# Mafu Yishen Formula ameliorates membranous nephropathy by promotion of regulatory T cell differentiation: a multi-omics study

## TL;DR

A traditional Chinese formula called Mafu Yishen improves kidney disease by boosting immune cells that reduce inflammation and tissue damage.

## Contribution

This study reveals that Mafu Yishen Formula promotes regulatory T cell differentiation via the PI3K/AKT pathway in membranous nephropathy.

## Key findings

- MFYS reduces urinary protein and improves kidney pathology in PHN rats.
- MFYS increases Treg cells and modulates cytokine levels (IL-2, IL-10 up; IL-6, IL-17 down).
- MFYS activates PI3K/AKT and mitochondrial oxidative phosphorylation while inhibiting pSTAT3.

## Abstract

Primary membranous nephropathy (PMN) is one of the main causes of nephrotic syndrome in adults, with edema as the primary symptom. The Mafu Yishen Formula (MFYS) is derived from a combination of classical prescriptions for treating edema diseases recorded in the Treatise on Cold Damage and Miscellaneous Diseases. While clinical efficacy of MFYS in treating PMN has been supported by evidence-based studies, its specific mechanisms remain unclear. This study aims to investigate the therapeutic effects and molecular mechanisms of MFYS in the treatment of membranous nephropathy, with a focus on whether MFYS promotes regulatory T cells (Treg) differentiation and modulates immune responses.

The passive Heymann nephritis (PHN) rat model was employed to simulate human PMN. Rats were treated with either low- or high-dose Mafu Yishen Formula (MFYS) or cyclosporine A (CsA) as a positive control. Urinary protein levels, serum biochemical parameters, renal pathological changes, and podocyte injury were evaluated. Immunofluorescence and flow cytometry were used to assess renal IgG deposition, B cell proliferation, and the proportion of Treg. Serum cytokine levels were measured using appropriate assays. The absorbed components of MFYS were identified via metabolomic analysis. Integrated strategies including network pharmacology, spleen transcriptomics, and proteomics were applied to identify key targets and signaling pathways. In vitro Treg polarization assays, supplemented with pathway inhibitors, were conducted to validate mechanistic findings. Molecular docking simulations were performed to explore interactions between active components of MFYS and potential target proteins.

MFYS significantly reduced urinary protein levels in PHN rats, improved biochemical indicators such as serum albumin and blood lipids. Pathological examination revealed that MFYS alleviated glomerular and podocyte injury, while also reducing intrarenal IgG deposition and suppressing splenic B cell activation. Serum tests indicated that MFYS increased levels of IL-2 and IL-10 while decreasing levels of IL-6 and IL-17. Notably, MFYS significantly increased the proportion of Tregs. Integrated network pharmacology and multi-omics analysis consistently revealed that MFYS upregulates the PI3K/AKT signaling pathway and enhances mitochondrial oxidative phosphorylation. Furthermore, MFYS intervention down regulated pSTAT3 expression while promoting PGC-1α expression. In vitro experiments further confirmed that MFYS directly promotes the differentiation of naïve T cells into Tregs, an effect that was counteracted by a PI3K/AKT pathway inhibitor. Molecular docking results suggested that some active components of MFYS can bind to AKT1.

MFYS modulates the immune response in PHN rats, reduces IgG deposition, and ameliorates renal and podocyte injury. It promotes Treg differentiation, regulates the cytokine network, and exerts multi-target effects on both inflammation and metabolism.

The online version contains supplementary material available at 10.1186/s13020-025-01272-1.