Background: The pathological hallmarks of Alzheimer's disease (AD) include microglial polarization toward the pro-inflammatory M1 phenotype and significant neuronal apoptosis. Overactivation of transient receptor potential vanilloid 4 (TRPV4) channels has been identified as a common feature in AD animal models associated with microglial activation and neuronal injury. However, the downstream molecules and signaling pathways regulated by TRPV4 remain incompletely understood. Therefore, this study aimed to elucidate the mechanisms by which TRPV4 promotes microglial M1 polarization and its subsequent effect on hippocampal neuronal apoptosis.

Methods: BV-2 cells were employed as an in vitro model to mimic microglial function, while HT22 cells represented hippocampal neurons. BV-2 cells were treated with the TRPV4 activator GSK1016790A, the TRPV4 antagonist HC-067047, and exogenous recombinant human transforming growth factor-beta 1 (TGF-β1) protein. Following treatment, phagocytic activity was evaluated by co-culturing BV-2 cells with fluorescent microspheres. Enzyme-linked immunosorbent assay was performed to quantify cytokine levels in the supernatant. The proportion of M1 phenotype cells was determined by immunofluorescence staining. Additionally, TGF-β1 protein expression and NF-κB pathway activation were evaluated via Western blot analysis. Subsequently, supernatants from BV-2 cells were collected as a conditioned medium to culture HT22 cells. Cell viability of HT22 cells was assessed using the MTT assay, and apoptosis was measured via Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.

Results: TRPV4 activation significantly enhanced the phagocytic activity and pro-inflammatory cytokine release in BV-2 cells, elevated M1 phenotype marker expression, reduced intracellular TGF-β1 protein levels, and activated the NF-κB signaling pathway (p < 0.05). Additionally, TRPV4 activation impaired HT22 cell viability and significantly promoted apoptosis (p < 0.01). However, treatment with HC-067047 or exogenous TGF-β1 partially reversed the adverse effects of TRPV4 activation on BV-2 and HT22 cells.

Conclusion: In vitro models demonstrated that TRPV4 activation downregulates TGF-β1 expression and activates the NF-κB signaling pathway, thereby enhancing BV-2 microglial polarization toward the M1 phenotype and accelerating neuronal apoptosis. These findings provide a theoretical foundation for developing novel AD therapies targeting TGF-β1, highlighting its potential clinical significance.