右美托咪定对内毒素致小鼠巨噬细胞凋亡时HIF-1α表达的影响

Effect of dexmedetomidine on expression of hypoxia-inducible factor-1α during endotoxin-caused apoptosis in macrophages of mice

目的评价右美托咪定对内毒素致小鼠巨噬细胞凋亡时缺氧诱导因子-1(HIF-1α)表达的影响。方法体外培养小鼠巨噬细胞系RAW264.7细胞,将细胞接种于6孔板或96孔板,待融合度为60%~70%时,采用随机数字表法分为4组(n=16):对照组(Con组)、右美托咪定组(Dex组)、脂多糖组(LPS组)和LPS+右美托咪定组(LPS+Dex组)。Con组加入PBS缓冲液;Dex组加入1μmol/L右美托咪定;LPS组和LPS+Dex组均给予1μg/ml的LPS;LPS+Dex组于加入LPS后立即给予1μmol/L右美托咪定,各组孵育24h。采用TUNEL法测定细胞凋亡率,采用JC-1法测定线粒体膜电位水平,采用荧光酶标仪法测定ROS含量,采用酶标仪法测定ATP含量,采用Westernblot法测定HIF-1α、细胞色素c(Cytc)、caspase-9和cleavedcaspase-3的表达水平。结果与Con组相比,LPS组巨噬细胞凋亡率和ROS含量升高,ATP含量和线粒体膜电位水平降低,HIF-1α、Cytc、caspase-9和cleavedcaspase-3表达上调(P<0.05),Dex组上述指标差异无统计学意义(P>0.05);与LPS组相比,LPS+Dex组巨噬细胞凋亡率和ROS含量降低,ATP含量和线粒体膜电位水平升高,HIF-1α表达上调,Cytc、caspase-9和cleavedcaspase-3表达下调(P<0.05)。结论右美托咪定可减轻内毒素致巨噬细胞氧化应激损伤,改善线粒体功能,抑制线粒体途径凋亡,其机制可能与上调HIF-1α表达有关。

Objective To evaluate the effect of dexmedetomidine on expression of hypoxia-inducible factor-1α(HIF-1α)during endotoxin-caused apoptosis in macrophages of mice. Methods Mouse macrophage cell line RAW264.7 cultured in vitro were seeded in 6-well or 96-well plates and divided into 4 groups(n=16 each)when cell confluence reached 60%-70% using a random number table method: control group(group Con), dexmedetomidine group(group Dex), lipopolysaccharide(LPS)group, and LPS plus dexmedetomidine group(group LPS+ Dex). Phosphate buffer solution was added in group Con.Dexmedetomidine 1 μmol/L was added in group Dex.LPS 1 μg/ml was added in LPS and LPS+ Dex groups.Dexmedetomidine 1 μmol/L was added immediately after adding LPS in group LPS+ Dex.Cells were then cultured for 24 h in each group.Cell apoptosis was measured using TUNEL, mitochondrial membrane potential using JC-1, reactive oxygen species(ROS) content by ROS kit, and ATP content by ATP kit.The apoptosis rate was calculated.The expression of HIF-1α, cytochrome C(Cyt-c), caspase-9 and cleaved caspase-3 was detected by Western blot. Results Compared with group Con, the apoptosis rate and ROS content were significantly increased, ATP content and mitochondrial membrane potential were decreased, the expression of HIF-1α, Cyt-c, caspase-9 and cleaved caspase-3 was up-regulated in group LPS(P<0.05), and no significant change was found in the parameters mentioned above in group Dex(P>0.05). Compared with group LPS, the apoptosis rate and ROS content were significantly decreased, ATP content and mitochondrial membrane potential were increased, the expression of HIF-1α was up-regulated, and the expression of Cyt-c, caspase-9 and cleaved caspase-3 was down-regulated in group LPS + Dex(P<0.05). Conclusion Dexmedetomidine can reduce endotoxin-caused oxidative stress injury to macrophages, improve mitochondrial function and inhibit mitochondrial apoptosis, and the mechanism may be related to up-regulating the expression of HIF-1α in mice.