| Naringin is a natural flavonoid with antioxidant,antibacterial,anti-inflammatory and other biological activities.In this experiment,We used network pharmacology to develop the "PPI" network and identify the biological functions and signaling pathways of naringin’s key targets in the treatment of diabetic retinopathy.In vitro,human retinal pigment epithelial-19(ARPE-19)cells were to explore the protective effect and potential mechanism of naringin on methylglyoxal(MGO)-induced cell damage.We hope to provide a direction for the development and application of naringin in the field of health products.In the network pharmacology experiment,Symmap,SWISS,Pharmmaper databases and literature search were used to identify naringin-related targets.Similarly,Genecards,TTD and Grug Bank databases were used to identify diabetic retinopathy-related targets.After screening,the key targets of naringin in the treatment of diabetic retinopathy were obtained.Subsequently,The “network analyzer” plugin was used to identify three core targets from the key targets,and Discover Studio 3.5 software was used to confirm their binding ability to naringin.In addition,the biological processes and potential pathways of key targets were analyzed using GO and KEGG functions in the Metascape database.In vitro experiment,a model of MGO-induced ARPE-19 cell injury was established.Then various concentrations of naringin(25,50,75 μmol/L)were used to pretreat cells for 2 h,followed by MGO damage for 36 h.MTT was used to measure cell viability,lactate dehydrogenase(LDH)assay was used to detect cell cytotoxicity,and 2’,7’-dichlorofluorescein(DCFH-DA)was used to assess ROS levels.In addition,related assay kits were used to assess the activity of endogenous antioxidant enzymes(SOD,CAT and GSH-Px),the fluorescent dye of Hoechst33342 and JC-1 were used to detect cell apoptosis and mitochondrial membrane potential,respectively.Moreover,we used a powerful realtime RT-PCR technique to examine the m RNA expression levels of related genes.The results of the network pharmacology showed that a total of 384 naringin-related targets and 2043 diabetic retinopathy-related targets were collected,and we screened 47 key targets of naringin in the treatment of diabetic retinopathy.According to the PPI network of 47 key targets,signal transducer and activator of transcription 3(STAT3),serine/threonine protein kinase(AKT1),and epidermal growth factor receptor(EGFR)are the core targets which have strong binding force with naringin that was verified by molecular docking.In addition,the results also showed that naringin in the treatment of diabetic retinopathy is mainly involved in biological processes such as inflammation and apoptosis,and may play a role in neurodevelopment by regulating AGE-RAGE,PI3K/Akt,MAPK and other related pathways.The results of in vitro experiments showed that naringin pretreatment increased the viability of ARPE-19 cells and and alleviated cytotoxicity induced by MGO in a concentration-dependent manner.Meanwhile,naringin reduced the level of cellular oxidative stress by reducing ROS production,increasing SOD,CAT and GSH-Px antioxidant enzyme activities,and reducing MDA content.Moreover,naringin increased mitochondrial membrane potential and alleviated the situation of apoptosis.In addition,naringin also increased the m RNA expression levels of PI3 K and Akt genes,following regulated the expressions of NF-κB,TNF-α,IL-6 inflammation-related genes and Bcl-2,Bax,and Caspase-3 apoptosis-related genes.In summary,naringin has antioxidant effects and may protect ARPE-19 cells from MGO-induced damage by regulating downstream inflammation and apoptosis-related genes through the PI3K/Akt signaling pathway. |
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