西红花苷通过过氧化物酶体增殖物激活受体γ-半胱氨酸天冬氨酸蛋白酶3-聚腺苷酸二磷酸核糖基聚合酶通路保护大鼠心肌缺血再灌注损伤

Crocin protects rats from myocardial ischemia-reperfusion injury through peroxisome proliferator-activated receptorγ/cysteine containing aspartate specific protease-3/polyadenylic acid diphosphate ribosyl polymerase pathway

目的探讨西红花苷通过过氧化物酶体增殖物激活受体γ(PPARγ)-半胱氨酸天冬氨酸蛋白酶3(caspase-3)-聚腺苷酸二磷酸核糖基聚合酶(PARP)通路对心肌缺血再灌注(MIR)大鼠的保护作用。方法将72只大鼠随机分为假手术组、模型组、西红花苷低、中、高剂量[20、40、80 mg/(kg·d),灌胃]组、西红花苷+PPARγ抑制剂T0070907组[西红花苷80 mg/(kg·d)+T00709071.5 mg/(kg·d)]。结扎冠状动脉前降支法构建MIR大鼠模型。建模成功后,测定大鼠血流动力学参数左心室舒张压(LVDP)、左心室舒张末压(LVEDP)、左心室内压最大上升和下降速率(±dp/dt_(max));试剂盒检测大鼠血清和心肌组织中乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、丙二醛水平;苏木精-伊红(HE)染色法检测大鼠心肌组织病理学变化;原位末端标记(TUNEL)法检测大鼠心肌细胞凋亡情况;蛋白免疫印迹(Western-blot)法检测大鼠心肌组织中PPARγ-caspase-3-PARP通路蛋白表达。结果假手术组大鼠心肌纤维排列整齐,间质无炎症细胞浸润;模型组大鼠心肌纤维排列紊乱,间质伴有大量炎症细胞浸润;与模型组比较,西红花苷低、中、高剂量组大鼠心肌组织病变得到改善,西红花苷中、高剂量组心肌纤维排列较整齐,存在少量炎症细胞浸润;与西红花苷高剂量组比较,西红花苷+T0070907组大鼠心肌组织病变严重。模型组大鼠血流动力学参数LVDP、+dp/dt_(max)、-dp/dt_(max)、PPARγ、B淋巴细胞瘤-2(Bcl-2)表达较假手术组降低,LVEDP、血清和心肌组织中LDH[血清LDH(2762.74±317.69)比(1105.68±286.45)U/L,q=18.605,P<0.001;心肌组织LDH(4852.34±244.82)比(2456.84±315.63)U/mg,q=29.820,P<0.001]、CK-MB、丙二醛水平,心肌细胞凋亡数目、Bcl-2相关X基因(Bax)、活化的半胱氨酸天冬氨酸蛋白酶3(Cleaved caspase-3)、活化的PARP(Cleaved PARP)蛋白表达较假手术组升高(P<0.05);与模型组比较,西红花苷低、中、高剂量组LVDP、+dp/dt_(max)、-dp/dt_(max)及PPARγ、Bcl-2蛋白表达升高,LVEDP、LDH、CK-MB、丙二醛水平、心肌细胞凋亡数目及Bax、Cleaved caspase-3、Cleaved PARP蛋白表达降低,且随着西红花苷剂量的增加,呈一定的剂量依赖性变化(P<0.05);T0070907可逆转西红花苷对心肌缺血再灌注大鼠的保护作用。结论西红花苷可能通过激活PPARγ-caspase-3-PARP通路预防MIR大鼠心肌损伤。

Objective To explore the protective effect of crocin on myocardial ischemia reperfusion(MIR)rats through peroxisome proliferator-activated receptorγ(PPARγ)-cysteine containing aspartate specific protease-3(caspase-3)-polyadenylic acid diphosphate ribosyl polymerase(PARP)pathway.Methods Seventy-two rats were randomly divided into 6 groups:sham operation group,model group,low-,medium-and high-dose[20,40,80 mg/(kg·d)]crocin groups,crocin+PPARγinhibitor T0070907 group[crocin 80 mg/(kg·d)+T00709071.5 mg/(kg·d)].The MIR rat model was constructed by ligating the anterior descending coronary artery.After successful perfusion,the hemodynamic parameters of rats were determined,including left ventricular diastolic pressure(LVDP),left ventricular end diastolic pressure(LVEDP),maximum rate of increase/decrease of left ventricular pressure(dp/dt_(max)),the levels of serum and myocardial tissue lactate dehydrogenase(LDH),creatine kinase isoenzyme(CK-MB).Malondialdehyde(MDA)of rats were detected with kits.Pathological changes of rat myocardium were detected with hematoxylin-eosin(HE)staining method.Cardiomyocyte apoptosis of rats was detected with TdT-mediated dUTP nick and labeling(TUNEL)method.The expression of PPARγ-caspase-3-PARP pathway proteins in rat myocardial tissue was detected with Western blot.Results The myocardial fibers of rats in the sham operation group were arranged neatly,with no inflammatory cell infiltration in the interstitium,while the arrangement of myocardial fibers in the model group was disordered,with a large number of inflammatory cell infiltration in the interstitium.Compared with the model group,rats in the low-,medium-,and high-dose crocin groups had myocardial tissue pathology improvements,and the myocardial fibers in the middle-and high-dose crocin groups were arranged relatively neatly,with small amount of inflammatory cell infiltration.Rats in the crocin+T0070907 group had more severe damage of the myocardial tissues than those in the high-dose crocin group.Compared with the sham operation group,the hemodynamic parameters(LVDP,+dp/dt_(max),-dp/dt_(max)),and the expression of PPARγand B cell lymphoma 2(Bcl-2)in the model group were significantly reduced,while the LVEDP and the levels of serum and myocardial tissue LDH[(2762.74±317.69)vs(1105.68±286.45)U/L,q=18.605,P<0.001;myocardial tissue(4852.34±244.82)vs(2456.84±315.63)U/mg,q=29.820,P<0.001],CK-MB and MDA,the cardiomyocyte apoptotic number,the expression of Bax,Cleaved caspase-3 and Cleaved PARP proteins were significantly increased(P<0.05).Compared with the model group,the LVDP,+dp/dt_(max),-dp/dt_(max),the expression of PPARγand Bcl-2 proteins in the low-,medium-,and high-dose crocin groups were significantly increased,while the LVEDP,the levels of serum and myocardial tissue LDH,CK-MB and MDA,cardiomyocyte apoptotic number,and the expression of Bax,Cleaved caspase-3 and Cleaved PARP proteins were significantly decreased.Furthermore,with the increase of the dose of crocin,the changes appeared to be dose-dependent(P<0.05).T0070907 could reverse the protective effect of crocin on myocardial ischemia-reperfusion in rats.Conclusion Crocin may prevent myocardial injury in MIR rats by activating the PPARγ-caspase-3-PARP pathway.