Phlorizin attenuates lupus nephritis via upregulating PI3K/Akt pathway-mediated Treg differentiation

Background: Lupus nephritis (LN) leads to widespread kidney damage and nephron loss, establishing it as a major contributor to acute and chronic kidney injury, which can progress to end-stage renal disease. Phlorizin (PHZ), a major pharmacologically active constituent derived from Lithocarpus polystachyus Rehd., has been shown to exhibit significant immunomodulatory and anti-inflammatory properties. Growing evidence indicates that PHZ may exert a protective influence on kidney function. However, the therapeutic effect and mechanism of PHZ in treating LN need to be elucidated.

Methods: The PHZ-associated targets were identified through tools such as PharmMapper, SwissTargetPrediction, SuperPred and Targetnet. Simultaneously, LN-associated target spots were retrieved fromOMIM, DisGeNET, GeneCards, and GEO databases. Additionally, Venny 2.1.0 was employed to analyze the overlap between drug targets and disease targets. Following this, the DAVID software was employed to perform enrichment analyses for GO terms and KEGG pathways on the shared drug-disease target sites. Following this, the construction of protein-protein interaction (PPI) networks for these intersecting targets was carried out using the STRING database and Cytoscape software, aiming to pinpoint critical targets. Ultimately, molecular docking alongside dynamic simulations was used to evaluate the binding affinity between PHZ and the critical genes. Based on these findings, PHZ or Dexamethasone (DXSM) was administered to female MRL/lpr mice, which are predisposed to lupus. The therapeutic effects of PHZ on LN were evaluated by assessing renal function and the degree of kidney inflammation. Concurrently, flow cytometry was employed to measure the percentage of CD4⁺ T cell subsets. Additionally, relevant signaling pathways were examined through western blot analysis. Furthermore, CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells were induced in vitro. Flow cytometry and immunoblotting were performed to confirm the role and mechanism of PHZ in Treg cell differentiation.

Results: The PHZ compound specifically targeted 161 genes associated with LN. PPI analysis revealed that among all the target genes, Akt1, ALB, MMP9, HSP90AA1, and NF-κB1 exhibited the highest centrality. KEGG pathway analysis suggested that the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway could play a crucial role in the treatment of LN. Molecular docking revealed that PHZ exhibits a strong affinity for binding with Akt1. Experimental studies, both in vitro and in vivo, showed that PHZ might alleviate LN by promoting Treg differentiation via activation of the PI3K/Akt signaling pathway.

Conclusions: Integrating network pharmacology, bioinformatics, and experimental validation, our study systematically deciphers the therapeutic efficacy and molecular mechanisms of PHZ against LN. Network pharmacology analysis and bioinformatics suggested PI3K/Akt signaling as the pivotal pathway to treat LN, while subsequent in vivo and in vitro experiments confirmed that PHZ exerts its therapeutic effects through activating the PI3K/Akt signaling pathway, ultimately driving FOXP3-dependent regulatory T cell differentiation.

Lupus nephritis (LN); Network pharmacology; PI3K/AKT; Phlorizin (PHZ); Treg cells.