Systems-Level Molecular and Immunological Evidence Identifies Th17/Treg Modulation as a Key Mechanism of CRSJ's Neuroprotection in Parkinson's Disease

Xun Li¹Xiyu Li²ShiYa Chen²Lin Wang¹Jinyan Xia¹Meiling Zheng¹Chutian Zhang³XiaoQian Chen²Jing Cai^4,5,6*

• ¹School of Integrative Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, China
• ²The Third Affiliated People's Hospital, Fujian University of Traditional Chinese Medicine, Fuzhou, China
• ³Department of Neurology, Hubei Provincial Hospital of Traditional Chinese Medicine, Wu Han, China
• ⁴The Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou, China
• ⁵The Third Affiliated People's Hospital, Fujian University of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, China
• ⁶Clinical Research Center for Traditional Chinese Medicine on Glucolipid Metabolic Disorders of Fujian Province, Fuzhou, China

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder in which neuroinflammation plays a central role. Congrong Shujing Granules (CRSJ), a traditional Chinese medicine formula, have shown clinical benefits in PD, yet their immunomodulatory mechanisms remain unclear. Methods: We investigated the effects of CRSJ on Th17/Treg immune balance. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used to identify representative chemical constituents of CRSJ. Representative CRSJ compounds were characterized, and their binding affinities were evaluated by molecular docking and molecular dynamics simulations. An MPTP-induced PD mouse model was established and treated with CRSJ. Behavioral outcomes, dopaminergic neuroprotection, immune cell subsets, transcriptomic profiles, and cytokine networks were assessed using flow cytometry, RNA sequencing, multiplex assays, immunofluorescence, and Western blotting. Results: HPLC analysis identified 44 representative compounds in CRSJ spanning multiple chemical classes associated with immunomodulatory, neuroprotective, and antioxidant activities. Molecular-level prioritization of CRSJ-derived serum constituents highlighted paeoniflorin as a key Th17/Treg balance immunoregulatory candidate, exhibiting stable interactions with RORγt, Foxp3, and α-synuclein in molecular docking and molecular dynamics simulations. In an MPTP-induced Parkinson's disease mouse model, CRSJ treatment dose-dependently improved motor performance, preserved dopaminergic neurons, and reduced striatal α-synuclein accumulation. Transcriptomic profiling revealed CRSJ-associated shifts toward regulatory immune programs, characterized by attenuation of Th17-related signatures and enhancement of Treg-associated pathways, accompanied by consistent modulation of the TGF-β/SMAD3 signaling axis. These molecular changes were supported by protein-level validation. CRSJ further alleviated neuroinflammation by promoting microglial M1/M2 polarization and partially normalizing dysregulated cytokine and chemokine profiles. Integrated immunological analyses demonstrated restoration of Th17/Treg balance and suppression of CX3CL1/CX3CR1–Th17 signaling, collectively supporting an immuno-neuroprotective profile of CRSJ in PD. Conclusion: CRSJ exerts neuroprotective effects in PD by restoring Th17/Treg homeostasis and suppressing neuroinflammatory pathways, supporting its potential as an immunomodulatory therapy.