大黄素对脓毒症大鼠肝细胞线粒体水通道蛋白8表达的影响

Effects of emodin on aquaporin 8 expression of hepatocellular mitochondria membrane in the septic rats

目的 探讨大黄素对脓毒症大鼠肝细胞线粒体水通道蛋白8(AQP-8)表达的影响。方法 成年雄性SD大鼠96只,分为4组:对照组、脓毒症组、预处理组、治疗组,每组24只,采用盲肠结扎穿孔法构建脓毒症大鼠模型,对照组探查盲肠后回纳腹腔。预处理组、治疗组采用大黄素悬浊液连续喂养5d,治疗组大鼠建模2h后腹腔内注射大黄素。18h后随机每组处死12只大鼠,采集标本,剩余12只大鼠观察生存时间。生化分析仪测定外周血ALT、AST、m-AST含量;ELISA法测定外周血IL-1β、IL-6、TNF-α、HMGB-1、NO含量;肝组织行ATP含量测定,肝细胞线粒体膜Na^(+)-K^(+)-ATP酶、Mg^(+)-ATP酶、Ca2+-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶的活性测定;RT-PCR法测定线粒体内膜AQP-8mRNA含量,蛋白免疫印迹法测定AQP-8蛋白水平。结果 对照组、预处理组和治疗组大鼠存活时间较脓毒症组长(P<0.01);与对照组相比,脓毒症组血清ALT、AST和m-AST水平显著升高,肝细胞ATP含量显著降低,肝细胞线粒体膜ATP酶活性、肝细胞线粒体AQP8 mRNA和AQP8蛋白表达量明显下降(P<0.01)。与脓毒症组相比,预处理组和治疗组血清ALT、AST和m-AST水平显著升高,肝细胞ATP含量显著降低,肝细胞线粒体膜ATP酶活性、肝细胞线粒体AQP8 mRNA和AQP8蛋白表达量明显上升(P<0.01),预处理组和治疗组间无差异(P>0.05);与对照组相比,脓毒症组血清IL-1β、IL-6、TNF-α、HMGB-1、NO含量明显上升(P<0.01);与脓毒症组相比,预处理组和治疗组血清IL-1β、IL-6、TNF、HMGB-1、NO含量明显下降(P<0.01),预处理组和治疗组间无差异(P>0.05)。结论 脓毒症可以导致肝细胞线粒体膜AQP8 mRNA和蛋白表达下降。大黄素可以上调脓毒症大鼠肝细胞线粒体膜AQP8 mRNA和蛋白的表达,延缓大鼠CLP手术后出现脓毒症症状的时间,延长脓毒症大鼠的生存时间。

Objective To investigate the effects of emodin on aquaporin8 expression of hepato-cellular mitochondria membrane in the septic rats.Methods A total of 96 adult SD rats were andom-ly divided into four groups:sham operation group,septic group Lestablished by cecal ligation and puncture(CLP)J,emodin preventive group and emodin treatment group(5 days before CLP,emod-in suspension was fed daily for 35 mg/kg),emodin treatment group(injection of emodin 20 mg/kg in-to abdominal cavity after CLP 2 hours),and there were 24 rats in cach group.12 rats in each group were randomly sarificed,abdominal aortic blood and liver were harvested at 18 hours after operation.The survival time of the remaining 12 rats in each group were observed.The content of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and mitochondrial aspartate aminotransferase(m-AST)were determined by enzyme coupling rate method.The content of inflammatory factors(IL-1β,IL-6,TNF,HMGB-1,NO)in peripheral blood was determined by ELISA.The content of ATP was detected by colorimetric and chemical fluorescein method.The activity of mitochondrial ATPase was detected by phosphorus quantification.The content of mRNA AQP8 in mitochondrial membrane of hepatocytes were determined by RT-PCR,and the expressions of mitochondrial membrane aquaporin8(AQP8)were detected by Western Blot.Results The survival time of rats in emodin preventive group and emodin treatment group was longer than that in septic group(P<0.01).Compared with sham operation group,the levels of serum ALT,AST and m-AST were significantly increased in septic group,and the content of ATP was reduced,the activity of mitochondrial membrane ATPase,the expressions of AQP8 and the content of mRNA-AQP8 were significantly decreased(P<0.01).Compared with septic group,the levels of serum ALT,AST and m-AST were significantly decreased in emodin preventive group and emodin treatment group,and the content of ATP,the activity of mitochondrial membrane ATPase,the expressions of AQP8 and the level of AQP8 mRNA were significantly increased(P<0.01),no significant difference between emodin preventive group and emodin treatment group(P>0.05).The levels of serum inflammatory factors(IL-1β,IL-6,TNF,HMGB-1,NO)in septic group were increased significantly than that in sham operation group(P<0.01),while the levels of serum inflammatory factors(IL-1β,IL-6.TNF,HMGB-1.NO)in emodin preventive group and emodin treatment group were lower than that in septic group(P<0.01),no significant difference between emodin preventive group and emodin treatment group(P>0.05).Conclusions Sepsis could lead to a decrease in the expression of AQP8 in the mitochondrial membrane of hepatocytes.Emodin could up-regulate the expression of AQP8 in hepatocyte mitochondria of sepsis rats,delayed the time of sepsis symptoms after CLP operation,and prolonged the survival time of sepsisrats.