Mechanistic Insights into Shenzhuo Formula for Diabetic Retinopathy: Integrating UPLC-Q-TOF-MS/MS, Network Pharmacology, Single-Cell RNA Sequencing Data, and Experimental Validation.

PURPOSE: For early-stage Diabetic retinopathy (DR), various pharmacological agents and neuroprotective factors have been developed. However, these treatments often show limited efficacy, especially when initiated after retinal damage, and may cause adverse effects. Therefore, there is an urgent need to develop safer and more effective therapeutic strategies for early-stage DR. Shenzhuo Formula (SZF), a modified classical traditional Chinese medicine prescription, has shown promising clinical efficacy in early-stage DR treatment. This study aims to investigate the underlying mechanisms of SZF to expand treatment strategies for DR. METHODS: SZF components were analyzed using Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry/Mass Spectrometry (UPLC-Q-TOF-MS/MS). Db/db mice received three different SZF doses for 12 weeks. Physiological parameters, including water and food consumption, body weight, and urine output, were monitored. Blood samples were analyzed for fasting blood glucose and other relevant parameters. Ocular changes were assessed using fundus photography (FP), fundus fluorescein angiography (FFA), optical coherence tomography (OCT) and hematoxylin and eosin (H&E). Network pharmacology analysis (NP) identified potential SZF targets, while immunofluorescence staining evaluated SZF's mechanism in delaying DR progression. The distribution of SZF pharmacological targets in critical DR target cells was analyzed using single-cell data from the GSE245561 dataset. Molecular docking predicted SZF-target interactions. RESULTS: SZF improved diabetic symptoms, increased retinal thickness, and reducedvascular leakage and microcirculation issues. The HIF-1α-VEGFA axis was suggested as a potential core target. Single-cell analysis of clinical samples suggested macrophages as a common target cell for HIF-1α and VEGFA. Molecular docking identified effective SZF components. CONCLUSION: Results indicate that SZF may impede the progression of DR by inhibiting the HIF-1α-VEGFA signaling pathway in macrophages, with quercetin and apigenin identified as significant contributors, though further experimental validation is needed to confirm these mechanistic.