| Multiple sclerosis(MS)is an inflammatory demyelinating disease of the central nervous system(CNS).Its main pathological manifestations are destruction of blood-brain barrier(BBB),infiltration of peripheral T cells/macrophages,causing a large number of inflammatory reactions to destroy myelin sheath and axons.The integrity of myelin sheath is an important factor to maintain the normal physiological function of CNS.The maintenance of the healthy myelin sheath and the regeneration of myelin sheath are very important for the functional recovery of CNS after demyelinating injury.The mobilization of oligodendrocyte precursor cells(OPCs)and the differentiation/maturation of oligodendrocytes(OLs)are the two key stages of the remyelination,which is regulated by positive and negative factors.Because myelin debris produced after demyelination play a votal role in aggravating the inflammatory reaction of CNS,inducing the degeneration and necrosis of neurons and inhibiting remyelination,it is particularly important to remove myelin debris.In MS and its models,microglia,an innate immune cell in CNS,mainly focused on the polarization in inflammatory state,and its influence on OLs,astrocytes(AST)or neurons.However,few people have studied the phagocytosis of microglia on myelin debris and the effect of phagocytosis on remyelination.Therefore,it is of great potential to explore cost-effective drugs that target microglia to phagocytize myelin debris and promote remyelination for the treatment of CNS demyelinating diseases.Fasudil,a novel potent vasodilator,has been approved effective for the treatment of vasospasm induced by subarachnoid hemorrhage.Studies on demyelinating models have shown that Fasudil improve the function of vascular endothelial cells,inhibit the infiltration of peripheral immune cells,and reduce the inflammatory response of CNS,thereby delaying demyelination and regenerating neuronal synapses.Therefore,in this study,we first observed the effect of Fasudil on microglial phagocytosis of myelin debris at the cellular level.Secondly,we explored the potential of Fasudil treatment,its influence on the microglial polarization in CPZ-induced mice,and further analyzed the relationship between microglial polarization and phagocytosis in vitro.Then,we identified the signaling pathway of Fasudil promoting microglial phagocytosis of myelin debris in vivo and in vitro.Finally,the role of microglial phagocytosis in the protection and regeneration of myelin sheath was explored in vivo and in vitro.Part I Effect of Fasudil on the phagocytosis of BV-2 microgliaObjective:To observe the effect of Fasudil at different concentrations and time on the phagocytosis of myelin debris by BV-2 microglia.Methods:Mouse-derived BV-2 microglia were cultured and subcultured by conventional methods.When the growth status and cell density were suitable,drug intervention was carried out.Firstly,5 mg/ml Fluorescent myelin debris(FMD)was added to each group.BV-2 microglia were cultured with different concentrations of Fasudil(?g/ml: 0,7.5,15,30,and 60,respectively)for 48 h.The phagocytosis of BV-2 microglia was analyzed by flow cytometry,fluorescence microscope and multifunctional microporous plate reader.Then,BV-2 microglia were intervened with 15 ?g/ml Fasudil and PBS for different time(hr: 6,12,24,48 and 72,respectively),and the phagocytosis of BV-2 microglia was detected by flow cytometry.Results:1.Compared with the 0 ?g/ml intervention group,the phagocytosis of BV-2 cells was not gradually increased with the change of Fasudil concentration.Relatively,under the intervention of 15?g/ml Fasudil,the ability of microglia to phagocytize FMD was the strongest,and the number of FMD+ microglia was the largest.2.With the extension of time,the phagocytic ability of BV-2 microglia in PBS group and Fasudil intervention group was gradually increased.Compared with PBS group,the number of BV-2 microglia phagocytizing FMD was significantly increased after Fasudil intervention.Conclusion:Fasudil induced BV-2 microglia to phagocytize FMD in a concentration-independent and time-dependent manner.Part ?Fasudil exerts anti-inflammatory effects and enhances phagocytic effects through inducing microglial transformation to M2 phenotypeObjective:To observe the effect of Fasudil on the polarization of microglia induced by myelin debris,and to explore the relationship between polarized microglia and the phagocytosis of myelin debris.Methods:Experiment in vivo: C57BL/6 mice were divided into three groups,Control group received standard diet for 6 weeks;CPZ+NS group received 0.2% CPZ diet for 6 weeks;CPZ+Fasudil group received 0.2% CPZ diet for 6 weeks,and injected intraperitoneally Fasudil(40 mg/kg/d)at the beginning of the fifth week.The mice in Control and CPZ+NS groups were injected 0.9% Na Cl.MWM,EPM and FST were used to measure the cognition,anxiety and depression of mice.Fluoromyelin staining was used to evaluate the extent of demyelination.The intensity of i NOS and Arg-1 in microglia was observed by immunofluorescence staining.Then,the protein expression of i NOS/Arg-1 was analyzed by Western blot.Experiment in vitro: BV-2 microglia were cultured in 4 groups,Fasudil group added with 15 ?g/ml Fasudil;PBS group added with PBS;Myelin+Fasudil group added with 5mg/ml FMD and 15 ?g/ml Fasudil;Myelin group added with 5 mg/ml FMD and PBS.All groups were incubated in incubator for 48 h.Immunocyte fluorescent and Western blot were used to analyze the level of i NOS/Arg-1 in BV-2 microglia.Flow cytometry was used to analyze M1(CD16/32,i NOS,IL-12)and M2(CD206,Arg-1,IL-10)miacroglia after FMD stimulation.Furthermore,the phagocytosis of M1/M2 microglia was analyzed by gating FMD-and FMD+ cells.The concentrations of IL-6,TNF-?,IL-10 and TGF-? in the supernatant were measured by ELISA.Results:1.Results in vivo:(1)Weight change: body weight has no difference between the three groups at 0 week(P>0.05).During the experiment,average weight in Control group increased continuously to 28.85±0.67 g at the end of 6 weeks.Compared with the normal mice(25.18±0.71 g),the average weight in CPZ mice decreased on the second weekend(19.67±0.61 g,P<0.0001),and then increased slowly.On the sixth weekend,the weight of CPZ mice increased to 22.78±0.74 g;Compared with the CPZ mice,Fasudil improved the weight loss(P=0.0027).(2)Behavioral changes: the results of MWM found that,compared with the Control group,the latency/distance to reach the platform of the CPZ mice were prolonged(P<0.001,P<0.01),while the proportion of time spent in target quadrant decreased(P<0.05).Fasudil effectively reversed these parameters(P<0.05,P<0.01,P<0.05).The results of EPM showed that,compared with the normal mice,the total resting time/distance of the CPZ mice were increased in closed arm(P<0.01),which shortened by Fasudil treatment(P<0.05).The results of FST showed that,compared with the normal mice,the mean swimming speed/total active distance were shortened in the CPZ mice(P<0.001,P<0.01),and the swimming rest time lengthened(P<0.0001).However,Fasudil effectively reversed these parameters(P<0.05,P<0.001,P<0.001).(3)Pathological observation: the demyelination of the corpus callosum in CPZ+NS group was worsen than that of the Control group,while Fasudil significantly inhibited the loss of myelin sheath of the CPZ mice(P<0.05).(4)Compared with the normal mice,immunofluorescence staining displayed that the number of Iba-1+i NOS+ cells in CPZ mice was enhanced.Fasudil inhibited it and increased Iba-1+Arg-1+ cells in brains.(5)The results of Western blot exhibited that,compared with the normal mice,the protein level of i NOS was enhanced(P<0.01)and Arg-1 inhibited(P<0.05)in CPZ mice brains,which were reversed by Fasudil treatment(P<0.05,P<0.001).2.Results in vitro:(1)Western blot showed that FMD stimulated BV-2 microglia to express i NOS(P<0.01),which was inhibited by Fasudil treatment(P<0.01).At the meantime,Fasudil enhanced the protein level of Arg-1(P<0.01).(2)Immunocyte fluorescent staining showed that FMD enhanced the level of i NOS,while Fasudil inhibited i NOS and promoted the expression of Arg-1.(3)Compared with the PBS group,the expression of M1 microglia markers(CD16/32,i NOS)in Myelin+PBS group were up-regulated(P<0.0001),while M2 microglia markers(CD206,Arg-1)were down-regulated(P<0.05,P<0.01).Fasudil effectively down-regulated the expression of CD16/32 and i NOS(P<0.0001,respectively),and up-regulated the expression of CD206 and Arg-1(P<0.0001,respectively).(4)The secretion of cytokines in the culture supernatant were measured by ELISA.Compared with PBS group,FMD increased the concentrations of TNF-? and IL-6 in Myelin+PBS group(P<0.05,P<0.001),which were inhibited by Fasudil intervention(P<0.001,respectively).Similarly,compared with Myelin+PBS group,the level of IL-10 and TGF-? was enhanced in Myelin+Fasudil group(P<0.05,respectively).(5)FMD-and FMD+ cells in Myelin+Fasudil group were measured by flow cytometry.Compared with FMD-cells,the expression of CD16/32,i NOS and IL-12 in FMD+ cells decreased(P<0.001,P<0.0001,P<0.0001),but CD206,Arg-1 and IL-10 increased(P<0.001,P<0.01,P<0.0001).Conclusion:1.Fasudil improved the weight change and behavioral abnormality of CPZ-induced demyelinating mice;2.Fasudil not only promoted the polarization of microglia to M2 phenotype in vitro and in vivo,but also enhanced the phagocytosis of M2 microglia to myelin debris,thereby improving the inflammatory microenvironment and protecting myelin sheath of CNS in CPZ mice.Part ?Mechanism of Fasudil promoting microglial phagocytosis of myelin debrisObjective:To explore the molecular mechanism of Fasudil promoting microglial phagocytosis of myelin debris.Methods:Experiment in vivo: C57BL/6 mice were divided into three groups,Control group received standard diet for 6 weeks;CPZ+NS group received 0.2% CPZ diet for 6 weeks;CPZ+Fasudil group received 0.2% CPZ diet for 6 weeks,and injected intraperitoneally Fasudil(40 mg/kg/d)at the beginning of the fifth week.The mice in Control and CPZ+NS groups were injected 0.9% Na Cl.Brain tissues were collected at the end of six weeks.The microglial expression of TREM2 and DAP12 was analyzed by immunofluorescence staining and Western blot.Experiment in vitro: BV-2 microglia were cultured in 4 groups,Fasudil group added with 15 ?g/ml Fasudil,PBS group added with PBS,Myelin+Fasudil group added with 5mg/ml FMD and 15 ?g/ml Fasudil,Myelin+PBS group added with 5 mg/ml FMD and PBS.All groups were incubated for 48 h.The expression of TREM2 and DAP12 in BV-2microglia was analyzed by RT-PCR,fluorescent staining and Western blot.Then,BV-2microglia were transfected with TREM2-Si RNA1-3.The silencing effect of TREM2 on BV-2 microglia was analyzed by RT-PCR.Under optimum conditions,BV-2 microglia were divided into four groups: Normal+FMD+Fasudil group added with 5mg/ml FMD and15 ?g/ml Fasudil;Normal+FMD group added with 5 mg/ml FMD and PBS;TREM2siRNA+FMD+Fasudil group with 5 mg/ml FMD and 15 ?g/ml Fasudil after TREM2 silence on BV-2 microglia;TREM2 siRNA+FMD group added with 5 mg/ml FMD and PBS after TREM2 silence on BV-2 microglia.Subsequently,the phagocytosis of BV-2microglia was analyzed by flow cytometry.Results:1.Results in vivo: immunofluorescence staining dispalyed that,compared with the normal and CPZ mice,Fasudil promoted the expression of TREM2 and DAP12 in Iba-1+microglia of the corpus callosum.Furthermore,Western blot showed that Fasudil significantly increased the expression of TREM2 and DAP12 proteins in the brain compared with other groups(P<0.001,P<0.0001).2.Results in vitro:(1)Western blot analysis showed that,compared with Myelin+PBS group,the protein expression of TREM2 and DAP12 was significantly enhanced in Myelin+Fasudil group(P<0.05,P<0.01).Similarly,fluorescence staining exhibited that the intensity of TREM2/DAP12 in Myelin+Fasudil group was higher than that of Myelin+PBS group.(2)RT-PCR assay displayed that the expression of TREM2 m RNA was inhibited by TREM2-siRNA1(P<0.0001),TREM2-siRNA2(P<0.001)and TREM2-siRNA3(P<0.01).TREM2-siRNA1,the strongest inhibition effect,was selected for the following experiment.(3)The effect of Fasudil on phagocytosis of TREM2-silenced BV-2 microglia was analysed by flow cytometry.The results showed that,compared with Normal+FMD group,the phagocytic ability of TREM2-silenced BV-2microglia was significantly decreased(P<0.01).Compared with Normal+FMD+Fasudil group,BV-2 microglia significantly reduced the phagocytosis of FMD in TREM2-siRNA+FMD+Fasudil group(P<0.0001).Compared with TREM2-siRNA+FMD group,the phagocytosis of TREM2-silenced BV-2 microglia was slightly enhanced by Fasudil intervention(P<0.05).Conclusion:Fasudil promoted the phagocytosis of myelin debris by exogenous and exogenous microglia by activating TREM2/DAP12 pathway.Part IV Fasudil enhances the phagocytosis of myelin debris by microglia and the expression of neurotrophic factors in CPZ-induced demyelinating miceObjective:To observe the phagocytosis of myelin debris by microglia in CPZ-induced demyelinating mice,and to explore the effects of microglia on the expression of neurotrophic foctors and remyelination.Methods:Experiment in vivo: C57BL/6 mice were divided into three groups: Normal+FMD group received standard diet for 6 weeks;CPZ+FMD group received 0.2% CPZ diet for 6weeks;CPZ+FMD+Fasudil group received 0.2% CPZ diet for 6 weeks,and injected intraperitoneally Fasudil(40 mg/kg/d)at the beginning of the fifth week.The Normal+FMD and CPZ+FMD groups were injected 0.9% Na Cl.Four weeks after CPZ feeding,FMD was stereotaxically injected into all mice brain.The mice were weighed every two days,and brain tissue were obtained at the end of six weeks.The intensity of FMD in corpus callosum was analyzed by microscope.The phagocytosis and BDNF/GDNF expression of microglia was analyzed by immunofluorescence staining.Western blot analyzed the protein level of BDNF/GDNF.Immunofluorescence staining and Western blot measured the protein level of Oligo2,PDGFR? and MBP in brains.Experiment in vitro: BV-2 microglia were cultured in 4 groups,Fasudil group added with 15 ?g/ml Fasudil;PBS group added with PBS;Myelin+Fasudil group added with 5mg/ml FMD and 15?g/ml Fasudil;Myelin+PBS group added with 5 mg/ml FMD and PBS.All groups were incubated in culture incubator for 48 h.The expression of BDNF/GDNF m RNA was analyzed by RT-PCR.The protein expression of BDNF and GDNF in the brain was analyzed by immunofluorescence staining and Western blot.The concentrations of BDNF and GDNF in culture supernatant were measured by ELISA.Furthermore,OPCswere intervened with conditioned medium of four groups.Then,the expression of MBP was observed by immunofluorescence staining.Results:1.Compared to the Normal+FMD group,the intensity of FMD in the CPZ+FMD group increased(P<0.01),which was decreased significantly by Fasudil treatment(P<0.01).Immunofluorescence staining displayed that Iba-1+MBP+ cells increased in CPZ+FMD+Fasudil group and existed around the myelin sheath.2.Immunofluorescence staining showed that Fasudil treatment significantly up-regulated the expression of BDNF and GDNF in Iba-1+ microglia in corpus callosum compared with CPZ+FMD group.Western blot showed that,compared to the Normal+FMD group,the expression of BDNF decreased in CPZ-fed mice(P<0.05),while Fasudil significantly up-regulated the expression of BDNF and GDNF in the brain(P<0.01,P<0.001).3.RT-PCR exhibited that the expression of BDNF and GDNF m RNA in Myelin+Fasudil group was up-regulated compared to Myelin+PBS group(P<0.001,P<0.0001).Similarly,Western blot displayed that,in Myelin+Fasudil group,the protein level of BDNF/GDNF was enhanced compared to Myelin+PBS group(P<0.05,P<0.001).Additionally,FMD inhibited the expression of GDNF m RNA/protein in BV-2 microglia compared with that in PBS group(P<0.01,P<0.05).Immunocyte fluorescent staining displayed that Fasudil induced BV-2 microglia to express BDNF and GDNF.In Myelin+Fasudil group,an analysis showed that BDNF and GDNF expression was mostly localized in FMD+ microglia compared with FMD-microglia.The concentration of BDNF/GDNF in Myelin+Fasudil group was higher than that in Myelin+PBS group by ELISA assay(P<0.01,P<0.001).4.Immunofluorescence staining showed that,compared with CPZ+FMD group,Fasudil increased the expression of Oligo2,PDGFR? and MBP in the corpus callosum of CPZ-induced mice.Similarly,the results of Western blot showed that Fasudil significantly up-regulated the protein expression of OPCs marker(Oligo2,PDGFR?)and OLs marker(MBP)compared with CPZ-induced mice(P<0.01,P<0.01,P<0.05).5.Immunocyte fluorescent staining showed that,compared with the other three groups,the conditioned medium of BV-2 microglia in Myelin+Fasudil group increased the branches of OPCs and enhanced the expression of MBP.Conclusion:Fasudil promoted the expression of neurotrophic factors and remyelination in the process of phagocytizing myelin debris by microglia.Conclusion1.Fasudil promoted the phagocytosis of myelin debris by microglia in a concentration-independent and time-dependent manner.2.Fasudil improved the clinical symptoms of CPZ-induced demyelinating mice.Also,Fasudil shifted microglia from M1 to M2 phenotype,and enhanced the phagocytic ability of M2 microglia.3.Fasudil enhanced microglial phagocytosis of myelin debris by up-regulating TREM2/DAP12 pathway.4.Fasudil increaseed the expression of neurotrophic factor by myelin debris-engulfed microglia,which promoted the differentiation/maturation of OPCs and remyelination. |
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