Sanqi Qushi formula alleviates podocyte damage in passive Heymann Nephritis rats by inhibiting GPX4 deficiency-mediated ferroptosis via the JNK/FoxO1 signaling pathway.

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Citation abstract

BACKGROUND: The Sanqi Qushi Formula (SQQS) is a traditional Chinese medicine comprising seven herbs, namely Astragali Radix, Curcumae Rhizoma, Notoginseng Radix Et Rhizoma, Radix Padoniae Rubra, Smilacis Glabrae Rhizoma, Atractylodis Macrocephalae Rhizoma, and Cordyceps Sinensis. While SQQS is widely used in clinics to treat membranous nephropathy (MN), the molecular mechanisms responsible for its therapeutic effects remain unclear.

PURPOSE: This study sought to explore the nephroprotective activity of SQQS, as well as its molecular mechanism in treating MN.

METHODS: The nephroprotective effects of SQQS were assessed in experimental Passive Heymann nephritis (PHN) rats. The chemical components of SQQS's freeze-dried powder and rat plasma were identified using UPLC-Q-Exactive-MS. For the mechanism exploration, network pharmacology was integrated with RNA-seq transcriptomics to explore its underlying multi-dimensional correlations. To investigate whether SQQS could alleviate podocyte damage by inhibiting GPX4 deficiency-mediated ferroptosis, a RSL3-induced podocyte model was established. The protein expression in the candidate signaling pathway was evaluated. Molecular docking experiments, molecular dynamics simulation analyses (MDS), bioactivity tests, and surface plasmon resonance technology (SPR) were conducted to explore the pharmacodynamic molecule of SQQS.

RESULTS: SQQS administration significantly ameliorated 24 h proteinuria, elevated serum albumin (ALB), and reduced the levels of serum creatinine (Scr), blood urea nitrogen (BUN), and total cholesterol (TC). Besides, SQQS alleviated glomerular pathological damage in PHN rats, decreased IgG and C3 deposition, and reversed the aberrant expression of podocyte damage-related protein. Moreover, SQQS treatment alleviated mitochondrial injury, upregulated the levels of glutathione peroxidase 4 (GPX4), cystine/glutamate antiporter xCT, glutathione (GSH), and superoxide dismutase (SOD), and reduced malondialdehyde (MDA) content and Acyl-CoA synthetase long chain-family member 4 (ACSL4) expression, as well as iron deposition in PHN rats. A total of 248 compounds were detected in the freeze-dried powder of SQQS, with 105 compounds identified in the rat plasma, of which 41 components were common to both in vitro and in plasma samples. Integrative investigations using network pharmacology and RNA-seq transcriptomics identified the JNK/FoxO1/GPX4 signaling pathway as a candidate therapeutic target. Further studies exhibited that SQQS reduced nuclear FoxO1 and p-JNK, and elevated p-FoxO1 and total FoxO1. In vitro experiments with SQQS-containing serum (SQQSCS) increased cell viability and reduced Fe 2+ levels, oxidative stress, lipid peroxidation, and mitochondrial injury. Furthermore, our results indicated that SQQSCS yielded a significant regulatory effect on the proteins of the JNK/FoxO1/GPX4 signaling pathway. A core protein intervention experiment showed that SQQS promotion of GPX4 expression was related to the nuclear translocation of FoxO1, regulated by p-JNK. Finally, molecular docking, molecular dynamics simulations, bioactivity tests, and SPR studies showed that Baicalein and Paeoniflorin, two major compounds in SQQS, upregulated the expression of GPX4 to inhibit ferroptosis in PHN rats by targeting JNK1.

CONCLUSIONS: These results suggest that SQQS may alleviate podocyte damage by inhibiting GPX4 deficiency-mediated ferroptosis through targeting JNK1 to decrease the nuclear translocation of FoxO1.