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. 2019 Jun 14:10:1360.
doi: 10.3389/fimmu.2019.01360. eCollection 2019.

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

Jianan Lu  1 , Zeyu Sun  1 , Yuanjian Fang  1 , Jingwei Zheng  1 , Shenbin Xu  1 , Weilin Xu  1 , Ligen Shi  1 , Shuhao Mei  1 , Haijian Wu  1 , Feng Liang  1 , Jianmin Zhang  1   2   3
Affiliations

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

Jianan Lu et al. Front Immunol. .

Abstract

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.

Keywords: A20; intracerebral hemorrhage (ICH); melatonin; microglia; necroptosis.

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Figures

Figure 1
Figure 1
Effects of melatonin on neurologic deficit score, neurological functions, and brain edema. (A) Representative photographs of brain slices in the sham and ICH groups (72 h after ICH). (B) Quantification of brain water content at 72 h after ICH. *P <0.05 vs. sham group, &P <0.05 vs. ICH+vehicle group (n = 6 in each group). (C) Comparison of neurologic deficit scores among ICH+vehicle and ICH+melatonin groups before ICH and at 1, 3, and 7 days after ICH. (D) Comparison of adhesive removal test results among the ICH+vehicle and ICH+melatonin groups before ICH and at 1, 3, 7 days after ICH. (E) Comparison of foot-fault test results among the ICH+vehicle and ICH+melatonin groups before ICH and at 1, 3, 7 days after ICH. (F) Comparison of rotarod test results among the ICH+vehicle and ICH+melatonin groups before ICH and at 1, 3, 7 days after ICH. *P <0.05.
Figure 2
Figure 2
Melatonin suppresses necroptosis in microglia after ICH. (A) RIP1, RIP3, and MLKL protein expression was significantly enhanced at 1 and 3 days after ICH. (B) RIP1 expression, *P <0.05 vs. sham group (n = 6/group). (C) RIP3 expression. *P <0.05 vs. sham group (n = 6/group), &P <0.05 vs. ICH 1-day group (n = 6/group). (D) MLKL expression. *P <0.05 (n = 6 in each group), &P <0.05 vs. ICH 1-day group (n = 6/group). (E,K) PI staining around ICH hematoma. A significant increase in PI+ microglia was observed in the ICH group. *P <0.05 vs. sham group (n = 6/group). Melatonin treatment significantly decreased PI+ microglia compared with the levels in the ICH+vehicle group. &P <0.05 vs. ICH+vehicle group (n = 6/group). (F) Expression of A20, RIP1, RIP3, and MLKL proteins in sorted microglia of each group. (G) A20 expression. (H) RIP1 expression. (I) RIP3 expression. (J) MLKL expression. *P <0.05 vs. sham group (n = 6/group), &P <0.05 vs. ICH 3-day group (n = 6/group).
Figure 3
Figure 3
Melatonin suppresses microglial necroptosis at the early stage after ICH. (A,B) Western blots for RIP1, RIP3, and MLKL proteins in mice pretreated or not with melatonin via intraperitoneal injection, at 1 day and 3 days after ICH. (C) RIP1 expression. *P <0.05 vs. corresponding sham group (n = 6/group), &P <0.05 vs. corresponding ICH+vehicle group (n = 6/group). (D) RIP3 expression. *P <0.05 vs. corresponding sham group (n = 6/group), &P <0.05 vs. corresponding ICH+vehicle group. (E) MLKL expression. *P <0.05 vs. corresponding sham group (n = 6/group), &P <0.05 vs. corresponding ICH+vehicle group. (F–I) Immunofluorescence staining showing the distribution of RIP1/RIP3/MLKL+ microglia surrounding hematoma at 3 days after ICH. Corresponding individually stained fluorescence images of higher magnification (40×) are shown in Supplementary Figure S3. *P <0.05 vs. sham group (n = 6/group), &P <0.05 vs. ICH+vehicle group (n = 6/group).
Figure 4
Figure 4
Mitochondrial damage after ICH. (A) TEM images of mitochondrial ultrastructure (black arrow) in basal ganglion area. (B) Representative photographs of JC-1-staied BV2 cells. (C) Quantification of JC-1 fluorescence intensity using a fluorometric microplate reader at dual wavelengths. Treatment with 10 μM OxyHb significantly decreased the Δψm of BV2 cells, whereas pre-incubation with melatonin attenuated the OxyHb-induced collapse of the Δψm. *P <0.05 vs. control group (n = 6 in each group), &P <0.05 vs. OxyHb+vehicle group (n = 6/group).
Figure 5
Figure 5
OxyHb decreases cell viability, exerts cytotoxicity, and promotes inflammatory factor production in BV2 cells, thus inducing necroptosis in HT22 cells. Pre-incubation with melatonin effectively reduces the occurrence of these effects. (A) Representative photographs of BV2 cells in the control and OxyHb group (72 h). BV2 cells in the OxyHb group showed cell swelling and plasma membrane rupture. (B) Results of cell viability assay. (C) Result of LDH assay. (D) Results of ROS production assay. (E) Results of TNF production assay in BV2 cell supernatant. OxyHb increased TNF production in BV2 cells, whereas melatonin significantly suppressed this. (F–H) Flow-cytometric analysis of HT22 cells cocultured with BV2 cells exposed to different treatments. HT22 cells cocultured with BV2 cells treated with OxyHb showed a higher necroptotic population (defined as PI+/FITC−), whereas melatonin significantly reduced the necroptotic population. *P <0.05 vs. control group (n = 6/group), #P <0.05 vs. OxyHb+vehicle group (n = 6/group), &P <0.05 vs. OxyHb+melatonin+con-siRNA group.
Figure 6
Figure 6
Role of A20 in the regulation of necroptosis after ICH by melatonin. (A) Western blots for A20 proteins in mice pretreated or not with melatonin via intraperitoneal injection, at 3 day after ICH. (B) A20 expression. *P <0.05 vs. sham group (n = 6/group), &P <0.05 vs. ICH+vehicle group (n = 6/group). (C,D) Immunofluorescence staining showing the distribution of A20+ microglia surrounding hematoma at 72 h after ICH. A significant increase in A20+/Iba-1+ cells was observed in the ICH+vehicle group. *P <0.05 vs. sham group (n = 6/group). Melatonin treatment significantly enhanced the increase in A20+/Iba-1+ cells. &P <0.05 vs. ICH+vehicle group (n = 6/group). Corresponding individually stained fluorescence images of higher magnification (40×) are shown in Supplementary Figure S3. (E) A20 knockdown attenuated the inhibitory effects of melatonin on microglial necroptosis. (F) A20 expression, (G) RIP1 expression, (H) RIP3 expression, (I) p-RIP3 expression, (J) NLRP3 expression, (K) MLKL expression, and (L) p-MLKL expression. *P <0.05 vs. sham group (n = 6/group), &P <0.05 vs. ICH+melatonin+con-siRNA group.
Figure 7
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.
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