Melatonin Suppresses Microglial Necroptosis by Regulating Deubiquitinating Enzyme A20 After Intracerebral Hemorrhage

Cell death is deeply involved in pathophysiology of brain injury after intracerebral hemorrhage (ICH). Necroptosis, one of the recently discovered forms of cell death, plays an important role in various diseases, including ICH. Previous studies have suggested that a considerable number of neurons undergoes necroptosis after ICH. However, necroptosis of microglia after ICH has not been reported to date. The present study demonstrated for the first time that necroptosis occurred in the microglia surrounding the hematoma after ICH in C57 mice, and melatonin, a hormone that is predominantly synthesized in and secreted from the pineal gland, exerted a neuroprotective effect by suppressing this process. When we further explored the potential underlying mechanism, we found that melatonin inhibits RIP3-mediated necroptosis by regulating the deubiquitinating enzyme A20 (also known as TNFAIP3) expression after ICH. In summary, we have demonstrated the role of microglial necroptosis in the pathogenesis of ICH. More importantly, A20 was identified as a novel target of melatonin, which opens perspectives for future research.


INTRODUCTION
Intracerebral hemorrhage (ICH) is a significant cause of morbidity and mortality worldwide. As one of the most serious forms of stroke, ICH affects ∼2 million people worldwide each year (1)(2)(3). Primary injuries after ICH are usually caused by the mechanical damage of the hematoma to the surrounding brain tissues. Following the primary injuries, secondary injuries, including inflammation and cell death, are extensively involved in the pathological processes following hemorrhagic events (4). Treatment of secondary injuries is one of the important interventions after ICH.
Cell death is a hallmark of secondary brain injury after ICH (5). Numerous experimental and clinical observations indicate a variety of cell death forms and mechanisms take place during hemorrhagic stroke (6). Among them, programmed cell death is highly correlated with the homeostatic mechanisms of the nervous system. Necroptosis, also known as programmed necrosis, shares upstream signaling elements with apoptosis, such as tumor necrosis factor (TNF), Fas-associated death domain (FADD), and Toll-like receptor (TLR) (7). However, apoptosis and necroptosis have distinct outcomes.
Apoptotic cells usually induce a non-inflammatory response, whereas necroptosis triggers an inflammatory response (8) due to the rapid loss of plasma membrane integrity prior to the exposure of phagocytic signal (9), which in turn causes the release of intracellular damage-associated molecular patterns (10), which induce inflammation. Microglia, which are primary immune cells of the central nervous system (CNS), undergo necroptosis in various pathological processes, such as ischemic stroke (11), retinal degeneration (12), and spinal cord injury (12). Therefore, it may be beneficial to inhibit necroptosis to reduce neuroinflammation and improve neuronal survival in the context of disease, particularly in microglia (13).
Melatonin (N-acetyl-5-methoxytryptamine) is a hormone that is predominantly synthesized in and secreted from the pineal gland. (14) In 1958, Lerner et al. were the first to isolate melatonin from bovine pineal gland extracts and named it according to its ability to aggregate melanin granules (15). Initial research on melatonin was focused on its regulation of circadian and circannual cycles (16). Later, the multifunctional roles of melatonin were further explored. Melatonin serves as an effective antioxidant in scavenging the highly toxic hydroxyl radical and other oxygen-centered radicals (17), and it promotes the immune response (18). No serious side effects or risks have been reported in association with the ingestion of melatonin (17). These findings suggest that melatonin might play a protective role in various pathological conditions. Therapeutic effects of melatonin in various diseases, including diabetes (19), cancers (20), cardiovascular diseases (21,22), and CNS diseases (23) have been reported.
In CNS diseases, melatonin has been shown to contribute to the maintenance of cell homeostasis and survival by regulating inflammation, apoptosis, or autophagy after different types of brain injury (24). Melatonin reportedly has a neuroprotective effect after stroke, either ischemic (25,26) or hemorrhagic (27). Melatonin has also been shown to inhibit necroptosis during myocardial ischemia and liver fibrosis (28)(29)(30), and this mechanism might explain its therapeutic effects after stroke. However, whether melatonin can inhibit necroptosis of microglia after ICH and how much protection can be achieved by inhibiting this process remain to be elucidated.
Serine/threonine kinase receptor interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3) are important molecules in the process of necroptosis (7). A previous study revealed that RIP1 expression was significantly increased 1-3 days after ICH (31), suggesting that necroptosis occurs in the acute phase after ICH. Upon inhibition of caspase activity, especially that of caspase 8 by genetic or chemical methods, RIP1 forms a necrosome with RIP3 by interacting via their homotypic interaction motif domains and activates their kinase activities (32)(33)(34). Subsequently, the pseudokinase mixed lineage kinase domain-like protein (MLKL), is activated as an executive molecule of necroptosis (35). In turn, MLKL translocates to the plasma membrane, where it forms pores and disrupts the plasma membrane integrity (36). Although the necrosome, the characteristic complex of necroptosis, is composed of both RIP1 and RIP3, RIP3 is considered to have a more critical role than RIP1 in the process of necroptosis (37)(38)(39). Previous studies have suggested that melatonin inhibits necroptosis mainly by inhibiting the kinase activity of RIP3 (28,29). However, the specific mechanism of RIP3 regulation by melatonin has not been elucidated. A20, also known as TNFAIP3, is a deubiquitinating enzyme (40). A20 is widely recognized as a potent anti-inflammatory protein linked to multiple human brain autoimmune diseases, neuro-degenerative diseases, and brain tumors, as well as stroke (41)(42)(43)(44). Voet et al. reported that A20 controls microglial activation to regulate neuroinflammation, a function closely related to inhibition of NLR family pyrin domain-containing protein 3 (NLRP3) (45). Moreover, Onizawa et al. discovered that A20 restricts RIP3-dependent necroptosis (46). Thus, we hypothesized that melatonin can upregulate the expression of A20 in the brain, especially in microglia, after ICH, which in turn inhibits the expression of RIP3, thereby exerting a protective effect by inhibiting necroptosis.
In this study, we aimed to demonstrate the occurrence of microglial necroptosis after ICH and to evaluate the effect of melatonin on this process as well as the underlying mechanism, to improve the recovery of neurological function after ICH.

Animals
C57 mice were purchased from SLAC Laboratory Animal Company Limited (Shanghai, China). In total, 214 male mice (8-10 weeks, 20-25 g) were used in this study. The mice were housed in a temperature-and humidity-controlled room under a standard 12-h light/dark cycle and had free access to food and water. The animal protocol was approved by the Institutional Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine. The procedures were conducted according to the National Institutes of Health's Guide for the Care and the Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines. Experimental grouping was shown in Supplementary Figure S1.

ICH Model
The ICH model was established as previously described (47) (Figure 1A). Briefly, mice were anesthetized with 40 mg/kg 1% pentobarbital sodium via intraperitoneal injection. Under stereotactic guidance, a small cranial burr hole was made at a precise location (bregma coordinates: 0.5 mm anterior and 2.5 mm lateral to the midline). Autologous blood (30 µL) from the femoral artery was injected 3.5 mm deep into the right basal ganglia at a rate of 3 µL/min using a microinfusion pump, and the syringe was pulled out after 10 min.

Drug Administration
As described previously (48,49), melatonin (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% normal saline. A dose of melatonin (20 mg/kg) or vehicle (5% DMSO) was given to mice randomly via intraperitoneal injection 30 min before ICH induction.

Neurobehavioral Function Assessment
Four evaluation methods were used to assess the voluntary activities and motor function of mice at 24 h, 72 h, and 7 days after ICH impairment.
Neurologic function was tested before and 1, 3, 7 days after ICH by assessing body symmetry, gait, climbing, circling behavior, front limb symmetry, and compulsory circling (50). Each test result was graded from 0 to 4, with a maximum deficit score of 24.
An adhesive removal test was conducted as previously described (51). Briefly, mice were accustomed to the experimental environment for 30 min. Then, an adhesive tape strip was placed on the left hairless part of the forepaws of the mice. Mice were then put into the testing cage and the time to feel and time to remove the strip by any behavior of the mice were recorded.
For the foot-fault test, mice were individually placed on a wired grid (50 × 55 × 52 cm length/width/height) with their paws. Behavior of the mice while they were moving was recorded for 1 min. Each successful foot placement onto the bar was recorded as a step. A foot fault was recorded when a paw slipped through the grid hole. The percentage of foot faults was calculated as: 100 × faults/(successful steps + faults) (52).
A rotarod test was performed as previously described (53). Mice were placed on a Rotamex 5 apparatus (Columbus Instruments). During the test, the speed was increased from 4 rpm to 40 rpm within 5 min. After adaptation for two consecutive days, mice were tested twice daily with at least 30 min between tests. The period to fall off the rotating rod was recorded, and the data were expressed as the mean from three trials.

Brain Water Content
We used a wet-dry method as previously described (54) to evaluate the brain water content at 72 h after ICH. Briefly, after euthanasia, mice (n = 6/group) were sacrificed and the brain hemispheres were collected and weighed immediately (wet weight). They were then dried at 100 • C for 48 h and weighed again to obtain the dry weight. The brain water content was calculated as follows: [(wet weight-dry weight)/(wet weight)] × 100% (55).

Propidium Iodide (PI) Staining in vivo
PI staining in vivo to identify microglial necroptosis was conducted as previously described (31). For all experiments, PI (Beyotime, Shanghai, China) was administered intraperitoneally (10 mg/kg) 1 h before the mice were sacrificed. The brain tissue was cryoprotected by immersion in 15 and 30% sucrose solution. Then, brain sections (10 µm) were cut along the anterior-posterior lesion and placed on poly-L-lysine-coated glass slides. Sections were visualized under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co.). PI+/Iba-1+ cells were counted by observers blinded to the experimental groups. Necroptotic microglia were counted in six microscopic fields per section, and the average number in each section was calculated.

Immunofluorescence
After the mice were anesthetized, transcardial perfusion was performed with 0.1 M PBS, followed by perfusion with 4% paraformaldehyde. The whole brain was immersed in 4% paraformaldehyde for 24 h, then cryoprotected in serial 15 and 30% sucrose solutions. The brain samples were then cut into coronal slices (9 µm) and fixed on slides. After preprocessing with 5% BSA and 0.3% triton X-100, the sections were incubated at 4 • C overnight with primary antibodies, including anti-Iba-

Small Interfering (si)RNA and Intracerebroventricular Injection
A20 siRNA or scramble siRNA (Genomeditech, Shanghai, China) mixed with transfection reagent (Engreen Biosystem, Auckland, New Zealand) was delivered via intracerebroventricular injection as previously described (57). Briefly, the injections into the right ventricle were performed using the following coordinates relative to bregma (0.2 mm posterior, 1.0 mm lateral, and 2.0 mm deep) at 48 h prior to surgery. After the injection was completed, the needle was left in the brain for 10 min, and the burr hole was blocked with bone wax.

Single Cell Sorting
Single cell sorting was performed as described previously (58). In brief, brain samples were fixed by transcardial perfusion with PBS before extraction. After dissociation, cells were separated by 30% Percoll density gradient separation by centrifugation at 800 ×g for 30 min at 18 • C. Then, the cells were passed through a 70-µm nylon mesh. Cell populations were sorted on an Aria SORP instrument (BD Biosciences, San Jose, CA, USA). The gating strategy is shown in Supplementary Figure S2. CD45+ (PerCP CD45, 1:200, BD Biosciences, 557235) and CD11b+ (FITC CD11b, 1:200, BD Biosciences, 557396) cells were isolated as microglia. Immediately after sorting, the cells were stored at −80 • C until processed.

Cell Lines and Coculture
The mouse microglial cell line BV2 and the mouse hippocampal cell line HT22 were cultured (37 • C, 5% CO 2 ) in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. As secondary damage in ICH is mainly caused by oxidized hemoglobin, we used 100 µM OxyHb to simulate the pathological process of ICH in vitro, as previously described (31). Melatonin was used at 1 mM to study the effect of melatonin on necroptosis in vitro. A Transwell coculture system was used to investigate the effect of BV2 cells on HT22 cells. BV2 cells were cultured at a density of 5 × 10 4 in Transwell inserts (pore size 0.4 µm; Corning, Corning, NY, USA) placed above the HT22 neuronal layer.

Cell Viability Assay and Cytotoxicity Assay
As necroptosis is a type of cell death, cell viability and cytotoxicity can reflect its occurrence in vitro. Cells were cultured in 96-well plates. The CCK-8 cell counting kit (Beyotime, Shanghai, China) was used to evaluate cell viability according to the manufacturer's instructions. Twenty microliters of CCK-8 solution was added to 200 µl of cell culture medium, after which the plates were incubated at 37 • C for 2 h. Then, the absorbance at 450 nm was measured. Cytotoxicity was evaluated using an LDH lactate dehydrogenase cytotoxicity test kit (Beyotime). Briefly, cells were cultured in serum-free medium for 24 h. Then, the plates were centrifuged at 400 ×g for 5 min. One hundred twenty microliters of supernatant of each well was transferred to a new 96-well plate, and the absorbance at 490 nm was measured immediately.

ELISA
To explore changes in inflammatory factors, a TNF ELISA KIT (Abcam, ab100747) was used according to the manufacturer's instructions to quantify the levels of TNF in BV2 cell supernatant.

Reactive Oxygen Species (ROS) Assay
ROS can be both an inducer and a product of necroptosis. ROS levels in cells were examined using a ROS Assay Kit (JianCheng, Nanjing, China) according to the manufacturer's instructions. In brief, cells were trypsinized, collected, and incubated with 10 µM 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate at room temperature for 30 min. After two washes, intracellular ROS production was measured by fluorescence detection using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Protein levels in the cells were measured using a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA, USA). ROS levels are reported as fluorescence/mg protein.

Transmission Electron Microscopy
Mice were sacrificed and perfused with 0.9% saline and 4% PFA. Fragments of peri-hematoma tissues (∼1 mm 3 ) were collected and immersed in glutaraldehyde (2.5%) at 4 • C overnight. The tissues were preprocessed as previously described (59,60). The samples were sliced into 100-nm sections and stained with 4% uranyl acetate and 0.5% lead citrate. The ultrastructure of the tissues was evaluated by transmission electron microscopy (Philips Tecnai 10, Netherlands).

Statistical Analysis
All data are expressed as the mean ± standard deviation (SD) and were analyzed in GraphPad Prism v. 6.0. Data were tested for normality of distribution by the Kolmogorov-Smirnov test. Oneway ANOVA was applied for comparing multiple groups. The analyses were conducted in SPSS v. 22.0 (SPSS Inc.). Statistical significance was defined as P < 0.05.

Melatonin Reduces Brain Edema, Improves Neurological and Motor Function After ICH
We evaluated the brain water content to explore the effects of melatonin treatment on ICH-induced brain edema. No significant differences were noted in the contralateral cortex, contralateral basal ganglia, or cerebellum between sham, ICH+vehicle, and ICH+melatonin groups. However, compared to the sham group, the brain water content was increased in the ipsilateral cortex and ipsilateral basal ganglia of mice in the ICH+vehicle group, whereas melatonin treatment significantly reduced the brain water content (Figure 1B). To explore the effect of melatonin on the neurological function of mice after ICH, we carried out various neurological tests before ICH and at 24 h, 72 h, and 7 days after ICH. Melatonin or vehicle (5% DMSO) was given to mice randomly via intraperitoneal injection 30 min before ICH induction. The neurological deficit scores were significantly increased after ICH and peaked at 72 h, whereas melatonin significantly improved neurological function ( Figure 1C). The results of motor function assessment showed trends similar to those of neurological deficit scores (Figures 1D-F).

Microglia Undergo Necroptosis After ICH
To explore the involvement of necroptosis of microglia after ICH, we performed a series of experiments. The expression of RIP1, RIP3, MLKL, which are executive molecules in necroptosis, was significantly increased at 24 h and 72 h after ICH (P<0.05 vs. sham group ; Figures 2A-D). Correspondingly, cells that were costained for these molecules and Iba-1 were also remarkably increased at 72 h after ICH (P < 0.05 vs. sham group; Figures 3F-I). To further illustrate the occurrence of microglial necroptosis after ICH, we isolated microglia, and we found that at 3 days after ICH, the expression of necroptosis-related proteins (RIP1, RIP3, MLKL) in microglia was significantly higher than that in sham group (P < 0.05 vs. sham group; Figures 2F-I).
We also counted PI+ microglia in frozen brain sections at 72 h after ICH. PI+/Iba-1+ cells were remarkably increased in brain tissues surrounding hematomas after ICH (P < 0.05 vs. sham group; Figures 2J,K). These results demonstrated that necroptosis occurred in microglia surrounding the hematoma after ICH.

Melatonin Plays a Protective Role by Suppressing Necroptosis in Microglia
To explore the effects of melatonin on necroptosis after ICH, the protein levels of RIP1, RIP3 and MLKL were detected by western blot analysis at 24 h and 72 h after ICH. As shown in Figures 3A-E, the expression of these proteins was significantly increased in the ICH+vehicle group compared to the sham group (P < 0.05), whereas melatonin treatment significantly suppressed the increases (P < 0.05 vs. ICH+vehicle group). Immunofluorescence staining revealed that cells costained for these molecules and Iba-1 were decreased in the ICH+melatonin compared to the ICH+vehicle group (P < 0.05; Figures 3F-I).
These results indicated that melatonin suppresses necroptosis of microglia in brain tissues after ICH.

Melatonin Reduces Mitochondrial Damage in Microglia After ICH
Mitochondrial damage has been shown to be involved in necroptosis (8,61). Therefore, we used TEM to detect mitochondrial damage at 72 h after ICH. In the brain tissues of the ICH group, mitochondria with obvious vacuolization and swelling were observed ( Figure 4A). Next, we determined the ψm of BV2 cells in vitro by JC-1 staining. As shown in Figure 4B, the green fluorescence intensity was increased in OxyHb+vehicle group compared to the sham group, indicating a decline of ψm; however, pre-incubation with melatonin attenuated the OxyHb-induced collapse of the ψm (P < 0.05). A microplate fluorescence read-out yielded similar results ( Figure 4C). These results suggested that melatonin reduces mitochondrial damage after ICH by suppressing necroptosis. Figure 5A shows representative photographs of OxyHb-treated and control BV2 cells. Swollen cells with ruptured plasma membrane can be observed in the OxyHb-treated group. We examined the effects of melatonin on necroptosis in vitro by cell viability and cytotoxicity assays. As shown in Figures 5B,C, cell viability was decreased in the OxyHb+vehicle group as compared to the control group (P < 0.05), whereas pretreatment with melatonin significantly improved cell viability (P < 0.05). The cytotoxicity assays suggested that OxyHb had a cytotoxic effect on BV2 cells (P < 0.05), which was prevented by melatonin pretreatment (P < 0.05). ROS were measured to investigate the effects of melatonin on ROS production in BV2 cells. As shown in Figure 5D, treatment with OxyHb remarkably promoted ROS production, which was suppressed by pretreatment with melatonin (P < 0.05). The above results indicated that melatonin improves BV2 cell viability and reduces cytotoxicity and ROS generation in vitro.

Melatonin Suppresses TNF Secretion by Microglia, Thus Reducing Neuronal Necroptosis
As the main immune cells in the nervous system, changes in microglia would affect the homeostasis of other cells, such as neurons. We firstly explored whether the inflammatory factor TNF, a common inducer of necroptosis, is involved in this process. The concentration of TNF was significantly higher in the culture medium of BV2 cells treated with OxyHb than that in that of the control group, whereas melatonin pretreatment significantly reduced TNF production ( Figure 5E, P < 0.05). Next, we explored the effects of necroptotic microglia on neurons after ICH by using a Transwell system. HT-22 neurons were cocultured with BV2 cells exposed to various treatments. The neurons were then stained with Annexin V and PI and analyzed by flow cytometry. The necroptotic population (defined as Annexin V-/PI+) was increased in neurons cocultured with BV2 cells treated with OxyHb when compared with the control group, whereas pretreatment with melatonin suppressed this increase (Figures 5F-H; P < 0.05). These results indicated that melatonin suppresses TNF secretion by reducing microglial necroptosis, which in turn reduces neuronal necroptosis.
A20 Is Expressed in the Acute Phase in Microglia After ICH, and Melatonin Promotes Its Expression and Thus Plays a Protective Role We analyzed the expression of A20 at 72 h after ICH; A20 expression was significantly increased after ICH (Figures 6A,B; P < 0.05 vs. sham group). Treatment with melatonin significantly enhanced the expression of A20 after ICH (Figures 6A,B; P < 0.05 vs. ICH+vehicle group). Accordingly, immunofluorescence analysis revealed that A20 was expressed in the microglia surrounding the hematoma after ICH, and A20 expression was significantly increased after treatment with melatonin ( Figures 6C,D, P < 0.05). To verify the above results, we measured A20 expression in microglia at 3 days after ICH. The results showed that A20 expression was significantly enhanced when compared with the sham group (Figures 2E,F; P < 0.05 vs. sham group). In line with the above results, melatonin treatment remarkably increased A20 expression after ICH (Figures 2E,F; P < 0.05 vs. ICH 3-day group). We investigated the relationship between A20 and RIP3-dependent necroptosis after ICH further by using A20 siRNA. As shown in Figure 6E, A20 siRNA transfection blocked the activating effect of melatonin on A20 expression (P < 0.05 vs. ICH+melatonin+con-siRNA group). Moreover, A20 siRNA blocked the effects of melatonin on RIP1, RIP3, MLKL expression (P < 0.05 vs. ICH+melatonin+con-siRNA group). Melatonin reduced the phosphorylation of RIP3 and MLKL, which was prevented by A20 siRNA transfection (Figures 6E-L; P < 0.05). In vitro, A20 siRNA abolished the effects of melatonin on viability and ROS production in BV2 cells (Figures 5B-D). As A20 is generally considered to be a negative regulator of NLRP3, we also detected NLRP3 expression. The results showed that A20 siNRA lowered the promotive effect of melatonin on NLRP3 expression (Figures 6E-L; P < 0.05). These results indicated that melatonin inhibits RIP3mediated necrotic apoptosis following ICH by regulating A20 expression.

DISCUSSION
In this study, we demonstrated that melatonin inhibits necroptosis of microglia after ICH, thereby reducing brain edema and improving neurological function in the acute phase of ICH.
In addition, we uncovered the potential underlying mechanism, in which melatonin regulates A20, thus affecting RIP3-dependent necroptosis (Figure 7). Increasing evidence indicates that necroptosis plays a crucial role in the pathogenesis of several diseases with an inflammatory component (13). Shen et al. (31) found that necroptosis is an important mechanism of neuronal death after ICH, and treatment with necrostatin-1, a specific inhibitor of RIP1, could rescue the neurons from necroptosis, revealing a potential therapeutic target for ICH. Microglia are associated with necroptosis in different disease models (62)(63)(64); however, the exact underlying mechanism remained unclear. Moreover, there are no reports on microglial necroptosis after ICH. This study for the first time demonstrated that a significant amount of microglia around the hematoma did undergo necroptosis around 24 and 72 h after ICH. Although the relationship between necroptosis and inflammation has not been fully elucidated, necroptosis is generally thought to induce inflammation as, unlike apoptosis, it is essentially a type of necrotic cell death. In addition to inflammation, necroptosis causes some other cascade reactions, including mitochondrial damage, which exacerbates the release of ROS (32), which induce necroptosis. In line with previous experimental results (65), the present study suggested that mitochondrial damage and ROS accumulation after ICH might be caused by necroptosis. Therefore, necroptosis can play an intermediate role in the induction of inflammation by microglia after ICH. As resident macrophages in the CNS, microglia exert a variety of neuroprotective functions, such as phagocytosis and elimination, secretion of anti-inflammatory factors, growth factors, and promotion of the recovery of neurological function (66,67). Thus, although microglia account for only approximately 10% of cells in the CNS, their death after ICH aggravates outcome. Therefore, we hypothesized that necroptosis plays an important role in the pathological processes after ICH.
A previous study revealed that A20 expression was increased after ICH and was associated with anti-inflammatory effects by inhibiting NF-κB activation (44). In addition to its NF-κB-regulatory function, A20 is a ubiquitin-regulating enzyme, and increasing evidence shows that the central importance of its functions can be attributed to its ability to modulate ubiquitin-dependent signaling cascades (42). Ubiquitination is one of most prevalent post-translational protein modifications that regulate a variety of important physiological functions. During ubiquitination, ubiquitin, a highly conserved 76-aminoacid protein, is conjugated to lysine residues of diverse cellular proteins (66,68). The E3 ubiquitin ligase activity of A20 has been shown to directly ubiquitinate RIP1, thus triggering proteasomal degradation of RIP1, which in turn inhibits TNF-induced NF-κB activation (69). Therefore, in the present study, we mainly focused on the role of A20 as a deubiquitinating enzyme. As RIP1 is involved in apoptosis, it seems plausible that A20 could inhibit cellular apoptosis by promoting RIP1 degradation. However, a previous studies indicated that its anti-apoptotic effect is cell type-and stimulus-specific; thus, its protective effect is controversial (70). One study reported that A20 tends to inhibit apoptosis via various mechanisms (41); however, other results indicated that in certain pathological conditions, such as IBD, A20 binds to RIP1 to enhance TNF-induced apoptosis (71). We did not further explore the effect of A20 on microglial apoptosis in this study. However, regardless of promotive or inhibitory effects, A20 is considered to directly FIGURE 7 | Signal pathway underlying the effect of melatonin on microglial necroptosis after ICH. After ICH, by upregulating A20 protein expression, melatonin suppresses RIP3 phosphorylation in microglia, which in turn inhibits the activation of MLKL, the executive molecule of necroptosis, thereby inhibiting inflammation, reducing mitochondrial damage, and reducing oxidative stress, ultimately playing a neuroprotective role after ICH.
interact with RIP1 rather than via regulating NF-κB (71). As mentioned earlier, the necrosome is composed of both RIP1 and RIP3. Because RIP1 is involved in various biological processes, RIP3 is more valuable than RIP1 when studying necroptosis specifically. Moreover, RIP1 is not considered to be ubiquitinated in the necrosome, which suggests that A20 might regulate necroptosis by regulating RIP3. Furthermore, RIP3 has been regarded a specific activator of necroptosis because it regulates RIP1 phosphorylation (32) and switches TNF-induced cell death from apoptosis to necroptosis (33). The potential of A20 to suppress TNF-induced necroptosis by affecting RIP3 was observed in L929 cells and in primary mouse T cells (46,72). In line herewith, in the present study, A20 regulated RIP3, thus inhibiting necroptosis.
Melatonin intervenes in different pathological processes through various mechanisms, e.g., it reduces inflammation (24,73,74), protects mitochondrial oxidoreductase and superoxide dismutase, thus improving ROS scavenging (75), and inhibits apoptosis (24,76). In myocardial ischemia, melatonin inhibits necroptosis in cardiomyocytes by inhibiting RIP3 (29). In our study, melatonin inhibited necroptosis of microglia after ICH. We further explored the possible underlying mechanism. A20 is a regulatory protein of the NF-κB signaling pathway, with effects quite similar to those observed after treatment with melatonin. Accordingly, we found that melatonin inhibits RIP3-mediated necroptosis by promoting A20 expression. Interestingly, the NLRP3-regulatory effect of melatonin could be abolished by A20 siRNA transfection in vitro. This opens a new perspective for future exploration of NLRP3-mediated inflammation.
This study had some limitations that cannot be ignored. Firstly, melatonin might exert its neuroprotective effects after ICH by regulating various functions; the present study only explored its effect on necroptosis. Secondly, we only used cell lines for investigating the effect of melatonin on necroptosis in vitro; considering some subtle differences between cell lines and primary cells, out data would be more convincing had we used primary cells.
In conclusion, this study revealed that melatonin exerts neuroprotective effects after ICH. We demonstrated that melatonin inhibits microglial necroptosis via the A20/RIP3 pathway, in turn reducing cell death, inflammation, and mitochondrial damage. These findings implicate A20 as a novel potential therapeutic target for ICH.

DATA AVAILABILITY
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal protocol was approved by the Institutional Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine. The procedures were conducted according to the National Institutes of Health's Guide for the Care and the Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines.

AUTHOR CONTRIBUTIONS
JiaZ is the principal investigator. JL, ZS, and FL contributed to the study design, performance, and manuscript draft. YF, JinZ, SX, and SM analyzed the experimental data. WX, LS, and HW revised the manuscript and polished the language.

ACKNOWLEDGMENTS
We thank Susu Lou for support to JL.