Managing circadian rhythm involved in alleviating membranous nephropathy by alkaloids of Alstonia scholaris

Background: Alstonia scholaris (L.) R. Br. is used in traditional Chinese medicine to treat kidney-related "water diseases"; however, studies on its use in treating membranous nephropathy remains limited.

Purpose: To explore the therapeutic components of A. scholaris and signal pathway in treating membranous nephropathy.

Methods: Bioinformatics was used to predict the therapeutic potential of major Alkaloids from A. scholaris, which were verified using a MPC5 inflammation model in vitro and mice membranous nephropathy model in vivo. Signal pathway of A. scholaris on membranous nephropathy was predicted using network pharmacology and then supported by transcriptome analysis of renal tissue. Additionally, molecular docking and dynamic simulations were selected to predict targets of Alkaloids binding, and validated using reverse transcription quantitative polymerase chain reaction and Western blotting at gene and protein levels.

Results: Four major Alkaloids (picrinine, vallesamine, scholaricine, and 19-epi-scholaricine) reduced urinary protein generation, decreased glomerular basement membrane thickness and immunoglobin G/C3 deposition, alleviated oxidative stress and inflammatory mediator release, and significantly mitigated podocyte Apoptosis in treating membranous nephropathy, in which compared with prednisone at 2 mg/kg, 19-epi-scholaricine (1 mg/kg) showed better therapeutic effect in vivo. Notably, the core circadian clock protein ARNTL showed strong binding affinity to Alkaloids with stable complex energy during 200 ns simulations in silico, which was further supported by Western blotting analysis, with significant up-regulation of ARNTL and CRY1, and down-regulation of phosphorylated Akt/mTOR and HSP90AA1. Additionally, after treatment with Alkaloids in vitro and in vivo, the expression of key genes Akt, mTOR, and Bcl-2/Bax in the Akt/mTOR pathway, and Arntl, Cry1, and Per3 in the circadian rhythm were increased, indicating that circadian rhythm regulation was involved in treating membranous nephropathy for the first time.

Conclusion: Following up experimental validation in vitro and in vivo after prediction in silico step by step, Alkaloids of A. scholaris exhibited protective effects on membranous nephropathy through the ARNTL/Akt/mTOR pathway to reduce urinary protein generation, alleviate oxidative stress and Apoptosis, and maintain normal renal physiological function.

AKT/mTOR pathway; Alstonia scholaris; Bioinformatics; Circadian rhythm; Membranous nephropathy.