Data Sheet 1_Astragaloside IV prevents calpain-1-mediated cardiac hypertrophy and fibrosis induced by diabetes.docx

ObjectiveAstragaloside IV (AsIV) has been reported to alleviate diabetes-induced endothelial dysfunction by inhibiting calpain-1. This study aimed to determine whether the same mechanism underlies its protective effect against diabetic cardiomyopathy (DCM).
MethodsAt the in vivo level, calpain-1 knockout mice with the genotype Capn1 EK684−/− (Capn1 EK684 knockout mice) were used to establish a type 2 diabetic cardiomyopathy model. At the in vitro level, H9c2 cells and cardiac fibroblasts were stimulated with high glucose to construct corresponding models. Meanwhile, a calpain-1 overexpression lentivirus was constructed to assess the effect of calpain-1 on myocardial cell injury. Different doses of AsIV were then used to intervene in diabetic mice and H9c2 cells. Body weight, blood glucose, myocardial hypertrophy, myocardial fibrosis, cardiac function, Ca2+ overload and its regulation, myocardial cell apoptosis and oxidative stress were evaluated in the current study.
ResultsAsIV could not completely normalize blood glucose in mice, but could significantly improve cardiac systolic and diastolic function, myocardial hypertrophy and fibrosis. The beneficial effect of calpain-1 gene knockout on diabetic cardiomyopathy was similar to that of AsIV, and calpain-1 knockout did not further enhance the beneficial effect of AsIV. Calpain-1 overexpression abolished the beneficial effect of AsIV on high glucose induced H9c2 cell injury and fibroblast proliferation. In addition, the intracellular Ca2+ overload, abnormal levels of sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), phosphorylation of phospholamban (p-PLN) and ryanodine receptor 2 (p-RyR2), apoptosis and oxidative stress associated with DCM were also improved by AsIV or calpain-1 knockout, and AsIV has the capacity to suppress the overactivation of calpain-1 and calcium/calmodulin-dependent protein kinase Ⅱ (CaMKII).
ConclusionsAsIV could ameliorate intracellular Ca2+ overload, apoptosis, and oxidative stress by regulating the calpain-1/CaMKII pathway, thereby improving myocardial hypertrophy and fibrosis caused by diabetes mellitus.

研究目的：已有研究表明，黄芪甲苷IV（Astragaloside IV，AsIV）可通过抑制钙蛋白酶1（calpain-1）缓解糖尿病诱导的内皮功能障碍。本研究旨在明确其对糖尿病心肌病（diabetic cardiomyopathy，DCM）的保护作用是否由相同机制介导。
实验方法：体内实验层面，本研究采用基因型为Capn1 EK684−/−的钙蛋白酶1基因敲除小鼠，构建2型糖尿病心肌病模型；体外实验层面，以高糖刺激H9c2细胞与心脏成纤维细胞构建对应疾病模型。同时，构建钙蛋白酶1过表达慢病毒，以评估钙蛋白酶1对心肌细胞损伤的影响。随后，使用不同剂量的AsIV对糖尿病小鼠及H9c2细胞进行干预。本研究评估的指标包括：小鼠体重、血糖水平、心肌肥厚、心肌纤维化、心功能、钙超载及其调控机制、心肌细胞凋亡与氧化应激水平。
实验结果：AsIV无法完全使糖尿病小鼠的血糖恢复至正常水平，但可显著改善其心脏收缩与舒张功能、心肌肥厚及纤维化症状。钙蛋白酶1基因敲除对糖尿病心肌病的保护作用与AsIV相似，且钙蛋白酶1敲除不会进一步增强AsIV的保护效果。钙蛋白酶1过表达可抵消AsIV对高糖诱导的H9c2细胞损伤及成纤维细胞增殖的保护作用。此外，AsIV或钙蛋白酶1基因敲除均可改善糖尿病心肌病相关的细胞内钙超载、肌浆网/内质网钙ATP酶2a（sarco/endoplasmic reticulum Ca2+-ATPase 2a，SERCA2a）水平异常、受磷蛋白磷酸化（phospholamban，p-PLN）与兰尼碱受体2磷酸化（ryanodine receptor 2，p-RyR2）异常、细胞凋亡及氧化应激。同时，AsIV可抑制钙蛋白酶1与钙/钙调蛋白依赖性蛋白激酶Ⅱ（calcium/calmodulin-dependent protein kinase Ⅱ，CaMKII）的过度激活。
研究结论：AsIV可通过调控钙蛋白酶1/钙/钙调蛋白依赖性蛋白激酶Ⅱ（calpain-1/CaMKII）通路改善细胞内钙超载、细胞凋亡与氧化应激，进而缓解糖尿病引发的心肌肥厚与纤维化。