Formononetin ameliorates depression-like behaviors through rebalancing microglia M1/M2 polarization and inhibiting NLRP3 inflammasome: involvement of activating PPARα-mediated autophagy

Background: The dysregulation of neuroinflammation triggered by imbalance of microglia M1/M2 polarization is a key pathogenic factor and closely associated with occurrence of depression. Formononetin (FMN), a natural non-steroidal isoflavonoid, has been confirmed to exhibit remarkable anti-inflammatory efficacy, but the impact of FMN on depression and the underlying antidepressant mechanisms are still not fully understood. This study aimed to investigate whether the antidepressant effect of FMN is involved in modulating microglia polarization, and if so, what are the underlying mechanisms.

Methods: Lipopolysaccharide (LPS)-induced depressive mice were used to study antidepressant mechanisms of FMN. Microglia cell line BV2 stimulated by LPS was employed to investigate pharmacological mechanisms of FMN. Effects of FMN on neuronal damage were detected by H&E, Nissl and Golgi staining. The efficacy of FMN were evaluated by immunostaining and western blots in vivo and vitro. In addition, molecular docking, luciferase reporter assay, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) were used to confirm the direct target of FMN.

Results: Our results showed that FMN significantly reverses depression-like behaviors, alleviates neuroinflammation and neuronal damage, rebalances M1/M2 polarization, inhibits NLRP3 inflammasome and enhances microglial Autophagy level in prefrontal cortex of LPS-induced depressive mice. In vitro assays, results unraveled that Autophagy inhibitor chloroquine (CQ) blocks effects of FMN on inhibiting NLRP3 inflammasome and rebalancing M1/M2 polarization. Moreover, PPARα is identified as a direct target of FMN and FMN can activate PPARα-mediated Autophagy. Furtherly, combination PPARα Agonist (WY14643) with FMN had no significant additive effects on inhibiting NLRP3 inflammasome and rebalancing M1/M2 polarization, whereas PPARα Antagonist (GW6471) abrogated these pharmacologic effects of FMN in BV2. Importantly, GW6471 exhibited similar pharmacologic effects to abolish antidepressant effect of FMN in LPS-induced depressive mice.

Conclusion: Our study firstly demonstrated that FMN can rebalance microglia M1/M2 polarization and inhibit NLRP3 inflammasome, with the involvement of activating PPARα-mediated Autophagy to ameliorate depression-like behaviors, which provides a novel view to elucidate antidepressant mechanisms of FMN and also offers a potential therapeutic target for depression.

Anti-depression; Autophagy; Formononetin; Microglia M1/M2 polarization; NLRP3 inflammasome; PPARα.