IDF21-0158 Aegeline mitigates high glucose induced renal cell fibrosis by alleviating ROS generation & augmenting hydrogen sulphide

Abstract

Background: Diabetic kidney disease is a complex multifactorial disease, characterised by excessive synthesis and deposition of extracellular matrix proteins (ECM), leading to the glomerulosclerosis and interstitial fibrosis. Innovative treatment strategies to combat DKD are needed in addition to the existing treatment methods as they are not very much effective in preventing the progression of the disease. Previous studies showed the protective role of Aegle marmelos (AM) in the treatment of diabetes mellitus and kidney injury. Aegeline, an active phytochemical identified in the leaves of AM has shown to have antihyperglycemic, antidyslipidemic, and antioxidant properties but no study has explored its molecular mechanisms in combating high glucose induced renal cell injury. So, we aimed to study the efficacy and the molecular mechanism of Aegeline, in high glucose induced renal cell fibrosis. Aim: We aimed to elucidate the role of aegeline in high glucose induced renal cell fibrosis and to study its mechanistic role in the regulation of hydrogen sulphide, a gasotransmitter which attenuates renal cell fibrosis. Method: Expression level of α-SMA (fibrotic marker) was analysed using western blotting. We also analysed fibrotic events such as cell migration, proliferation and trans-well invasion in kidney Proximal Tubular Epithelial Cells (NRK-52E) after Aegeline (10µM) and HG treatment (25mM). Generation of reactive oxygen species was measured using fluorescent probes and NADPH oxidase-4 (NOX4) protein expression was analysed by immunoblotting. Hydrogen sulphide (H2S) level was measured by methylene blue assay and its synthesising enzyme Cystathionine β synthase (CBS) expression level was analysed by immunoblotting. Also, we studied the protein interaction of aegeline with the NOX4 and other related proteins using molecular docking (Auto dock). The statistical analysis was performed using GraphPad Prism software (GraphPad Prism Software, USA). All the experiments were performed for a minimum of three independent experiments. Results: Aegeline treatment alleviates the HG induced fibrosis by significantly reducing the expression level of fibrotic marker (α-SMA) and also impedes the fibrotic events such as migration, invasion and cell proliferation. Additionally, aegeline treatment significantly reduced the high glucose induced ROS generation. Molecular docking studies showed that aegeline had high binding conformation with NOX4 and CBS protein and also exhibit good drug-likeness property. We showed that aegeline treatment significantly reduced the NADPH oxidase activity and NOX4 expression. Further, high glucose treatment significantly decreased the level of H2S which may be the reason for the increase in ROS generation and fibrosis. Aegeline treatment significantly increased the level of H2S and the CBS expression under high glucose condition. Discussion: Oxidative insult and ROS generation are important processes in the progression of renal cell fibrosis under high glucose conditions. H2S exerts promising protective role against renal cell injury by attenuating oxidative stress and inflammation. Hence, an increase in this gasotransmitter (H2S) will eventually diminish the progression of renal cell injury. Here we have observed a protective effect of aegeline in high glucose mediated kidney cell fibrosis by subjugating fibrotic events, ROS generation and by augmenting hydrogen sulphide.