Abstract
Background: Diabetic cardiomyopathy (DCM), which is characterised by myocardial fibrosis, is a major cause of mortality and morbidity in diabetic patients, and inhibition of cardiac fibrosis is a fundamental strategy for the treatment of DCM. Gastrodin (GAS), a compound extracted from Gastrodia elata, has a protective effect against DCM, but the molecular mechanism by which it exerts its antifibrotic effect has not been investigated.
Methods：In vivo, the effects of GAS on a high-sugar, high-fat (HSF) diet combined with streptozotocin (STZ)-induced DCM leading to DCM in C57BL/6 mice were investigated，this included small animal ultrasonography to assess cardiac function, detection of glucose-lipid metabolism, quantification of Sirius red staining, Mason staining, HE staining to observe histopathological changes in mouse heart tissue, protein immunoblotting, immunohistochemistry, and real-time fluorescence quantitative PCR to detect fibrosis-associated proteins as well as mRNA levels in mouse heart tissue. In vitro, neonatal rat cardiac fibroblasts (CFs) were transformed to myofibroblasts using high glucose combined with high palmitic acid (HG-PA) exposure, and CFs were induced by plasmid pEX-1 (pGCMV/MCS/EGFP/Neo) plasmid-mediated overexpression of KLK8 containing the rat KLK8 gene and by knockdown of KLK8 siRNA, to study the effect of GAS on CFs differentiation, collagen synthesis and cell migration by specific mechanisms of action of GAS.
Results：GAS attenuated pathological changes in the hearts of DCM mice, rescued impaired cardiac function, and attenuated cardiac fibrosis. Furthermore, based on molecular docking, GAS was found to bind to kinin releasing enzyme-related peptidase 8 (KLK8) to inhibit the increase of protease-activated receptor-1 (PAR-1) and thus attenuate the process of myocardial fibrosis. Specifically, GAS attenuated the transformation of neonatal rat cardiac fibroblasts (CFs) to myofibroblasts exposed to high glucose combined with high palmitic acid (HG-PA). KLK8 overexpression promoted CFs differentiation, collagen synthesis and cell migration, and KLK8 siRNA attenuated HG-PA-induced CFs differentiation, collagen synthesis and cell migration. Further studies revealed that PAR-1 antagonist but not PAR-2 antagonist attenuated CFs differentiation, collagen synthesis and cell migration. Importantly, GAS inhibited KLK8 upregulation and PAR1 activation thereby blocking differentiation, collagen synthesis and cell migration of HG-PA-exposed CFs and triggering TGF-β1/smad3 signalling.
Conclusion：GAS alleviated pathological changes in the heart of DCM mice induced by HSF diet combined with STZ, rescued impaired cardiac function and attenuated cardiac fibrosis. KLK8 mediates HG-PA-induced differentiation, collagen synthesis, and cell migration of CFs. GAS attenuated differentiation, collagen synthesis, and cell migration of CFs through inhibition of the KLK8-PAR1 signalling axis, a process in which TGF-β1/ smad3 are involved in this process.