ENTERIC GLIAL INFLAMMATION IN EARLY PARKINSON’S DISEASE: MODULATORY EFFECTS OF RAS INHIBITORS ON TLR4/NLRP3 SIGNALING THROUGH INTEGRATED <EM >IN VITRO</EM> AND <EM>IN SILICO</EM> ANALYSIS

Parkinson’s disease (PD) is increasingly recognized as a multisystem disorder where neuroinflammation within the enteric nervous system may serve as a critical initiator of pathology before the onset of motor symptoms. Given that enteric glial cells (EGCs) are central mediators of gut-derived inflammation, targeting their inflammatory response offers a promising neuroprotective strategy. This study aimed to evaluate the therapeutic potential of the renin-angiotensin system (RAS) inhibitors, losartan and captopril, against rotenone-induced inflammation in EGCs, specifically focusing on the TLR4/NLRP3 inflammasome axis. Following the determination of non-cytotoxic dose ranges (10-100 μM) via viability assays, EGCs were exposed to 1 μM rotenone. Caspase-1 and IL-1beta levels were quantified via ELISA, and mRNA expression of TLR4/NLRP3 was assessed by RT-qPCR. Molecular docking was performed to predict drug interactions with TLR4 and NLRP3 proteins. Rotenone significantly triggered the inflammatory cascade, as evidenced by elevated caspase-1 and IL-1beta levels alongside upregulated TLR4 and NLRP3 mRNA expression. Notably, losartan demonstrated superior potency, significantly attenuating NLRP3 expression at a low dose (10 μM), while captopril required a tenfold higher concentration to achieve comparable efficacy. Paradoxically, losartan and captopril alone increased caspase-1 and/or TLR4 expression, suggesting a complex regulatory role within the enteric environment. Molecular docking showed a stronger binding affinity for losartan compared to captopril toward TLR4/NLRP3 proteins, consistent with observed biochemical effects. Overall, these results underscore the potential of RAS modulation in mitigating early-stage neuroinflammatory signaling, positioning the gut-brain axis as a critical therapeutic target for prodromal PD intervention.