| Most of the tissue damage after cerebral ischemia is necrosis,mainly manifested by edema of organelles,decreased ATP,increased reactive oxygen species(ROS),raised intracellular Ca2+,and the final rupture of organelles and membranes.In the past,necrosis was considered irreversible and uncontrollable,so there was less research on necrosis than apoptosis.However,recent studies have found a new type of cell death which is precisely regulated,known as necroptosis.Necroptosis is mainly triggered by tumor necrosis factor receptor(TNFR)family and toll-like receptors(TLRs)family.When apoptosisis inhibited,necrosis as “substitute” is activated.Necroptosis signaling is activated by receptor interacting protein kinase(RIPK)1 and RIPK 3,and phosphorylating mixed lineage kinase domain like protein(MLKL),which formed necrosome,eventually leading to cell death.Because of losing the membrane integrity in necrotic cells after cerebral ischemic injury,necroptotic cells release a mass of damage associated molecular patterns(DAMPs),which can trigger a violent inflammatory response.Apoptosis and autophagy are generally considered as non-inflammatory or only to trigger a limited inflammatory response.Indepth study of the molecular mechanism of necroptosis and its relationship with inflammation has become a new hotspot and trend in the field of cell death research.The problems such as the main way and mechanism of cell death in injured area in cerebral ischemia,dynamic changes and type of dead cells,have yet clear.Because of the blood brain barrier(BBB)damage after cerebral ischemia,the monocytes/macrophages from vessels infiltrate into brain tissue.Moreover,microglia cells,the resident macrophages in central nervous system(CNS)are activated.Migration and accumulation of macrophages/microglia immediately cause inflammation,further aggravating tissue damage.However,whether necroptosis is involved in regulating macrophages/microglia M1/M2 polarization after cerebral ischemia,and its underlying mechanism is be explored.Elucidating the relationship between necroptosis and inflammatory response after cerebral ischemia is important for the understanding of the ischemic neurological damage and functional recovery.Therefore,we designed the following experiments to investigate the effects and mechanism of necroptosis on the polarization of local macrophages/microglia in cerebral ischemia.This project consists of three parts.Part 1: The investigation of necroptosis in the injured area after cerebral ischemia in mice.Objective: To study the cell types and dynamic changes of necrotic cells in cerebral ischemia of mice.Methods: Focal cerebral ischemia is induced by photothrombosis of the cortical microvessels.Dead cells in damaged area and the surrounding in ischemic cortex are stained by propidium iodid(PI).We evaluated the number of necrotic cell respectively in 1 d,3 d,5 d and 7 days post injury(dpi).Double-staining of PI with Neu N,GFAP,NG2 or Iba-1 were done to determinethe cell types and dynamic changes of necrotic cells in cerebral ischemia.Western blot was used to detect the expression of RIPK3,MLKL and p MLKL protein in control side(Con)and ischemic cortex respectively at 1d,3d,5d and 7 dpi.Double immunostaining and quantification of p MLKL with Neu N,GFAP and Iba-1 in the ischemia cortex was done to estimate necroptotic cell type.Immuno-electron microscope was adopted to study the ultrastructural distribution of RIPK3 and MLKL protein in ischemic areas.In addition,we used RIPK3 and MLKL gene knockout mice to observe the changes in the infarct area of ischemic cortex respectively in 7 dpi and 14 dpi.The functional recovery of the forelimb movement of wide-type mice(WT),RIPK3-/-and MLKL-/-mice was detected by behavioral experiments,such as forelimb activity(FLA)ability test,slide scores,foot faults test and asymmetrical index respectively in 1,3,7 and 14 dpi.The protective effects of RIPK3-/-and MLKL-/-on neuronal apoptosis in the injured area after cerebral ischemia were observed by TUNEL staining with Neu N immunostaining.Results: 1.Three days after the cerebral ischemia,the number of PI positive cells reached peak,and then gradually decreased.PI positive cells were mainly co-labeled with Neu N at 3 dpi,and switched to be mainly co-labeled with GFAP positive cells at 7 dpi.2.The expression of RIPK3,MLKL and p MLKL in the ischemia cortex of mice at 3,5and 7 dpi were significantly increased as compared with control side(Con)(P<0.05,P<0.01).3.Phosphorylated MLKL-immunoreactivities were mainly detected in Neu N positive cells in the ischemia cortex of mice at 1-3 dpi.RIPK3 and MLKL nano-gold particles were mainly deposited in neurons at 3 dpi.Whereas p MLKL is mainly associated with GFAP positive cells at 5-7 dpi.RIPK3 and MLKL nano-gold particles are mainly distributed in astrocyte cytoplasm or cell membrane.4.Behavioral experiments show that the FLA test,sliding scores,foot faults test and asymmetric index of RIPK3-/-and MLKL-/-mice are significantly improved than that of WT mice in 7 dpi and 14 dpi(P<0.05,P<0.01).5.The lesion area in ischemia cortex of RIPK3 and MLKL gene knockout mice were significantly reduced compared to WT mice at 7 and 14 dpi(P<0.05,P<0.01).6.Compare with WT mice,TUNEL positive cells in RIPK3 and MLKL gene knockout mice were significantly reduced in the lesion area after cerebral ischemia(P<0.05,P<0.01).Conclusion: 1.Necroptosis that was mainly composed of necrotic neurons occurs in the ischemia cortex of mice at 1-3 dpi,and the necroptosis was dominated by astrocytes in the lesion area at 5-7 dpi.2.Knockout of RIPK3 and MLKL gene can reduce the lesion area of cerebral ischemia,promote the recovery of sensorimotor function and therefore have obviously neuroprotective effectsPart 2: The effect of necroptosis on the polarization of local macrophages/microglia after cerebral ischemia of mice.Objective: To study the effects of necroptosis of ischemic cortex on the M1/M2 polarization of macrophage/microglia in the lesion area.Methods: In vivo,expression and distribution of Iba-1 in the lesion area of cerebral ischemia were detected by immunostaining and Western blot.Double-immunostaining and quantification of i NOS with F4/80 or Arginase 1(Arg-1)with Iba-1 were done to estimate M1/M2 polarization of macrophages/microglia.Western blot and q PCR were used to detect the expression of i NOS and Arg-1 at protein and mRNA level.Moreover,the expression of M1-type factors,such as TNF?,IL-12,IL-18 and other M2-type factors,such as IL-4 and IL-10 were detected by q PCR.In vitro,the oxygen and glucose deprivation(OGD)of neurons and astrocytes was performed,and conditioned medium(CM)was collected separately.The expression of RIPK3 and p MLKL after OGD treatment were detected by Western blot in order to evaluate the establishment of cellular necropotosis model in vitro.The primary cultured bone marrow-derived macrophages(BMDMs)from WT and RIPK3-/-mice were stimulated by the CM respectively.Effect of these CM on the M1/M2 polarization state of BMDMs was observed by Western blot.The expressions of M1-type and M2-type associated factors by neurons after OGD were examined by q PCR.Results: 1.Expression of Iba-1 in the lesion area of cerebral ischemia in RIPK3-/-and MLKL-/-mice was increased obviously than that in WT mice(P<0.05).2.Compared with WT mice,the expression level of i NOS in the lesion area of cerebral ischemia in RIPK3-/-and MLKL-/-mice was decreased significantly(P<0.05,P<0.01).mRNA levelsof i NOS?TNF?,IL-12 and IL-18 in RIPK3-/-and MLKL-/-mice also were significantly reduced than those in WT mice(P<0.05,P<0.01).3.Compared with WT mice,the expression level of Arg-1 in the lesion area of cerebral ischemia in RIPK3-/-and MLKL-/-mice was increased significantly(P<0.05,P<0.01).The mRNA levels of Arg-1?IL-4 and IL-10 in RIPK3-/-and MLKL-/-mice also were significantly increased than those in WT group(P<0.05,P<0.01).4.In vitro,the expression of RIPK3 and MLKL incultured astrocytes or neurons treated with OGD were significantly higher than that in Con group(P<0.01).5.CM from astrocytes with OGD treatment has little influence on the expression of i NOS and Arg-1 of bone marrow-derived macrophage(BMDMs)(P>0.05).6.CM from WT-mice neurons with OGD treatment(OGD-WT N)can cause higher expression of i NOS of BMDMs(P<0.01).7.Compared with CM from OGD-WT N,the protein and mRNA of i NOS,and the mRNA of CD86,TNF?and IL-18 in BMDMs stimulated by CM from RIPK3-/--mice neurons with OGD treatment(OGD-RIPK3-/-N)were lower(P<0.05,P<0.01).The protein and mRNA of Arg-1,as well as the mRNA of CD206,IL-4 and IL-10 were significantly increased(P<0.05,P<0.01).8.The expression of BDNF and the number of BDNF/Iba-1 in the lesion area of cerebral ischemia in RIPK3-/-and MLKL-/-mice was increased significantly than that in WT mice(P<0.01).9.RIPK3 or MLKL knockout had no significant effect on the expression of CD86 or CD206 in CD11 b positive cells in the peripheral blood(P>0.05).10.Compared with WT mice,the expression of i NOS in the lesion area of cerebral ischemia in Caspase 3-/-mice was obviously increased(P<0.05),and the expression level of Arg-1did not change significantly(P>0.05).Conclusion:1.Necroptosis in cerebral ischemia or necroptotic neurons with OGD treatment can induce the M1 polarization of macrophages/microglia cells,while the effect of necroptotic astrocytes is not significant.2.Knockout of RIPK3 or MLKL can induce the M2 polarization of macrophages/microglia cells in the ischemic cortex.3.RIPK3 or MLKL knockout is advantageous to repair of lesion area in cerebral ischemia may be due to BDNF released by M2 macrophages/microglia.4.RIPK3 or MLKL knockout had little influence on the M1/M2 type polarization of monocyte / macrophage in the blood.5.Caspase-3 knockout does not induce the M2 polarization of macrophages/microglia cells.Part 3: Invovlement of TLRs/My D88 pathway in the regulation of macrophages/ microglia polarization induced by necroptosis in cerebral ischemia.Objective: To explore the role of TLRs/My D88 signaling pathway in the regulation of macrophages/microglia polarization after cerebral ischemia.Methods: In vivo,the expression of TLR2,TLR4 and My D88 in the lesion area of cerebral ischemia were observed by immunohistochemical staining and Western blot.The changes of TLR2,TLR4 and My D88 expression in RIPK3-/-and MLKL-/-mice were detected by Western blot and q PCR.In addition,we blocked the TLRs/My D88 pathway by using a My D88 inhibitory peptide(MIP)in order to acess the effects of MIP on local macrophages/microglia polarization after cerebral ischemia.In vitro,CM of OGD treated neurons from WT and RIPK3-/-mice were separately collected.The CM was used to stimulate cultured bone marrow-derived macrophages(BMDMs)for observing the expression difference of My D88 and polarization change.Furthermore,MIP was used to stimulate BMDMs.In the end,the effect of MIP on the M1/M2 polarization of BMDMs was investigated.Results: 1.The expression of TLR2 and TLR4 in RIPK3-/-and MLKL-/-mice was significantly lower than that in WT mice(P<0.05).2.The expression of My D88 in the lesion area of cerebral ischemia was significantly higher than that of non-injured side(P<0.01).Compared with WT mice,the expression of My D88 in RIPK3-/-and MLKL-/-mice was significantly reduced(P<0.05,P<0.01).3.The expression of My D88 was significantly increased in neurons treated with OGD(P<0.05).Compared with OGD-WT N group,the expression of My D88 in OGDRIPK3-/-N group decreased significantly(P<0.05).4.Compared with control peptide(CP)group,the protein level of i NOS,the mRNA levels of i NOS and CD86 of BMDMs after MIP treatment decreased significantly(P<0.05),and the protein level of Arg-1,and the mRNA levels of Arg-1and CD206 significantly increased(P<0.05).5.After injection of MIP,the protein level of i NOS,the mRNA levels of i NOS CD86,TNF? and IL-18 in lesion area of cerebral ischemia were significantly decreased than that of CP group(P<0.05,P<0.01).The protein level of Arg-1,the mRNA levels of Arg-1,CD206,IL-4 and IL-10 mRNA in MIP group was significantly increased compared with that in the CP group(P<0.05,P<0.01).6.MIP can significantly reduce the number of TUNEL positive cells around the area of cerebral ischemia injury,and the number of TUNEL/Neu N co-labeled cells decreased significantly compared with CP group(P<0.05).7.Compared with the CP group,the expression of BDNF in the ipsilateral injured cortices after MIP treatment were obviously increased(P<0.01),and the BDNF/Iba-1 doublepositive cells increased significantly.Conclusion: 1.The expression of TLR4,TLR2 and My D88 in the injured area after cerebral ischemia of mice was significantly increased.2.The expression of My D88 in macrophages/microglia was up-regulated by the CM of necropototic neurons induced by OGD.3.RIPK3 or MLKL gene knockout can block the increase of My D88 induced by cerebral ischemia or OGD treatment.4.Antagonizing the function of MyD88 could phenocopy the effects of RIPK3-and MLKL-knockout on the polarization of microglia/macrophages. |
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