姜黄素对脓毒症大鼠肝细胞线粒体膜通透性转换的作用机制研究

Study of the mechanisms of curcumin on mitochondrial permeability transition of hepatocytes in rats with sepsis

目的 研究线粒体膜通透性转换（MPT）与肝细胞凋亡及线粒体损伤之间的关系,初步探讨姜黄素对MPT的影响及其可能机制.方法 将15只健康雄性SD大鼠按随机数字表法分为假手术组、脓毒症组及姜黄素组,每组5只.采用盲肠结扎穿孔术（CLP）制备脓毒症大鼠模型;假手术组仅开腹翻动盲肠,不进行结扎、穿孔.姜黄素组术前以100 mg·kg-1·d-1姜黄素溶于生理盐水至10 mL/kg,连续灌胃7d,其余组灌胃等量生理盐水.术后12h取肝组织,透射电镜下观察肝细胞线粒体形态学改变;采用钙离子荧光探针Fluo-3/AM检测细胞内游离Ca2＋浓度;采用蛋白质免疫印迹试验（Western Blot）检测活化天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）和B细胞淋巴瘤-2基因（Bcl-2）及其相关X蛋白（Bax）的蛋白表达;采用逆转录-聚合酶链反应（RT-PCR）检测肝细胞活化caspase-3、Bcl-2、Bax的mRNA表达.结果 透射电镜下观察假手术组细胞膜完整,胞质均匀,线粒体正常、清晰;脓毒症组肝细胞线粒体明显肿胀,内膜嵴断裂或消失,双层膜结构消失;姜黄素组线粒体轻度肿胀,少数细胞出现线粒体肿胀,双侧膜结构不清.假手术组、姜黄素组、脓毒症组荧光强度指数依次增高,分别为417.33±15.88、772.95±42.37、1 560.84±160.78 （F=184.149,P=0.000）.脓毒症组活化caspase-3和Bax的蛋白及mRNA表达最高,姜黄素组次之,假手术组最低[活化caspase-3蛋白（灰度值）：1.698±0.061、0.694±0.045、0.246±0.027,F=1 289.667,P=0.000;活化caspase-3 mRNA（2-ΔΔα）：1.031±0.135、0.578±0.144、0.183±0.036,F=66.958,P=0.000; Bax蛋白（灰度值）：1.826±0.126、1.254±0.140、0.623±0.901,F=94.536,P=0.000; Bax mRNA（2-△△Ct）：2.774±0.338、1.661±0.226、0.656±0.114,F=124.710,P=0.000],且各组间两两比较差异均有统计学意义（均P＜0.01）;而假手术组、脓毒症组、姜黄素组Bcl-2的蛋白及mRNA表达依次升高[Bcl-2蛋白（灰度值）：0.716±0.091、1.328±0.147、1.656±0.104,F=84.918,P=0.000; Bcl-2 mRNA（2△△Ct）：0.617±0.118、1.393±0.096、1.650±0.167,F=83.846,P=0.000].脓毒症组Bcl-2/Bax的蛋白及mRNA表达最低,假手术组次之,姜黄素组最高[Bcl-2/Bax蛋白（灰度值）：0.726±0.055、1.150±0.043、1.333±0.163,F=46.265,P=0.000; Bcl-2/Bax mRNA（2-ΔΔCt）：0.505±0.041、0.944±0.097、1.006±0.168,F=12.211,P=0.001].结论 MPT可导致线粒体功能损伤,并进一步引起肝细胞凋亡;姜黄素通过降低细胞内Ca＋浓度、促进抗凋亡基因Bcl-2表达以及抑制caspase-3活化和Bax基因表达,从而发挥调节MPT的作用.

Objective To study the effects of mitochondrial permeability transition （MPT） on hepatocyte apoptosis and mitochondrial damage,and investigate the effects of curcumin on MPT and the related mechanisms in septic rat.Methods Fifteen healthy male Sprague-Dawley （SD） rats were randomly divided into three groups：sham group,sepsis group and curcumin group,with 5 rats in each group.Septic rat model was reproduced by cecal ligation and puncture （CLP）.The rats in sham group were flipped the cecum without perforation and ligation.The rats in the curcumin group were treated with curcumin 100 mg·kg-1·d-1 （dissolved in saline to 10 mL/kg） by oral gavage for 7 days,while the other groups were treated with normal saline.Tissue samples were harvested in each group at 12 hours after operation.Pathological changes in hepatic mitochondria were observed under electron microscopy,concentration of free calcium was examined with confocal laser scanning microscope.After Fluo-3/AM staining,protein and mRNA expression of active caspase-3,Bcl-2 and Bax were examined by Western Blot and reverse transcription-polymerase chain reaction （RT-PCR）.Results Under the transmission electron microscope,intact cell membrane,adqulis cytoplasm,and normal and clear mitochondrion was found in the sham group.Mitochondria in sepsis group swelled obviously with mitochondrial cristae broken or disappearance,unclear bilateral membrane structure,while the curcumin group showed much less pathological changes,with few mitochondria swell,and smear bilateral membrane structure.The fluorescence intensity index of sham group,sepsis group and curcumin group was raised successively （417.33 ± 15.88,772.95 ± 42.37,1 560.84 ± 160.78,respectively,F=184.149,P=0.000）.The protein and mRNA expression of active caspase-3 and Bax had the highest level in sepsis group,followed by the curcumin group,and that in the sham group was the lowest [active caspase-3 protein （gray scale）：1.698 ± 0.061,0.694 ± 0.045,0.246 ± 0.027,F=1 289.667,P=0.000; active caspase-3 mRNA （2-Δ△Ct）：1.031 ±0.135,0.578 ± 0.144,0.183 ±0.036,F=66.958,P=0.000; Bax protein （gray scale）：1.826 ±0.126,1.254 ±0.140,0.623 ±0.901,F=94.536,P=0.000; Bax mRNA （2-ΔΔCt）：2.774 ±0.338,1.661 ±0.226,0.656 ±0.114,F=124.710,P=0.000],all of these values had statistical significance among the three groups （all P〈0.01）.While Bcl-2 protein and mRNA had the highest level in curcumin group and lowest level in the sham group [Bcl-2 protein （gray scale）：0.716 ±0.091,1.328 ±0.147,1.656 ±0.104,F=84.918,P=0.000; Bcl-2 mRNA （2-△ΔCt）：0.617 ±0.118,1.393 ±0.096,1.650 ±0.167,F=83.846,P=0.000].The protein and mRNA expressions of Bcl-2/Bax ratio were lowest in sepsis group,then sham group,and highest in curcumin group [Bcl-2/Bax protein （gray scale）：0.726 ± 0.055,1.150 ± 0.043,1.333 ± 0.163,F=46.265,P=0.000; Bcl-2/Bax mRNA （2-Δ△Ct）：0.505 ±0.041,0.944±0.097,1.006 ±0.168,F=12.211,P=0.001].Conclusions MPT can lead to mitochondrial dysfunction and further cause hepatocyte apoptosis.Mechanism of effect of curcumin on MPT may be related to reduction of intracellular calcium concentration,promotion of anti-apoptotic Bcl-2 gene expression,inhibition of caspase-3 activation and Bax gene.