氢气对脓毒症小鼠肠组织Rho/ROCK信号通路的影响

Effect of hydrogen gas on intestinal Rho/ROCK signaling pathway in septic mice

目的 评价氢气对脓毒症小鼠肠组织Ras同源基因（Rho）/Rho相关螺旋卷曲蛋白激酶（ROCK）信号通路的影响.方法 雄性ICR小鼠64只,体重20～ 25 g,6周龄,采用随机数字表法,将其分为4组（n=16）：假手术组（SH组）、氢气组（H2组）、脓毒症组（S组）和脓毒症＋氢气组（S＋H2组）.采用盲肠结扎穿孔（CLP）法制备脓毒症模型.于CLP术后1和6h时H2组和S＋H2组分别吸入2％氢气1h.于CLP术后20 h时取8只小鼠,向胃内灌注异硫氰酸荧光素标记的右旋糖酐（FITC-右旋糖酐）,4h后心脏穿刺取血样,测定血清FITC-右旋糖酐的浓度.于CLP术后24 h时取8只小鼠,取血液、肝脏、脾脏和肾脏进行细菌培养,观察菌群移位情况;取小肠组织,透射电镜下观察肠上皮细胞超微结构,光镜下观察肠组织病理学改变并进行评分,采用Western blot法测定肠组织Rho、ROCK1和ROCK2的表达水平.结果 与SH组比较,S组和S＋H2组血清FITC-右旋糖酐浓度和肠损伤评分升高,血液、肝脏、脾脏和肾脏的菌群移位增加,肠组织Rho、ROCK1和ROCK2的表达上调（P＜0.05）,H2组上述各指标差异无统计学意义（P＞0.05）;与S组比较,S＋H2组血清FITC-右旋糖酐浓度和肠损伤评分降低,血液、肝脏、脾脏和肾脏的菌群移位减少,肠组织Rho、ROCK1和ROCK2的表达下调（P＜0.05）,肠组织病理学损伤减轻.结论 氢气减轻脓毒症小鼠肠损伤的机制与抑制Rho/ROCK信号通路激活有关.

Objective To evaluate the effect of hydrogen gas （H2） on intestinal Ras homolog gene （Rho） /Rho-associated coiled coil-forming protein kinase （ROCK） signaling pathway in septic mice.Methods Sixty-four male ICR mice,weighing 20-25 g,aged 6 weeks,were randomly divided into 4 groups （n =16 each） using a random number table：sham operation group （group SH）,H2 group （group H2）,sepsis group （group S） and sepsis＋H2 group （group S＋H2）.Sepsis was produced by cecal ligation and puncture （CLP）.H2 and S＋H2 groups inhaled 2％ H2 for 1 h starting from 1 and 6 h after CLP operation,respectively.Eight mice of each group were selected at 20 h after CLP operation,and gavaged with fluorescein-isothiocyanate-conjugated dextran （FITC-dextran）,4 h later blood samples were obtained by cardiac puncture,and the concentration of FITC-dextran in serum was measured.The left 8 mice in each group were sacrificed at 24 h after CLP operation.After anesthesia,the sterile samples of blood,liver,spleen and kidney were obtained and cultured for bacterial growth to evaluate the condition of bacterial translocation.The intestinal tissues were obtained for examination of the epithelial ultrastructure （by transmission electron microscope）,and of the pathological changes which were scored （by light microscope） and for determination of the expression of Rho,ROCK1 and ROCK2 （by Western blot）.Results Compared with group SH,the serum concentration of FITC-dextran and pathological scores were significantly increased,the colony-forming units in bacterial culture plates of blood,liver,spleen and kidney were increased,and the expression of Rho,ROCK1 and ROCK2 was up-regulated in S and S＋H2 groups,and no significant change was found in the parameters mentioned above in H2 group.Compared with group S,the serum concentration of FITC-dextran and pathological scores were significantly decreased,the colony-forming units in bacterial culture plates of blood,liver,spleen and kidney were decreased,and the expression of Rho,ROCK1 and ROCK2 was down-regulated,and the pathologic changes of intestines were mitigated in group S＋H2.Conclusion The mechanism by which H2 alleviates the intestinal injury is related to inhibition of the activation of Rho/ROCK signaling pathway in septic mice.