【作者】 吴健；

【导师】 赵韧；

【作者基本信息】 安徽医科大学 ， 内科学（心血管）， 2025， 硕士

【摘要】 背景高血压病是最常见的心血管疾病之一,也是心力衰竭最主要的病因。高血压合并心力衰竭患者的预后差,生存率低下,严重危害患者生活质量。而心脏重构(Cardiac Remodeling,CR)是高血压病常见的靶器官损害,也是心力衰竭病理生理的关键机制。心脏重构会加重心肌细胞凋亡,凋亡的细胞又会激活成纤维细胞并促进胶原纤维沉积,这种恶性循环会推进心血管疾病的进展,最终引起心力衰竭。达格列净(Dapagliflozin,Dapa)作为一种钠-葡萄糖共转运蛋白2抑制剂(sodium-glucose cotransporter-2 inhibitors,SGLT2i),最初是为治疗2型糖尿病而研发的。然而,最近的研究表明,其心脏保护作用与其降血糖特性无关。这些效应可能与细胞内钙离子浓度的降低有关,但确切的机制尚不清楚。作为一种重要的钙离子通道,钙库操纵性钙内流(store-operated calcium entry,SOCE)主要有由Orai1和钙感受器基质相互作用分子1(stromal interaction molecule 1,STIM1)构成,在心血管疾病的发生进展中起着至关重要的作用。然而目前达格列净是否通过抑制SOCE发挥心脏保护作用并不明确。目的本研究旨在通过构建血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)诱导的心肌重构大鼠模型,研究CR心室的结构改变和Orai1、STIM1、Bax、Bcl-2、Caspse 9蛋白的变化。探讨达格列净对血管紧张素Ⅱ诱导的心脏重构的影响,并研究其潜在机制。方法在动物实验中,将20只大鼠随机分为Sham组、Dapa组、AngⅡ组和AngⅡ+Dapa组,Sham组仅进行假手术,术后每日灌胃生理盐水,Dapa组进行假手术,术后每日灌胃达格列净,AngⅡ组在皮下植入微渗透泵持续注入AngⅡ(200ng/kg/min),术后每日灌胃生理盐水;AngⅡ+Dapa组植入AngⅡ微渗透泵,术后每日灌胃达格列净;28天后完成构建心脏重构模型。通过超声心动图评估大鼠心脏功能和结构,使用Masson染色评估心室纤维化程度,使用TUNEL染色分析心室肌凋亡率,使用Western blot检测SOCE相关蛋白Orai1和STIM1以及凋亡相关蛋白Bax、Bcl-2、Caspase 9的表达水平的变化。在细胞实验中,培养H9C2细胞,以合适的AngⅡ和Dapa浓度处理细胞,使用Western Blot验证上述蛋白表达水平变化,使用钙成像检测H9C2细胞内钙离子浓度的变化,使用免疫荧光检测H9C2细胞上Orai1和STIM1的蛋白聚集现象。结果达格列净改善大鼠心室重构和心肌凋亡的作用与血压和代谢因素无关。达格列净缓解大鼠心肌重构,减轻了心肌纤维化和凋亡。并且降低了心室肌组织中AngⅡ诱导的SOCE相关蛋白Orai1、STIM1及凋亡相关蛋白Caspase 9和Bax/Bcl-2的表达水平(p<0.05)。在AngⅡ诱导的H9C2细胞中,Orai1、STIM1、Caspase 9和Bax/Bcl-2的水平与体内研究结果一致(P<0.05)。此外,达格列净抑制了STIM1和Orai1的聚集,并降低了细胞内钙离子浓度(P<0.01)。结论以上研究结果表明,达格列净缓解了AngⅡ诱导的心肌重构和凋亡,其心脏保护机制与抑制SOCE有关。更多还原

【Abstract】 BackgroundHypertension is one of the most common cardiovascular diseases and the leading cause of heart failure(HF).Patients with hypertension complicated by HF have poor prognoses and low survival rates,significantly impairing their quality of life.Cardiac remodeling(CR)is a common target organ damage associated with hypertension and a key pathological mechanism underlying HF.Cardiac remodeling exacerbates cardiomyocyte apoptosis,and the apoptotic cells further activate fibroblasts,promoting collagen fiber deposition.This vicious cycle accelerates the progression of cardiovascular diseases,ultimately leading to heart failure.Dapagliflozin(Dapa),a sodium-glucose cotransporter 2 inhibitor(SGLT2i),was initially developed to treat type 2 diabetes.However,recent studies have demonstrated that its cardioprotective effects are independent of its glucose-lowering properties.These effects may be associated with a reduction in intracellular calcium ion concentrations,although the precise mechanisms remain unclear.Store-operated calcium entry(SOCE),a critical calcium ion channel primarily mediated by Orai1 and stromal interaction molecule 1(STIM1),plays a pivotal role in the development and progression of cardiovascular diseases.However,whether Dapagliflozin exerts its cardioprotective effects by inhibiting SOCE remains unknown.ObjectiveThis study aimed to investigate the structural changes in cardiac remodeling(CR)induced by angiotensin Ⅱ(AngⅡ)in a rat model,focusing on the alterations in ventricular structure and the expression levels of Orai1,STIM1,Bax,Bcl-2,and Caspase 9 proteins.Furthermore,the study explored the effects of Dapagliflozin on AngⅡ-induced CR and its potential underlying mechanisms.MethodsIn the animal experiments,20 rats were randomly divided into four groups: Sham group,Dapa group,AngⅡ group,and AngⅡ+Dapa group.The Sham group underwent a sham operation followed by daily gavage with saline.The Dapa group also underwent a sham operation but received daily gavage with dapagliflozin post-surgery.The AngⅡ group had subcutaneous implantation of a mini-osmotic pump for continuous infusion of AngⅡ(200 ng/kg/min)and was administered saline daily via gavage post-surgery.The AngⅡ+Dapa group underwent AngⅡ pump implantation followed by daily gavage with dapagliflozin.After 28 days,the cardiac remodeling model was successfully established.Masson’s staining was performed to assess the degree of myocardial fibrosis,while TUNEL staining was used to analyze myocardial apoptosis.Western blot was utilized to detect the expression levels of SOCE-related proteins(Orai1 and STIM1)and apoptosis-related proteins(Bax,Bcl-2,and Caspase 9).In the cellular experiments,H9C2 cells were cultured and treated with appropriate concentrations of AngⅡ and Dapagliflozin(Dapa).Western blot was used to confirm the changes in protein expression levels mentioned above.Calcium imaging was performed to measure intracellular calcium ion concentration in H9C2 cells.Immunofluorescence was used to observe the aggregation of Orai1 and STIM1 proteins on the H9C2 cell membrane.ResultsDapagliflozin’s effects on improving ventricular remodeling and cardiomyocyte apoptosis in rats were independent of blood pressure and metabolic factors.Dapagliflozin alleviated cardiac remodeling,reduced myocardial fibrosis and apoptosis,and decreased the levels of AngⅡ-induced SOCE-related proteins Orai1,STIM1,and apoptosis-related proteins Caspase 9 and Bax/Bcl-2 in ventricular muscle tissue(p < 0.05).In AngⅡ-induced H9C2 cells,the levels of Orai1,STIM1,Caspase 9,and Bax/Bcl-2 were consistent with the in vivo findings(p < 0.05).Moreover,dapagliflozin inhibited the aggregation of STIM1 and Orai1 and reduced intracellular calcium ion concentration(p < 0.01).ConclusionsThese findings suggest that dapagliflozin mitigates AngⅡ-induced myocardial remodeling and apoptosis,and its cardioprotective effects are associated with the inhibition of SOCE.更多还原