Traumatic brain injury (TBI) is responsible for significant morbidity and mortality worldwide, with considerable socioeconomic implications. Annually, over 69 million new TBI patients are reported, accumulating costs of up to 400 billion USD [1,2]. The pathophysiology of TBI characteristically involves two phases known as primary and secondary brain injury. Primary injury results directly from the traumatic event; secondary injury is caused by inflammation, edema, cell death, and systemic inflammatory comorbidities following the trauma [3]. Anti-inflammatory drugs currently approved for TBI target pathogenic cytokines and can remarkably improve treatment response. However, not all TBI patients respond to these therapies, and many lose their effectiveness over time.

Melatonin (MLT), a neuroendocrine hormone secreted by the autologous pineal gland, exerts major physiological functions, including anti-inflammatory and antioxidant effects [4,5]. The physiological functions of MLT are mediated by two seven-transmembrane G protein-coupled receptors, namely MT1 and MT2 [6]. MT1/2 are widely distributed and abundant in the brain [7] and TBI has been shown to cause a decrease in the expression of both MT1 and MT2. Increased expression levels of MT1 and MT2 by agonist MLT has been shown to attenuate the motor function, learning and memory ability deficits induced by TBI [8], suggesting that MT1 and MT2 play an important role in the neurological damage following TBI. Previous studies have found that other MT1/2 agonists alleviate neurological dysfunction by increasing the expression of IL-10 and reducing that of TNF-α [9]. In contrast, the MT1/2 antagonist luzindole increases TNF-α and IL-1β expression and exacerbates pathological damage [10,11], providing further evidence that MTR1/2 activation counteracts the inflammatory response. However, the specific mechanisms by which MLT mediates the MT1/2 regulation of the inflammatory response post-TBI, remains unclear.

As an important inflammatory mediator, IL-33 exerts neuroprotective functions in various brain injury models including TBI [[12], [13], [14]]. IL-33 can attenuate the neuropathological damage (e.g., brain edema and cell death) after brain injury by inhibiting the expression of pro-inflammatory factors (i.e., TNF-α, L-1β, and IL-6) [12,15], and increasing anti-inflammatory factors (i.e., IL-5, IL-10, and IL-13) expression [16,17]. Further, IL-33 can activate the transformation of anti-inflammatory macrophages such as CD206+ macrophages and T helper 2 (Th2) cells [18,19], suggesting that IL-33 plays an important role in the anti-inflammatory effects post-TBI. Interestingly, a previous study found that MLT alleviated diabetic nephropathy through regulating the expression of IL-33 [20]. However, whether MLT regulates IL-33 and exerts its neuroprotection effects through the IL-33 signaling pathway after TBI, remains unknown.

In addition to anti-inflammation, MLT can also suppress oxidation and ferroptosis [21]. Ferroptosis is a newly identified lipid peroxidation-induced cell death that occurs by hallmark mechanisms of iron metabolism disturbance and iron overload. Iron metabolism includes iron transport, utilization, and storage [22]. Ferritin, an important iron storage protein, improves neurological deficits caused by TBI. Ferritin is composed of light chain-ferritin (FtL) and heavy-chain ferritin (Fth) and can catalyze the conversion of the ferrous form (Fe2+) to iron form (Fe3+) for storage in ferritin nanocages. This allows for the formation of a stable iron pool, thereby limiting iron-mediated reactive oxygen species (ROS) catalysis [23,24]. In our previous study, we found that conditional knockout of neuronal Fth diminished the expression of glutathione peroxidase (GPX) 4, increased that of 4-hydroxynonenal (4-HNE), aggravated iron deposition, and lipid peroxidation, and ultimately increased susceptibility to ferroptosis [8,25]. Apart from ferroptosis, Fth suppresses inflammation; Fth overexpression upregulates anti-inflammatory cytokines IL-10 and IL-4, while Fth deletion upregulates pro-inflammatory cytokines IL-1β and TNF-α [26,27]. Considering that both IL-33 and Fth have anti-inflammatory and lipid peroxidation-inhibiting properties [27,28] and further, that they are both regulated by MLT [20,25,29], we suspect that IL-33 and Fth are likely to be involved in MLT-mediated suppression of inflammation and ferroptosis. However, the specific interactions and mechanisms by which this occurs, remains to be further explored.

In the present study, we analyzed the transcriptome of a post-TBI brain and identified that the differentially expressed genes Mt1, Mt2, Il33, and Fth1 were upregulated in the cortical region. Using inhibitors including Luzindole, 4-P-PDOT, anti-IL-33, and liproxstatin-1, we then demonstrated that MLT increased the expression of IL-33 via MT2 post-TBI and suppressed inflammation and ferroptosis via the activation of the MT2/IL-33 pathway. By establishing Fth knockout in vivo and in vitro, we further demonstrated that Fth deletion exacerbated the inflammatory response and counteracted the anti-inflammatory effect of MLT post-TBI. Thus, we reasoned that dynamic MT2/IL-33/Fth-mediated inhibition of inflammation and ferroptosis might be a promising therapeutic approach for TBI patients.