。与CO组比较,HBO组大鼠血清HMGB1浓度、脑组织HMGB1浓度、Caspase-3表达水平和海马神经细胞凋亡指数在3、7、14、21 d均较低、血清TNF-α浓度在3、7 d均较低、血清IL-6浓度在1、3 d均较低,差异均有统计学意义(P<0.05)。与NC组比较,CO组大鼠脑组织HMGB1和Caspase-3表达水平在3、7、14 d均较高,海马神经细胞凋亡指数在3、7、14、21 d均明显升高,差异均有统计学意义(P<0.05)。结论急性CO中毒可以诱发HMGB1、IL-6、TNF-α等炎症因子的释放,HBO通过下调HMGB1的表达,降低Caspase-3蛋白表达,抑制急性CO中毒后神经细胞的凋亡。

Objective To study the expression of high mobility group protein 1(HMGB1)in the brain of rats after hyperbaric oxygen(HBO)treatment of acute carbon monoxide poisoning(DEACMP),and to explore the mechanism of HBO in the prevention and treatment of DEACMP pathological process by regulating HMGB1.Methods 108 SD rats were randomly divided into control group(NC group)and co group(CO group).HBO treatment group(HBO group),48 rats in each group.Co group and HBO group were used to establish CO poisoning model,HBO group were treated with hyperbaric oxygen once a day.Water maze test was used to detect and analyze the memory retention ability of three groups of rats in 3 d,7 d,14 d.ELISA was used to detect the plasma concentration of HMGB1、IL-6、TNF-αin three groups of rats on the 1 d,3 d,7 d,14 d,21 d Concentration.Western blotting was used to detect the expression of HMGB1 and Caspase-3 in the brain of the three groups on the 1 d,3 d,7 d,14 d,21 d.TUNEL staining was used to detect the apoptosis of hippocampal neurons in the three groups.Results Compared with NC group,the average escape latency of rats in CO group and HBO group was significantly prolonged,and the activity time of platform quadrant in CO group was significantly shortened on 14 d and 21 d(P<0.05);compared with CO group,the average escape latency of HBO group on 7 d,14 d and 21 d was significantly shortened(P<0.05).Compared with NC group,plasma HMGB1 in CO group and HBO group were significantly increased(P<0.05);after 3 days,HBO group was significantly lower than co group,the difference was statistically significant(P<0.05).The levels of IL-6 and TNF-αin HBO group and co group increased rapidly and then decreased gradually.The increased levels of IL-6 and TNF-αin HBO group were significantly lower than those in CO group(P<0.05).Compared with NC group,the expression of HMGB1 and Caspase-3 in CO group was significantly increased on 3 d,7 d and 14 d(P<0.05);the expression of HMGB1 and Caspase-3 in HBO group was significantly increased on 3 d,7 d,14 d and 21 d(P<0.05);compared with CO group,the expression of HMGB1 and Caspase-3 in HBO group decreased significantly on 3 d,7 d,14 d and 21 d(P<0.05).The apoptotic index of nerve cells in CO group began to increase at 3 days,which was significantly different from that of NC group(P<0.05),and the difference was still statistically significant on 21 d(P<0.05);the apoptotic index of nerve cells in HBO group was slightly increased,but there was no significant difference compared with NC group(P>0.05),and the apoptotic index of 3 d,7 d,14 d and 21 d in HBO group was significantly lower than that in CO group(P<0.05).Conclusion acute CO poisoning can induce the release of HMGB1 and a variety of inflammatory factors.HMGB1 can promote the apoptosis of nerve cells after acute CO poisoning by up regulating the expression of caspase-3 protein,and participate in the pathological process of DEACMP.HBO can down regulate the expression of HMGB1,IL-6,TNF-αand caspase-3 protein,inhibit the apoptosis of nerve cells,and play a protective role on nerve cells.