Beta‐Arrestin2‐Biased Activation by Pilocarpine Suppresses Microglial Inflammatory Response

Microglia play a pivotal role in inflammatory regulation through multifarious signaling pathways within the central nervous system, and mitigating microglial inflammation is considered a promising strategy to delay the progression of neurodegeneration. However, the role of biased receptor signaling in modulating microglial inflammation remains largely unexplored. In this study, the anti-inflammatory effects and the underlying mechanism of muscarinic receptor agonists pilocarpine and iperoxo were explored. Our results showed that pilocarpine, rather than iperoxo, inhibited the expression of TNF-α and IL-6, as well as restored ramified morphology and physiological phagocytosis of over-activated microglia. RNA-seq revealed that pilocarpine-treated BV2 exhibited transcriptional profiles more similar to the control group, with upregulation of anti-inflammatory genes. β-arrestin2 knockdown attenuated the anti-inflammatory effect of pilocarpine by reversing the expression of inflammatory factors and activation of NF-κB. Furthermore, through chemogenetic DREADDs, activation of Gαq, Gαi, or β-arrestin pathways demonstrated that β-arrestin, but neither Gαq nor Gαi, inhibited the inflammatory response in microglia. Our findings proved that pilocarpine could abate the microglial inflammatory response via biased activation of the β-arrestin2 pathway, which could be considered a promising therapeutic approach for anti-neuroinflammation.