Experimental validation of the molecular mechanism of phlorizin in the treatment of diabetic retinopathy.

Abstract

This study conducted an experiment to scrutinize the effect of phlorizin (Phl) on diabetic retinopathy (DR) and to delve into the related molecular mechanisms. Within this investigation, DR was induced in rats with diabetes mellitus (DM) by subjecting them to a regimen involving a high-fat and high-sugar diet, coupled with intraperitoneal administration of streptozotocin (STZ) at a dosage of 45 mg/kg. Retinal damage in DR rats was assessed by means of hematoxylin and eosin (HE) staining. The serum levels of inflammatory and angiogenic factors were also measured. Additionally, the levels of tight junction proteins, angiogenic proteins, and inflammatory proteins in the retinas of DR model rats were assessed using western blot (WB),immunohistochemistry(IHC) and immunofluorescence(IF). Moreover, bioinformatics and network pharmacology methodologies were utilized to pinpoint intersecting genes linked to DR and to elucidate the mechanism of action of Phl. This involved screening with Venny, conducting Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)analyses, constructing a Protein-Protein Interaction (PPI) network, and performing molecular docking analysis. The results of this study demonstrated that Phl significantly normalized fasting glucose levels and reduced body weight, thereby alleviating obesity in DR rats after 12 weeks. Furthermore, the serum levels of inflammatory and angiogenic factors were considerably reduced in the drug-treated rats. WB, IHC and IF revealed increased expression of the tight junction proteins zonula occludens-1(ZO-1) and occludin in the retinas of drug-treated DR rats, validating the observed findings. Molecular biology validation experiments based on the predictions by network pharmacology indicated a substantial decrease in the expression levels of vascular endothelial growth factor (VEGF), notch homolog 1 (Notch1), and hypoxia inducible factor-1 (HIF-1α) in the retina upon treatment with Phl. This reduction resulted in the inhibition of neovascularization. Furthermore, Phl exhibited inhibitory effects on inflammatory pathways, leading to a decrease in cytokine release. The overexpression of VEGF was identified as a factor diminishing brain-derived neurotrophic factor(BDNF) expression while increasing the expression levels of inflammatory proteins. Therefore, the results of this research demonstrate that Phl has the potential to protect the retina of DR rats by inhibiting VEGF expression. This protective effect may be associated with the modulation of the VEGF/BDNF/NF-κB signaling pathway.