Background
Spontaneous intracerebral hemorrhage (ICH) is a common fatal stroke subtype that accounts for 10 to 15% of all stroke cases and associated with high mortality and morbidity [
1]. The hematoma formation and its expansion within the brain parenchyma is regarded as the primary brain injury to damage the brain tissue. Red blood cell lysis, blood products, and thrombin can then initiate inflammatory cell activation which contributes to neuroinflammation, a major contributor to secondary brain injury after ICH that results in brain edema, disruption of the blood-brain barrier, and cell death [
2,
3]. Moreover, numerous studies have demonstrated the critical role of inflammation in ICH-induced secondary brain injury, including microglia/macrophage activation and neutrophil infiltration [
4‐
6]. Therefore, anti-inflammatory strategies may have potential therapeutic effects against ICH and improve neurological functions after ICH [
7].
Melanocortin receptor 4 (MC4R) is a G protein-coupled receptor with seven transmembrane domains and an intronless gene that encodes a protein of 332 amino acids with four potential glycosylation sites and two potential palmitoylation sites [
8,
9]. Among the melanocortin receptors, MC4R is predominantly expressed in the central nervous system (CNS) including the thalamus, hypothalamus, cortex, hippocampus, and brainstem, although it is also detected in peripheral tissues [
10,
11]. Moreover, several studies have described that MC4R could be expressed by neurons, microglia, and astrocytes [
12,
13]. The binding of MC4R to its endogenous ligand, α-melanocyte-stimulating hormone (α-MSH), has demonstrated the protective, anti-inflammatory, and anti-apoptotic effects in experimental renal ischemia/reperfusion, cerebral ischemia, and traumatic brain injury [
14‐
16]. In recent years, some low molecular weight non-peptide compounds, selective MC4R agonists, appear to be suited for the treatment of immune-mediated inflammatory diseases, without having the side effects of corticosteroids [
17,
18]. However, the potential role of MC4R activation against neuroinflammation after ICH-induced brain injury still has not been studied.
c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) that belong to the MAPK family have already been widely investigated for their roles in response to various stress stimuli [
19]. Several studies have demonstrated that JNK and p38 MAPK signaling pathways were activated after ICH, leading to the upregulation of proinflammatory mediators including TNF-α, IL-6, and IL-1β [
20,
21]. A previous study indicated that activation of MC4R could significantly decrease the expression levels of JNK and p38 MAPK [
18]. Adenosine monophosphate-activated protein kinase (AMPK) plays an important role in energy homeostasis and regulation of inflammatory responses [
22]. In addition, MC4R activation was able to attenuate oxidative stress and mitochondrial dysfunction via increasing the expression of AMPK [
23]. Recent studies reported that activation of AMPK was shown to rapidly inhibit proinflammatory JNK and p38 MAPK [
24,
25].
Therefore, the aim of the present study was to investigate whether the selective MC4R agonist, RO27-3225, could improve neurological outcomes and attenuate neuroinflammation through AMPK/JNK/p38 MAPK pathway in an experimental ICH model.
Discussion
In the present study, we investigated the potential anti-inflammatory effects of activation of MC4R and its underlying mechanism after collagenase-induced ICH in mice. Our results showed that activation of MC4R with its selective agonist RO27-3225 improved the neurobehavioral functions, decreased brain edema, and inhibited microglia/macrophage activation and neutrophil infiltration in perihematomal areas after ICH. In addition, RO27-3225 treatment improved long-term neurobehavioral outcomes after ICH. Mechanistically, administration of RO27-3225 was associated with upregulation of MC4R and phosphorylated AMPK and downregulation of phosphorylated JNK, phosphorylated p38 MAPK, TNF-α, and IL-1β after ICH. However, blockage of MC4R and AMPK reversed the beneficial effects of RO27-3225 on neurobehavioral functions, brain edema, and the inflammatory protein expression. Finally, our findings suggested that activation of MC4R with RO27-3225 might attenuate neuroinflammation after ICH, which was, at least in part, mediated by the AMPK/JNK/p38 MAPK signaling pathway.
Melanocortin neuropeptides, including the adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormones (α-MSH), shorter fragments, and synthetic analogs, have significant influences on energy homeostasis, memory formation, cardiovascular regulation, neuroprotection, and inflammation [
9,
37]. These peptides act through five melanocortin G protein-coupled receptors MC1R to MC5R, among which MC4R is the predominant melanocortin receptor subtype in the CNS [
10]. It has been reported that MC4R was expressed on neurons, microglia, and astrocytes in previous studies [
12,
13]. Consistently, our double immunofluorescence staining results showed that MC4R was positively expressed on neurons, microglia, and astrocytes following ICH. Interestingly, we also observed that the expression level of MC4R was upregulated in the right hemisphere in a time-dependent manner after ICH. A previous study demonstrated that the mRNA level of MC4R was increased in the contralateral striatum after hypoxia-ischemic brain injury in rats [
38]. Likewise, the mRNA and protein expression of MC4R in the rat liver cells were dramatically increased during acute phase response and liver regeneration [
39,
40]. Taken together, these results indicate that MC4R may be increased as a response to deleterious stimuli to counteract cytokine production in the acute phase after ICH.
Numerous studies have indicated that activation of MC4R exerts anti-inflammatory effects by inhibiting the production of proinflammatory cytokines in various diseases, including circulatory shock, myocardial ischemia, and ischemic stroke [
18,
41‐
43]. In recent years, apart from α-MSH and its synthetic analogues, the selective MC4R small molecule agonist RO27-3225 was used to activate MC4R. A previous study reported that RO27-3225 attenuated the inflammatory and apoptotic responses and improved neuronal functionality after cerebral ischemia in gerbils [
18]. Moreover, the anti-inflammatory and protective effects of RO27-3225 have been shown in brain injuries induced by intraabdominal hypertension, pancreatitis severity, and hemorrhage shock [
17,
44‐
46]. Up to date, our study is the first to administrate RO27-3225 to determine whether it has protective effects in experimental ICH.
It is well known that brain edema is a pathological phenomenon associated with hematoma enlargement, which results in poor neurological outcomes of ICH [
5]. We found that RO27-3225 treatment ameliorated the neurobehavioral impairment and reduced brain edema at 24 and 72 h after ICH. Furthermore, the sensorimotor (foot fault test and Rotarod test) and cognitive (Morris water maze) assessments were also performed in our long-term study. Our results showed that the administration of RO27-3225 has the potential to improve long-term movement coordination ability, spatial learning, and memory abilities after collagenase-induced ICH. In addition, mounting evidence suggests that microglial activation and neutrophil infiltration after ICH exacerbated the release of proinflammatory mediators, such as TNF-α and IL-1β, reactive oxygen species (ROS), nitric oxide, and other potentially toxic factors, leading to neuroinflammation and ICH-induced secondary brain injuries [
5,
6,
47]. On the contrary, several studies have reported that activation of MC4R obviously inhibited the overexpression of TNF-α, IL-6, and IL-1β in cerebral ischemia, Alzheimer’s disease, and subarachnoid hemorrhage [
8,
18]. Consistent with previous findings, our data showed that RO27-3225 significantly inhibited microglia/macrophage activation and neutrophil infiltration and downregulated the expression of Iba-1, MPO, TNF-α, and IL-1β after ICH. In general, our present study indicated that activation of MC4R with RO27-3225 exerted an anti-inflammatory effect in ICH mice.
Next, we evaluated the possible mechanism by which MC4R activation reduces neuroinflammation after ICH. Recent studies have demonstrated the role of AMPK as a novel signaling molecule modulating inflammatory response and oxidative stress in various cell types and animal models [
48]. AMPK is generally activated under conditions, such as glucose deprivation, exercise, oxidative stress, CA
2+ overload, and ischemia [
22]. Activation of AMPK was shown to suppress the expression of JNK and p38 MAPK [
24,
25]. Moreover, a recent study showed that MC4R activation attenuated oxidative stress and mitochondrial dysfunction via increasing AMPK [
23]. Additionally, the activation of MC4R could also significantly decrease the expression levels of JNK and p38 MAPK [
18] and further inhibited the proinflammatory mediators TNF-α and IL-1β, as well as the proapoptotic marker Bax [
18,
49]. Consistently, our western blot results demonstrated that RO27-3225 treatment upregulated the expression of MC4R and phosphorylated AMPK, while downregulated the expression of phosphorylated JNK, phosphorylated p38 MAPK, TNF-α, and IL-1β after ICH. To further validate this pathway, HS024 and dorsomorphin were employed to investigate the neurobehavioral functions and the expression of downstream signaling proteins. Our results showed that pretreatment with HS024 and dorsomorphin abolished the protective effects of RO27-3225 on neurobehavioral functions. Furthermore, blockage of those two proteins significantly upregulated the expressions of phosphorylated JNK, phosphorylated p38 MAPK, and proinflammatory cytokines TNF-α and IL-1β. Thus, these findings suggested that activation of MC4R with RO27-3225 may alleviate the neuroinflammation through inhibition of JNK and p38 MAPK dependent on AMPK activation after collagenase-induced ICH.
There are still some limitations in this study. First, since we only focused on the anti-inflammatory effects of activation of MC4R after ICH, we cannot exclude the possibility that activation of MC4R may have also exerted other protective effects, such as anti-apoptosis, preservation of blood-brain barrier integrity, and synaptic plasticity [
18,
50]. Thus, further studies are needed to investigate those effects of activation of MC4R after ICH and its underlying signaling mechanisms. Second, previous studies have reported that MC4R also activates the Gs/cAMP/PKA pathway to alleviate the inflammation [
8]. We cannot rule out the possibility that other pathways may also contribute to downregulating inflammatory mediators with the activation of MC4R. Besides, the reason why activation of MC4R regulates the expression of AMPK after ICH is still unclear. Further studies are needed to clarify this mechanism. Last but not least, MC4R is involved in the regulation of food intake and body weight in a dose-dependent manner [
51]. Although the single administration of RO27-3225 did not significantly affect the body weight at 24 h after ICH in our study, whether long-term administration of this drug can change the body weight should be closely observed in the future.