Study of the Role and Mechanism of Asprosin/Spartin Pathway in Cardiac Microvascular Endothelial Injury Induced by Diabete Mellitus

Abstract

To detect the effects and mechanism of asprosin (Asp) and spartin on the injury of mice cardiac microvascular endothelial cells (CMECs) induced by high glucose.  Methods  The cultured CMECs were divided into 2 groups, one group is normal group (5.5 mmol/L glucose in the medium) and another is HG group (30 mmol/L glucose in the medium). Real-time PCR (qRT-PCR) and Western blot were respectively used to detect the mRNA level of spastic paraplegia 20 (SPG20) and protein expression of spartin in CMECs. Upregulation or downregulation of the expression of spartin was achieved via transfection with adenovirus (Ad) or small interfering RNA (siRNA) respectively. CMECs with downregulation of spartin expression were firstly treated with anti-oxidant N-acetylcysteine (NAC) or Asp respectively for 48 h, and then were interfered with 30 mmol/L glucose for 24 h afterward. The apoptosis of cell was detected by flow cytometry. Nitric oxide (NO) production was detected by NO probe and ELISA kit. The intracellular reactive oxygen species (ROS) levels were tested by DHE staining and ELISA kit. Type 2 diabetic model mice were established and then divided into T2DM group and T2DM+Asp group. After the model mice were established successfully (random blood glucose was more than 16.7 mmol/L), Asp (1 μg/g) was intraperitoneally injected once a day. After 2 weeks, mice echocardiography was performed to test cardiac diastolic function. The integrity of the microvascular endothelium was observed by scanning electron microscopy.  Results  Compared with the normal group, the mRNA level of SPG20 and protein expression of spartin in mice CMECs of HG group were significantly reduced (P < 0.05). Under the condition of high glucose, Ad transfection induced significant decrease of the intracellular ROS level and the apoptosis level of the CMECs (P < 0.05), while NO increased after Ad transfection. In contrast, siRNA intervention resulted in opposite effect. In addition, the antioxidant NAC partly reversed the above changes caused by downregulating spartin. Asp upregulated the level of SPG20 mRNA and spartin protein expression in CMECs, reduced ROS production, reduced apoptosis and increased NO production. However, intervention effects of Asp, such as decreasing of ROS production, inhibiting apoptosis of CMECs and increasing of NO production, were partly reversed in spartin downregulated cells. In vivo, we found that Asp can improve cardiac function and increase the integrity and smoothness of cardiac microvascular endothelium in type 2 diabetic mice.  Conclusion  Asp can inhibit oxidative stress in mice CMECs through upregulating spartin signaling pathway, thereby alleviating the damage of microvascular endothelium in diabetic heart.

Keywords: Diabetes, CMECs, Asprosin, Spartin, Oxidative stress