类法尼醇X受体拮抗剂可减轻高脂载脂蛋白E基因敲除小鼠心肌缺血再灌注损伤

Farnesoid-X-receptor blockade reduces myocardial reperfusion injury in cholesterol-fed apolipoprotein E knockout mice

目的探讨类法尼醇x受体（FXR）拈抗剂对高脂载脂蛋白E（ApoE）基因敲除（ApoE_-/-）小鼠心肌缺血再灌注（I／R）损伤的影响及其机制。方法取雄性ApoE_-/-小鼠，分为3组：（1）标准ApoE_-/-组（n=18）：予标准鼠料喂养12周后建立心肌I／R模型；（2）高脂ApoE_-/-组（n=22）：予高脂鼠料（含1．25％胆固醇）喂养12周后建立心肌I／R模型；（3）高脂ApoE_-/-＋FXR拮抗剂组（n=17）：予高脂鼠料喂养12周后建立心肌I／R模型，心肌I／R前30min给予FXR拮抗剂Z—guggulsterone（100mg／kg）。荧光实时定量PCR（RT—qPCR）检测FXR基因变化，伊文斯兰及氯化三苯基四氮唑（TFC）双染色法检测心肌梗死面积，TUNEL法检测心肌细胞凋亡情况，酶联免疫吸附试验（ELISA）法测定心肌线粒体细胞色素C释放，荧光底物法检测caspase-9、12、8和3的活性，RT—qPCR检测BAX、BCL-2、CCAAT／增强子结合蛋白同源蛋白（CHOP）、自杀相关因子（Fas）和自杀相关因子配体（FasL）等基因的表达水平。结果高脂ApoE_-/-组小鼠心肌组织FXRmRNA表达水平显著高于标准ApoE_-/-组（P〈0．01）。高脂ApoE_-/-组小鼠心肌I／R诱导心肌梗死面积显著高于标准ApoE_-/-组[（62．1±7．0）％比（33．8±5．8）％，P〈0．01]，心肌细胞凋亡指数亦显著高于标准ApoE_-/-组[（36．8±5．7）％比（17．2±3．8）％，P〈0．01]。高脂ApoE_-/-＋FXR拮抗剂组小鼠心肌梗死面积则显著小于高脂ApoE_-/-组[（24．4±4．7）％比（62．1±7．0）％，P〈0．01]，心肌细胞凋亡指数亦显著低于高脂ApoE_-/-组[（13．8±2．7）％比（36．8±5．7）％，P〈0．01]，细胞凋亡的线粒体通路激活的标志物（线粒体细胞色素c释放、caspase-9以及BAX／BCL-2表达比例）和内质网应激通路的标志物（caspase-12活性以及CHOP表达）均较高脂ApoE_-/-组低（P均〈0．01），而膜受体死亡通路的标志物（caspase．8活性以及Fas、FasL表达）与高脂ApoE_-/-组比较差异均无统计学意义。结论FXR拮抗剂可减轻高脂ApoE_-/-小鼠心肌I／R损伤，其机制与抑制细胞凋亡的线粒体通路和内质网应激通路有关。

Objective To investigate the effect of farnesoid-X-receptor （FXR） antagonist Z- guggulsterone in an in vivo high-fat fed apolipoprotein E knockout （ApoE -/- ） mice model of myocardial ischemia/reperfusion （I/R）. Methods Male ApoE -/ mice were randomly divided into three groups： standard ApoE-/- group （fed with standard mouse diet for 12 weeks before myocardial I/R procedure, n = 18）, high-fat ApoE /- group （fed with high-fat mouse diet for 12 weeks before myocardial I/R procedure,n = 22）, and high-fat ApoE-/- ＋ FXR antagonist group （fed with high-fat mouse diet for 12 weeks received FXR antagonist Z-Guggulsterone 30 minutes before myocardial I/R procedure, n = 17） and The expression of FXR was detected by real-time quantitative-PCR. Myocardial infarct size was determined by Evans blue/Trc double staining methods. Myocardial apoptosis was determined by in situ TUNEL technique. Markers of the mitochondrial-mediated apoptotic pathway （cytochrome c release, caspase-9 activity, and BAX and BCL-2 levels）, endoplasmic reticulum stress apoptotic pathway （caspase-12 activity and CHOP level） , and death receptor apoptotic pathway （caspase-8 aetivity, and Fas and FasL levels） were also measured. Result FXR expression （ 3.7-fold higher, P 〈 0. 01 ）, myocardial infarct size [ （ 62. 1 ± 7.0）% vs, （33.8±5.8）%, P〈0.01] and myocardial apoptosis index[ （36.8±5.7）% vs. （17.2± 3.8 ） % , P 〈 0. 01 ] were all significantly higher in high-fat ApoE -/- group than those in standard ApoE -/- group. Compared with high-fat ApoE / group, myocardial infarct size [ （ 24. 4 ± 4. 7 ） % vs. （ 62. 1 ± 7.0）%, P〈0.01] and myocardial apoptosis index [（13.8 ±2.7）% vs. （36.8±5.7）%, P〈0.011 were significantly reduced in high-fat ApoE / ＋ FXR antagonist group. Moreover, levels of mitochondrial- mediated apoptotic pathway markers （cytochrome c release, caspase-9 activity, and BAX/BCL-2 levels） and endoplasmic reticulum stress apoptotic pathway markers （caspase-12 aetivity and CHOP level） were significantly lower in high-fat ApoE / ＋ FXR antagonist group than those in high-fat ApoE-/- group （all P 〈0. 01）, Levels of death receptor apoptotic pathway markers （caspase-8 activity, and Fas and FasL levels） were similar between high-fat ApoE-/- group and high-fat ApoE / ＋ FXR antagonist group. Conclusion FXR antagonist alleviates myocardial repeffusion injury in cholesterol-fed ApoE / mice via inhibition of the mitoehondrial-mediated and endoolasmic-reticulum stress pathway.