Abstract

Introduction: Neuroinflammation, a key innate immune response in the CNS, is mediated by inflammasomes, notably nucleotide-binding oligomerization domain and leucine-rich repeat pyrin domain containing protein 3 (NLRP3). This NLRP3 inflammasome plays an important role in the activation of pro-inflammatory caspase-1, which further initiates secondary inflammatory response leading to neuronal injury. 5' adenosine monophosphate-activated protein kinase (AMPK), crucial for energy regulation, can modulate microglial phenotypes from pro-inflammatory to anti-inflammatory, potentially mitigating neuroinflammation-related diseases. However, the correlation between AMPK and NLRP3 in neuroinflammation remains unexplored. Targeting NLRP3 inflammasome holds therapeutic promise for neurodegenerative disorders, though research is ongoing.
Aim: The present study is aimed at studying the effect of AMPK activators on NLRP3 levels and other markers of neuroinflammation in lipopolysaccharide (LPS) induced BV-2 microglial cells activation.
Methodology: The murine microglial BV2 cell line was cultivated in Dulbecco's Modified Eagle Medium (DMEM) with high glucose, 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin and 100mg/ml streptomycin at 37 °C in a humidified incubator with 5% CO2. BV2 microglial cells were incubated with AMPK activators which includes Metformin, Quercetin, Resveratrol, cinnamonaldehydeand berberine, for 24 hours to assess the cytotoxicity by sulforhodamine B (SRB) Assay. The concentration showing 90% cells viability (IC10) of each drug was added to Bv2 cells for 1 hour and then incubated with LPS (1ug/ml) for 24 hours. Later, TNF-α and IL-1β were determined in the supernatant of Bv2 cells by enzyme-linked immunosorbent assay (ELISA). The protein levels of AMPK, P-AMPK, NLRP3, NF-κB, Caspase-1, CD86, and CD206 were analyzed by Western Blot.
Summary of results: Pre-treatment with AMPK activators with IC10 (Metformin- 1421μM, 1421 μM, Quercetin, 33.9 μM, Resveratrol, 3.56 μM, Cinnamaldehyde, 13.04 μM, and Berberine, 6.13 μM) significantly enhanced the p-AMPK/AMPK ratio in LPS-stimulated BV2 cells. This enhanced ratio prompted a shift in the microglial polarization from pro-inflammatory CD86 to anti-inflammatory CD206 in presence of LPS. Furthermore, the elevated ratio demonstrated a notable suppression of NF-κB and NLRP3 protein levels, along with downregulation of NLRP3 inflammasome-mediated caspase-1 activation and pro-inflammatory cytokines IL-1β and TNF-α levels in LPS-stimulated Bv2 cells. The values are represented as mean ± SEM. Statistical analysis was done using one-way ANOVA followed by Tukey’s multiple comparison test, where *p<0.05 compared to normal control and #p<0.05 compared to the LPS-treated cells (N = 3).
Conclusion: Our results indicate that AMPK activators might have neuroprotective effects by inhibiting the NF-κB/NLRP3 inflammasome axis via activation of AMPK signaling, thereby offering potential therapeutic strategies for managing neuroinflammatory conditions. This multifaceted approach underscores the intricate interplay between metabolic pathways and immune responses in the central nervous system. It emphasizes AMPK activators as promising candidates for precise pharmacological interventions in neurodegenerative diseases and associated conditions, illuminating potential pathways for targeted therapeutic development.