| Part one The neuroprotective effect of sphingomyelin synthase 2deficiency in the mouse model of cerebral ischemic reperfusionObjective: To observe the effect of sphingomyelin synthase 2(SMS2)in the mouse model of focal cerebral ischemic reperfusion through evaluating neurological deficit scores and infarct volumes.Methods:1 Male,healthy adult C57BL/6 mice(WT)and SMS2 konck out mice(on the background of C57BL/6)were used as the objects.Transient middle cerebral artery occlusion(t MCAO)in mice were established using the classic suture method.WT mice and SMS2 konck out mice(SMS2-/-)were then randomly divided into two subgroups: Sham group and t MCAO group.Mice in Sham group received the sham operation.Mice in t MCAO group received the operation of middle cerebral artery occlusion for 1 hour and then realized reperfusion.Neurological defect scores and cerebral infarct volumes of mice in each group were evaluated at 24 h and 72 h after the t MCAO.Neurological function was evaluated by neurological deficit scores.Cerebral infarction volumes of mice in each group were measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Results:1 Neurological deficit scores of t MCAO group were significantly higher than Sham group which was 0,the difference was statistically significant(P <0.05).Neurological deficit scores of SMS2-/-group were decreased significantly compared with WT group at 72 h after t MCAO,the difference was statistically significant(P < 0.05).There was no significant difference in neurological deficit scores at 24 h after t MCAO(P > 0.05).2 After TTC staining,the brain slices of Sham group were red uniformly,while the t MCAO group showed a large white infarct area on the ischemic hemisphere.Compared with WT group,the infarct volumes of SMS2-/-group were reduced at 72 h after t MCAO,the difference was statistically significance(P < 0.05).There was no significant difference in the infarct volumes at 24 h after t MCAO(P > 0.05).Part two Sphingomyelin synthase 2 deficiency suppressed the inflammatory in the mouse model of cerebral ischemic reperfuision.Objective: To evaluate the effect of SMS2 deficiency on inflammatory in the mouse model of focal cerebral ischemic reperfusion,we investigated the expression of activated microglia and the protein and m RNA level of related inflammatory mediators in the penumbra of ischemic hemisphere.Methods: Male,healthy adult C57BL/6 mice and SMS2 knock out mice(on the background of C57BL/6)were used as the objects.Transient middle cerebral artery occlusion(t MCAO)in mice were established using the classic suture method.C57BL/6 mice(WT)and SMS2-/-mice(SMS2-/-)were then randomly divided into two subgroups: Sham group and t MCAO group.Mice in Sham group received the sham operation.Mice in t MCAO group received the operation of middle cerebral artery occlusion for 1 hour and then realized reperfusion.At 24 h and 72 h after t MCAO,RT-PCR was used to detect the m RNA level of i NOS,Arg-1,IL-1? and galectin-3.Immunofluroence was utilized to dectect the expression of activated microglia in the penumbra of ischemic brain.Western-blot was used to measure the nucler translocation of NF-?B.Flow cytometry staining was used to detect the expression of TLR4 and TLR4/MD2 in microglia.Results:1 After RT-PCR analysis,the m RNA levels of i NOS,IL-1? and IL-6 in t MCAO group were significantly increased,while the m RNA levels of Arg-1was significantly decreased in t MCAO group compared with Sham group,the difference was statistically significant(P < 0.05).The m RNA levels of IL-1? and galectin-3 in SMS2-/-group were significantly decreased compared with WT group,while the m RNA levels of Arg-1 was significantly increased compared with WT group,the difference was statistically significant(P <0.05).2 Western-blot analysis showed that the protein level of galectin-3 was significantly increased at 48 h and 72 h after t MCAO(P < 0.05).When compared with WT group,the protein level of galectin-3 was significantly decreased at 72 h after t MCAO in SMS2-/-group(P < 0.05).3 There was no difference in the numbers Iba1+ cells between WT and SMS2-/-groups,while the amount of Iba1+galectin-3+ cells in the penumbra area of mice in SMS2-/-group were significantly decreased compared with WT group,the difference was statistically significant(P < 0.05).4 Western-blot analysis showed the nuclear translocation of NF-?B in t MCAO group were significantly increased compared with Sham group,the difference was statistically significant(P < 0.05).When compared with WT group,the nuclear translocation of NF-?B was decreased in SMS2-/-group both at 24 h and 72 h after t MCAO,the difference was statistically significant(P < 0.05).5 Flow cytometry analysis showed that SMS2-/-mice contained fewer TLR4/MD2 complexes on the cell surface than WT mice at 72 h after t MCAO.Part three Sphingomyelin synthase 2 inhibited the recruitment of TLR4 into lipid rafts throngh altering the SM content in lipid raft to exert anti-inflammation effect.Objective: To investigate the underlying mechanisms of the antiinflammation effect of SMS2 deficency in mice model of cerebral ischemic reperfusion,we detected the expression of TLR4 in lipid rafts and non-lipid rafts,and the sphingomyelin quantification in mice brain were measured by tandem mass spectrometry(LC-MS/MS).Methods: Male,healthy adult C57BL/6 mice and SMS2-/-mice(on the background of C57BL/6)were used as the objects.Transient middle cerebral artery occlusion(t MCAO)in mice were established using the classic suture method.C57BL/6 mice(WT)and SMS2 knock out mice(SMS2-/-)were then randomly divided into two subgroups: Sham group and t MCAO group.Mice in Sham group received the sham operation.Mice in t MCAO group received the operation of middle cerebral artery occlusion for 1 hour and then realized reperfusion.At 24 h and 72 h after t MCAO,Lipid raft isolation was used to detect the distribution of TLR4 in lipid raft fractions and non-lipid raft fractions in mice brain.LC-MS/MS was used to measure the SM content in the mice brain of WT mice and SMS2-/-mice.Results:1 Western blot analysis showed that the expression of TLR4 in the fractions of lipid rafts was increased after t MCAO.When compared with WT group,the expression of TLR4 in fractions of lipid rafts was decreased in SMS2-/-group.2 The results of LC-MS/MS showed that lower level of SM,such as C14-SM,C16-SM,C18:1-SM,C18-SM,C20:1-SM and C24:1-SM,was detected in the fractions of brain lipid raft in SMS2-/-group(P <0.05).Therefore,the alteration of SM level in the lipid rafts fractions was,at least partly,contributed to the impairment of TLR4 recruitment into lipid raft in SMS2-/-mice.Conclusions:1 The mouse model of cerebral ischemic reperfusion was successfully established by occlusing the middle cerebral artery for 1 hour using classic suture method.The neurological deficit scores and infarct volumes in SMS2-/-group were significantly improved at 72 h after t MCAO.2 SMS2 deficiency down-regulated the m RNA level of pro-inflammatory cytokines(IL-1? and galectin-3),and up-regulated the m RNA level of antiinflammatory cytokines(Arg-1).Meanwhile,SMS2 deficiency reduced the number of Iba1+galectin-3+ cells in the penumbra area at 72 h after t MCAO.Moreover,SMS2 deficiency also reduced the nuclear translocation of NF-?B.Moreover,SMS2 deficiency decreased the numbers of TLR4/MD2 complex on the cell surface.3 SMS2 deficiency decreased the SM levels in the fractions of brain lipid rafts,and thus inhibited the recruitment of TLR4 into lipid rafts.The above results indicated that the underlying mechanisms of the neuroprotective and anti-inflammation effect of SMS2 deficiency attributed to,at least partly,the inhibition of the TLR4 recruitment into lipid raftsa after cerebral ischemic reperfusion. |
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