Piezo1在不同周龄ApoE^(-/-)小鼠中的表达水平及对血管内皮功能的作用

Expression of Piezo1 in ApoE^(-/-) mice of different ages and its effect on vascular endothelial function

目的:探明ApoE^(-/-)小鼠中机械敏感性离子通道Piezo1的表达变化及Piezo1在动脉粥样硬化血管内皮功能中的作用。方法:选用24只SPF级8周龄雄性ApoE^(-/-)小鼠为模型组,24只SPF级8周龄雄性C57BL/6J小鼠为正常对照组,高脂饲料喂饲,于高脂饲料干预的第12、14、16和18周,每组各处死6只小鼠,检测小鼠主动脉病理情况、血脂水平、血管内皮功能及Piezo1的表达。进一步选用SPF级8周龄Piezo1^(flox/flox)Cdh5-Cre^(+)/ApoE^(-/-)(以下简称Cre^(+))小鼠与Piezo1^(flox/flox)Cdh5-Cre-/ApoE^(-/-)(以下简称Cre-)小鼠各10只,采用高脂饲料喂养12周,从小鼠病理染色、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)、内皮素1(endothelin-1,ET-1)、前列环素I_(2)(prostaglandin I_(2),PGI_(2))、血栓素A_(2)(thromboxane A_(2),TXA_(2))和离体血管环张力等多方面检测Piezo1缺失对动脉粥样硬化小鼠血管内皮功能的影响。结果:通过对高脂饲料喂养不同时间的小鼠进行观察显示,模型组小鼠主动脉斑块较正常组逐渐加重;血脂水平较正常组显著升高(P<0.01)。血清中eNOS与PGI_(2)的含量降低(P<0.01),ET-1与TXA_(2)含量增多(P<0.05)。免疫荧光染色显示模型组小鼠主动脉斑块组织中内皮细胞完整性受损,Piezo1的表达较正常组升高,但随着高脂饲料喂养周时的延长,模型组小鼠Piezo1的荧光强度及m RNA水平逐渐下降。通过Person相关性检验分析显示,Piezo1与小鼠内皮功能相关指标eNOS和PGI_(2)表达呈正相关,与ET-1和TXA_(2)表达呈负相关(P<0.01)。Piezo1表达水平与血管内皮功能有较强相关性。进一步在基因敲除小鼠中观察到,Cre^(+)小鼠主动脉斑块面积较Cre-小鼠显著降低(P<0.01);Cre^(+)小鼠血清eNOS和PGI_(2)含量较Cre-小鼠显著减少(P<0.05,P<0.01),TXA_(2)含量较Cre-小鼠显著增加(P<0.05)。血管细胞活力检测观察到Cre^(+)小鼠细胞活力较Cre-小鼠显著减弱(P<0.01)。小鼠离体主动脉张力检测显示Cre^(+)小鼠主动脉环对乙酰胆碱的内皮依赖性舒张作用较Cre-小鼠显著减弱(P<0.05)。免疫荧光染色检测到Cre^(+)小鼠主动脉斑块组织中内皮细胞完整性受损较Cre-小鼠严重,Piezo1的表达降低。结论:Piezo1参与了ApoE^(-/-)小鼠的病理进程,与血管内皮功能密切相关,Piezo1基因的缺失影响了ApoE^(-/-)小鼠斑块的形成,加重血管内皮功能的损伤。

AIM:To investigate the expression of the mechanosensitive ion channel Piezo1 in ApoE^(-/-)mice and the role of Piezo1 in the function of atherosclerotic vessels.METHODS:Twenty-four SPF 8-week-old male ApoE^(-/-)mice were used as the model group and 24 SPF 8-week-old male C57BL/6J mice were used as the control group.Both the model group and the control group were fed with high-fat diet.Mice were sacrificed at 12 weeks,14 weeks,16 weeks and18 weeks of high-fat diet intervention.The pathological changes of aorta,blood lipid levels,vascular endothelial function and Piezo1 expression were measured.Ten SPF 8-week-old Piezo1^(flox/flox)Cdh5-Cre^(+)/ApoE^(-/-)(hereafter referred to as Cre^(+)) mice and 10 Piezo1^(flox/flox)Cdh5-Cre-/ApoE^(-/-)(hereafter referred to as Cre-) mice were fed with high-fat chow for 12 weeks.The effects of Piezo1 deficiency on vascular endothelial function in ApoE^(-/-)mice were examined in various aspects,including the endothelial nitric oxide synthase (eNOS),endothelin-1 (ET-1),prostaglandin I_(2)(PGI_(2)),thromboxane A_(2)(TXA_(2)),and isolated vascular ring tension.RESULTS:The results slowed that the aortic plaque in the model group aggravated gradually at different time after high-fat diet feeding,and the blood lipid level was significantly higher than that of the control group (P<0.01).The content of eNOS and PGI_(2)was decreased (P<0.01),and the content of ET-1 and TXA_(2)was increased (P<0.05).Immunofluorescence staining showed that endothelial cell integrity was impaired in the aortic plaque tissues of the model group,and the expression of Piezo1 was increased,but the fluorescence intensity and m RNA level of Piezo1 decreased gradually in the model group as the weeks of high-fat diet feeding were extended.The results showed that Piezo1 was positively correlated with the expression of eNOS and PGI_(2),and negatively correlated with the expression of ET-1 and TXA_(2)by Person correlation test.The expression level of Piezo1 was strongly correlated with vascular endothelial function.It was further observed in knockout mice that aortic plaque area was significantly reduced in Cre^(+)mice compared with Cre-mice (P<0.01).The contents of eNOS and PGI_(2)in Cre^(+)mice were significantly lower than those in Cre-mice (P<0.05 or P<0.01),and the content of TXA_(2)was significantly higher than that of Cre-mice (P<0.05).By observing vascular cell activity assay,the cellular activity was significantly reduced in Cre^(+)mice compared with Cre-mice (P<0.01).Mouse isolated aortic tone assay showed endothelium-dependent diastolic effect of aortic rings on acetylcholine significantly was reduced in Cre^(+)mice compared with Cre-mice (P<0.05).Immunofluorescence staining demonstrated that the endothelial cell integrity in aortic plaque tissue of Cre^(+)mice was more damaged than that of Cremice,and the expression of Piezo1 was decreased.CONCLUSION:Piezo1 is involved in the pathological process of ApoE^(-/-)mice and is closely related to vascular endothelial function.The deletion of Piezo1 gene affects the formation of plaque and aggravates the impairment of vascular endothelial function in ApoE^(-/-)mice.