| Stroke is the second leading cause of death and the most frequent cause of permanent disability of worldwide.Significant advances have been made to lessen the immense public health problem of stroke.However,limited achievements have been made in developing therapies to decrease the detrimental results of cerebral ischemia.Minutes after brain ischemia,inflammatory mechanisms can be activated which have been known as a key target of current translational ischemic stroke research.Researches suggested the levels of proinflammatory cytokines and chemokines are increased after focal ischemia.While some cytokines may offer protection,many cytokines and chemokines have been shown to participate in neuronal damage processes.Pathophysiological process of cerebral ischemia included processes of upregulation of cerebral proinflammatory cytokines,activation of local microglia,astrocytes and systemic lymphocytes and invasion of leukocyte in the brain.MCPIP1(also known as ZC3H12A)is a recently identified protein in human peripheral blood monocytes treated with monocyte chemotactic protein1(MCP-1).In previous studies,MCPIP1 has known to be a negative regulator of macrophage activation.Researchers indicated that MCPIP1 can play a significant anti-inflammatory role by inhibiting the generation of a set of major proinflammatory cytokines.Melittin,the main component of honeybee(Apis mellifera)venom,is a cationic,hemolytic peptide comprising a small linear peptide composed of 26 amino acid residues.Many research has reported melittin having various biologic activities,such as anti-bacterial,anti-viral,and anti-inflammatory,anti-cancer activities.Recent study suggested that melittin has potential therapeutic effects asan agent for the prevention of neurodegenerative diseases and can inhibit neuroinflammation in an ALS animal model.In this study we used adult male C57BL/6 mice and subjected to occlusion of distal branches of middle cerebral artery(dMCAO).And we examined proinflammatory cytokines including IL-1β,IL-6,TNFα,TLR,MyD88,NF-κB and MCPIP1 gene expression and protein concentration in dMCAO mouse and in mouse brain under LPS treatment.We designed to investigate whether melittin improved neurological recovery and whether the TLR4/MyD88/NF-κB pathway and neurotrophic factors are involved.In order to provide more research evidence and research train of thought for the treatment and prevention of ischemic stroke.Part one Effect of MEL on neurological recovery,neuroprotection and anti-inflammatory in experimental cerebral infarction miceObjective: The neuroprotective effects of melittin on focal cerebral ischemia mice were evaluated and the effective dose was determined by evaluating the scores of nerve function defect,brain water content,cerebral infarction volume and cerebral blood flow in mice with focal cerebral ischemia after treatment with melittin.Then the anti-inflammatory effects of melittin were studied by detecting the transcription and translation levels of inflammatory factors.Methods:We used adult male C57BL/6 mice and subjected to dMCAO.All the mice were randomly assigned to the following groups: Sham group(Sham),d MCAO group,low dose group:0.1μg/g and medium dose group 0.2g/g and high dose group 0.4 g/g.MEL was administered intraperitoneally one day before operraton and once daily 1day until sacrificed.We assessed neurologic deficits with Rota-Rod test and modified neurological severity score(mNSS)at base,24 h,48h,72 h after d MCAO.We evaluated infarct volume with TTC staining at 72 h days,examined cerebral blood flow(CBF)using a laser speckle imaging at baseline,0,6h,12 h,24h,48 h,72h after stroke.We evaluated mRNA expression and protein concentration of IL-1β、IL-6、TNFα、TLR4、MyD88 and NF-κB.Results:1.Weight changes showed that the weight,24 h,48h and 72 h of mice in different groups decreased rapidly after dMCAO(P<0.01),and there was no significant difference between groups(P>0.05).2.Before cerebral ischemia,mice showed no neurological deficits.Neurological function of 24 h,48h and 72 h decreased significantly after dMCAO(P < 0.01).Neurologic deficit as assessed by mNSS was not significantly different between Vehicle and MEL groups on 24 h,48h,72h(P>0.05).3.In the Rota-Rod test,neurological function decreased significantly after dMCAO operation(P<0.01).In Vehicle group,neurological function declined more significantly than those in high dose MEL group(P<0.05).After 48 h,72h,medium and high dose MEL group,the neurological function recovery was more significant than Vehicle group(48h: P<0.05;72h: P<0.01).And the recovery of neurological function was in a dose-dependent manner(P<0.05).4.After TTC staining,brain infarct volume was significantly smaller in MEL-treated mice than in vehicle-treated mice on 24 h,48h,72 h after dMCAO(24h,72h: P<0.05;48h: P<0.01).5.Compared with Vehicle group,MEL group reduced the brain water content significantly both at 24 h,48 h and 72 h(P<0.05).6.Cerebral perfusion on the contralateral side showed a slight decrease after dMCAO,but on the ipsilateral side,there was a significant decrease in CBF flux to from the baseline(P<0.01).There was no significant difference between MEL group and Vehicle group on the lesion side of the focus side of6 h immediately after operation,but there was no significant difference between MEL group and Vehicle group,but the recovery of CBF was more obvious from 12 h to 72 h.7.qRT-PCR analysis showed melittin can reduce inflammatory factors IL-1β(Vehicle vs.Sham : P<0.01;MEL-L vs.Vehicle: P>0.05;MEL-M vs.Vehicle: P<0.05;MEL-H vs.Vehicle :P<0.01),IL-6,TNFα,TLR4,MyD88,NF-κB(Vehicle vs.Sham : P<0.001;MEL-L vs.Vehicle: P>0.05;MEL-M vs.Vehicle: P<0.05;MEL-H vs.Vehicle :P<0.05)mRNA in ischemic brain tissue.8.ELISA results showed melittin can reduce protein density of inflammatory factors IL-1β(Vehicle vs.Sham : P<0.001;MEL-L vs.Vehicle:P>0.05;MEL-M vs.Vehicle: P<0.01;MEL-H vs.Vehicle :P<0.01),IL-6(Vehicle vs.Sham : P<0.001;MEL-L vs.Vehicle: P>0.05;MEL-M vs.Vehicle: P<0.05;MEL-H vs.Vehicle :P<0.01)、TNFα(Vehicle vs.Sham :P<0.05;MEL-L vs.Vehicle: P>0.05;MEL-M vs.Vehicle: P<0.05;MEL-H vs.Vehicle :P<0.05).9.According to the above results,we selected the MEL high dose treatment group to represent the drug treatment group to carry out the Western blot test.The results showed that compared with group Vehicle,MEL treatment group could significantly reduce protein density of TLR4(Vehicle vs.Sham :P<0.01;MEL vs.Vehicle :P<0.05),MyD88(Vehicle vs.Sham: P<0.05;MEL vs.Vehicle: P <0.05)and NF-κB(Vehicle vs.Sham :P < 0.05;MEL vs.Vehicle:P<0.05),MEL also can continuously up-regulated MCPIP1 protein density(Vehicle vs.Sham:P<0.001;MEL vs.Vehicle:P<0.001).Part two Protective effect of MEL on rat microglial inflammatory injuryObjective:To establish an in vitro model of inflammatory reaction,to detect the changes in inflammatory factors of LPS at different treatment time and to detect the changes of inflammatory factors after the treatment of MEL,so as to investigate whether the neuroprotective effect of MEL is played through the anti-inflammatory effect.Methods:LPS induced BV2 microglia to construct an in vitro inflammatory injury model.According to the time of LPS treatment,BV2 cells were randomly assigned to the following groups: LPS 3h group,LPS 6h group,LPS 12 h group,LPS 24 h group,qRT-PCR detected IL-1β、IL-6、TNFα、TLR4、MyD88 and NF-κB mRNA expression level.BV2 treated with MEL-L(0.5μg/ml),MEL-M(1μg/ml),MEL-H(2μg/ml),the inflammatoryfactors IL-1β 、 IL-6 、 TNFα 、 TLR4 、 MyD88 and NF-κB mRNA expression and protein density were detected and analyzed by qRT-PCR,Western blot and ELISA.Cell immunofluorescence was used to detect the expression and localization of MCPIP1 and the nucleation of NF-κB after drug treatment.To evaluate the neuroprotective effect of MEL on BV2 inflammatory injury,the cell supernatants of the treated BV2 were collected and incubated with SH-SY5 Y to detect the cell supernatants.Results:1.QRT-PCR method was used to detect the effect of LPS treatment on mRNA after BV2 inflammatory injury,results showed compared with the control group.The mRNA levels of IL-1β,IL-6,TNFα,TLR4,MyD88,NF-B and MCPIP1 increased with the prolongation of LPS treatment time(P<0.05),shows the vitro model of inflammation was constructed successfully.2.ELISA method was used to detect the effect of melittin on the levels of inflammatory factors after LPS induced BV2 inflammatory injury,results showed compared with the LPS treatment group,the levels of IL-1β,IL-6 and TNFα in MEL medium and high concentration groups decreased significantly(P<0.05).3.The Western blot results show that compared with the LPS treatment group,the protein levels of TLR4,MyD88 and NF-κB decreased after treatment of low,medium and high MEL drugs(P<0.01).Compared with the LPS treatment group,the protein level of anti-inflammatory factor MCPIP1 in MEL drug treatment group continued to rise(P<0.01).4.Effect of melittin on nuclear localization of NF-kappa B after LPS induced BV2 inflammatory injury detected by cell immunofluorescence.The result shows that NF-B(p65 subunit)is mainly located in cytoplasm at resting state.After stimulation with LPS,the nuclear localization of NF-κB increased,indicating that the pathway was activated.After MEL treatment and LPS treatment,the migration of NF-κB into the nucleus was inhibited.5.In order to investigate the cytotoxic effects of cellular inflammatory metabolites after BV2,the pathophysiological process of cerebral ischemiawas simulated.In this experiment,the cell supernatant after BV2 cell treatment was incubated with SH-SY5 Y,and the changes of the cell viability and apoptosis related factors of SH-SY5 Y after incubation were detected,reflecting the effect of the inflammatory metabolites of microglia on the nerve cells.The results showed that compared with the Control group,The viability of SH-SY5 Y cells incubated with LPS treated supernatant of BV2 decreased significantly(P<0.01).The relative toxicity of BV2 cell supernatants decreased and cell viability increased after MEL treatment in the medium and high concentration group(P<0.05).QRT-PCR assay was used to detect the mRNA level of apoptosis related factors in SH-SY5 Y cells.The results showed that after LPS treatment,compared with group Control,the level of Bax and AIF mRNA decreased significantly(P<0.01),and Bcl-2 level increased significantly(P<0.01).Compared with the LPS group,the level of SH-SY5 Y Bax and AIF mRNA in the medium and high concentration MEL drug treatment group increased significantly(P<0.01),and Bcl-2 level decreased(P<0.05),suggesting that the cell viability of the drug treatment group was increased.Part three Protective mechanism of MEL on BV2 cell inflammatory injuryObjective:In this experiment,the MCPIP1 gene was knocked down by si-RNA transfected BV2 cells,and the changes of various inflammatory factors were detected,and whether the mechanism of anti-inflammatory action of MEL was related to the MCPIP1 protein.Methods:The MCPIP1 si-RNA was transfected into BV2 cells,then LPS treatment and MEL drug treatment were carried out.The inflammatory factors IL-1β,IL-6,TNFα and protein levels were detected by qRT-PCR,Western blot and ELISA methods.The cell supernatant of BV2 was collected and incubated with SH-SY5 Y to detect its neurotoxicity,so as to explore whether the anti-inflammatory mechanism of MEL is related to MCPIP1 protein.Results:1.As for the transfection efficiency detection,the transfection efficiency of si-RNA was detected by qRT-PCR and Western blot respectively.The results showed that the mRNA level and protein expression level of MCPIP1 were significantly decreased(P<0.01)compared with si-Control(P<0.01)after transfection of BV2 cells,which proved that the transfection was successful.2.In order to further study the effects of MCPIP on IL-1β,IL-6 and TNFα,and the relationship between MCPIP and NFκB,after successfully knocking down the expression of MCPIP1 and protein,LPS was induced to induce inflammatory reaction and MEL drug treatment,the expression of mRNA and protein in IL-1β,IL-6,TNFα,TLR4,MyD88,and NF-κB were detected by qRT-PCR,ELISA,and Western blot,respectively.The results showed that the levels of IL-1β mRNA and protein were not significantly different from those in the si-Control group(P>0.05),and the mRNA levels of IL-6,TNFα,TLR4,MyD88 and NF-κB increased(P>0.05).4,NF-κB: P<0.05;MyD88:P<0.01 vs.si-Control).The results of cell immunofluorescence showed that the expression of NF-κB in si-ZC3H12 A group was more than that in si-Control group,suggesting that MCPIP1 gene knocks.In addition,the antiinflammatory effect of MEL is weaker than before,indicating that the antiinflammatory effect of MEL is mediated by MCPIP1 protein,which affects the signal transduction of the TLR4/MyD88/NF-κB pathway.3.The cytotoxicity of cell supernatant to SH-SY5 Y after si-RNA transfection of BV2 cells showed that the results of CCK-8 and LDH showed that the activity of SH-SY5 Y in the cell supernatant of the si-ZC3H12 A group decreased(P<0.01)compared with the si-Control group.QRT-PCR indicated that the level of AIF and Bax apoptosis related factors mRNA decreased(P<0.01)and Bcl mRNA(P<0.01),compared with group si-Control.It is suggested that for the BV2 cells that knock out MCPIP1,the cytotoxicity of the cell supernatant induced by LPS induced inflammatory injury increases in spite of the MEL drug treatment,which confirms that the anti-inflammatory and neuroprotective effects of MEL are mediated by MCPIP1.Conclusions:1.It can promote the recovery of nerve function in mice with focal cerebral ischemia,reduce the volume of cerebral infarction,reduce the edema of brain tissue,promote the recovery of blood flow of ischemic brain tissue,and inhibit the expression of IL-1β,IL-6,TNFα,and TLR4/MyD88/NF-κB signaling pathway related factors of ischemic brain tissue,and promote the anti-inflammatory factors.The expression of sub MCPIP1 has antiinflammatory damage and neuroprotective effects.2.Melittin can inhibit the increase of gene and protein levels of the key factors such as IL-1β,IL-6,TNFα in LPS induced BV2 cells and the key factors of TLR4/MyD88/NF-κB signaling pathway,and inhibit the transfer of NFκB into the nucleus,which can reduce the toxicity of BV2 inflammatory metabolites to the nerve cells.It has neuroprotective effect through antiinflammatory reaction activity.3.Melittin can induce the continuous expression of MCPIP1 during cerebral ischemia and inflammatory injury in BV2 microglia and mouse brain tissue,and the inhibition effect of melittin treatment on inflammatory related factors IL-1β,IL-6,TNFα,and TLR4/MyD88/ NF-κB signaling pathway in BV2 cells of inflammatory injury.It indicates that the anti-inflammatory activity of melittin is mediated by MCPIP1,thereby inhibiting the transduction of TLR4/MyD88/NF-κB signaling pathway. |
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