The Effect And Mechanism Of CD21,a Novel Neuroprotectant,on TPA-Mediated Hemorrhagic Transformation After Ischemic Stroke

Background Stroke,including ischemic and hemorrhagic stroke,is the leading cause of death in China,and ischemic stroke accounts for > 80% of all stroke cases.It is the first choice for acute ischemic stroke treatment that intravenous administration with thrombolytic drugs,and tissue plasminogen activator(tPA)is the only drug approved by the U.S.food and drug administration for acute thrombolytic therapy clinically.However,thrombolysis with tPA easily lead to complications such as cerebral hemorrhagic transformation(HT),and increase the mortality and disability rate.It is reported that less than 5% of cerebral ischemic patients benefit from tPA thrombolytic therapy.Therefore,reducing the incidence or degree of HT is of great significance for clinical ischemic treatment.A large number of studies have proved that neuroinflammation plays an important role in the development of HT induced by tPA.Damage-associated molecular patterns(DAMPs),such as peroxiredoxins(Prxs)and high mobility group box-1 protein (HMGB1),are released from injured or dead brain cells after ischemic and hemorrhagic stroke.On the one hand,DAMPs recognize and bind to toll like receptors(TLRs)on membranes of glial cells and infiltrating immune cells.The activated TLRs promote the translocation of nuclear factor kappa-B(NF-κB)into nucleus,and thus promotes cytokines transcription and expression,leading to sterile inflammation in the brain.On the other hand,DAMPs(Prxs)can be recognized by macrophage scavenger receptor 1(MSR1)on the membrane of microglia/macrophages and degraded in lysosomal,resulting in alleviation of neuroinflammation.It has been reported that MSR1-mediated clearance of DAMPs(Prxs)is mainly demonstrated in M2 microglia/macrophages.Studies have shown that tPA increased the level of DAMP(HMGB1)in plasma of cerebral HT patients,and that extracellular DAMP(HMGB1)aggravated HT by inducing neuroinflammation in ischemic stroke animals.Therefore,promoting the clearance of DAMPs and reducing tPA-induced neuroinflammation after stroke is a novel therapeutic strategy to alleviate HT.Recently,clinical case analysis reported that combined treatment with tPA and 3-dl-butylphthalide(NBP)reduced the incidence of HT and improve prognosis in cerebral ischemic patients,and these beneficial effects were related to the alleviation of tPA-induced neuroinflammation.Our research group has been devoted to exploring the preventive and therapeutic effects of phthalides on stroke in recent years.Previous studies have found that natural phthalide compounds played a neuroprotective role by alleviating the neuroinflammatory reaction in stroke animals,and the phthalide derivative CD21 with stable physicochemical properties was obtained by structural modification of natural phthalide compounds.CD21 alleviated brain injury by alleviating DAMPs-induced neuroinflammation and promoting M2 polarization of microglia/macrophages in rats and mice after cerebral ischemia.Therefore,it is speculated that CD21,a novel neuroprotectant,might also play a neuroprotective role in tPA-mediated HT after ischemic stroke.Based on this conjecture,this study explored the effect and mechanism of CD21 on tPA-mediated cerebral HT in ischemic stroke mice.Methods and results Chapter 1.The effect of CD21 on tPA-induced cerebral hemorrhagic transformation in ischemic stroke miceMethods1.Permanent middle cerebral artery occlusion(p MCAO)model was induced by thrombus method in mice,and divided into five groups: normal(Sham)group,permanent cerebral ischemia(p MCAO)group,CD21 administration(CD21)group,tPA administration(tPA)group,and combined treatment with tPA and CD21(tPA+CD21)group.Different groups of mice were treated correspondingly.Cerebral hemorrhagic transformation(HT)was triggered by tPA(10 mg/kg)intravenous administration 3 hours after occlusion.CD21(13.78 mg/kg)or vehicle was injected intravenously immediately after tPA,and repeated on the first and second day after surgery,respectively.On the third day after operation,the overall neurobehavioral dysfunction and sensorimotor dysfunction of mice among each group were measured by modified neurological severity score(m NSS)and corner test,respectively.2,3,5-triphenyltetrazole chloride(TTC)staining was used to detect the cerebral infarct volume of mice among each group.The volume of cerebral hemorrhage of mice among each group was detected using Drabkin’s reagent.2.Transient middle cerebral artery occlusion(t MCAO,2 hours occlusion)model was induced by suture method in mice,and divided into five groups: normal(Sham)group,transient cerebral ischemia(t MCAO)group,CD21 administration(CD21)group,tPA administration(tPA)group,and combined treatment with tPA and CD21(tPA+CD21)group.Different groups of mice were treated correspondingly.Cerebral HT was triggered by tPA(10 mg/kg)intravenous administration following reperfusion.CD21(13.78 mg/kg)or vehicle was injected intravenously immediately after tPA,and repeated on the first and second day after surgery,respectively.On the third day after operation,the overall neurobehavioral dysfunction and sensorimotor dysfunction,the infarct volume,and the volume of cerebral hemorrhage of mice among each group were tested.3.The t MCAO mice were divided into four groups: Sham group,t MCAO group,tPA group,and tPA+CD21 group.On the third day after operation,Evans blue leakage was used to evaluate the degree of Blood-brain barrier(BBB)damage in the ipsilateral brain hemisphere of t MCAO mice.Western blot was used to detect the degradation of tight junction proteins(zonula occludens 1(ZO-1)and Claudin-5),the expression of TLR4,myeloid differentiation factor 88(My D88),Prx1,v-maf musculoaponeurotic fibrosarcoma oncogene homolog B(MAFB)and MSR1,and the nuclear translocation of NF-κB p65 in the ipsilateral brain hemisphere of mice among each group.The expression of matrix metalloproteinase 9(MMP-9)around microvessels in the peri-infarct area of mice among each group was evaluated by immunofluorescence staining.The activation of MMP-9 in the ipsilateral brain hemisphere of mice among each group was evaluated by gelatin zymography.Immunofluorescence staining was used to detect the activation of glial cells,the infiltration of blood-derived macrophages,the polarization of microglia/macrophages and the subcellular localization of DAMP(Prx1)in the peri-infarct area of mice among each group.Quantitative real-time polymerase chain reaction was used to detect the transcription of pro-inflammatory factors in the ipsilateral brain hemisphere of mice among each group.Results1.There were obvious neurobehavioral dysfunction,and infarcted brain tissues in p MCAO mice.Although these pathological changes of p MCAO mice showed a trend of reduction after tPA infusion,there were no significant differences.But,tPA induced cerebral HT in p MCAO mice.After CD21 was given alone,the volume of cerebral infarction,neurobehavioral dysfunction were significantly reduced,which were more obvious after combined treatment with tPA and CD21 in p MCAO mice.CD21 significantly reduced the cerebral hemorrhage induced by tPA in p MCAO mice.2.Compared to sham group,mice in t MCAO group displayed obvious neurobehavioral dysfunction,and infarcted brain tissues.After tPA administration,the neurobehavioral dysfunction was significantly aggravated,and the infarct volume tended to increase with obvious HT.CD21 significantly alleviated neurobehavioral dysfunction and cerebral infarction in t MCAO mice.Also,CD21 significantly ameliorated tPA-mediated neurobehavioral dysfunction,cerebral infarction and HT in t MCAO mice.3.BBB damage represented by the increased Evans blue extravasation,MMP-9 expression and activation,and tight junction proteins(ZO-1 and Claudin-5)degradation in the ipsilateral brain hemisphere of t MCAO mice was significantly aggravated by tPA.Combined treatment with tPA and CD21 significantly alleviated tPA-mediated BBB damage in t MCAO mice.4.The Neuroinflammation of tMCAO mice was enhanced by tPA administration.The transcriptions of pro-inflammatory cytokines(tumor necrosis factor α(TNF-α),Interleukin 1β(IL-1 β)and Interleukin 6(IL-6)),the expression of TLR4 and My D88,and the nuclear translocation of NF-κB p65 in the ipsilateral brain hemisphere of t MCAO mice were significantly upregulated by tPA.The activation of glial cells including Glial fibrillary acidic protein(GFAP)-positive astrocytes and Ionized calcium binding adaptor molecule(IBA-1)-positive microglia,the infiltration of blood-derived macrophages,and the M1 polarization of microglia/macrophages were significantly increased by tPA.Combined treatment with tPA and CD21 significantly attenuated tPA-mediated neuroinflammatory responses in t MCAO mice.5.After transient cerebral ischemia,the expression and extracellular distribution of Prx1,and the expression of MSR1 and its dependent nuclear transcription factor MAFB in the ipsilateral brain hemisphere of mice were upregulated,and tPA enhanced the upregulation.CD21 significantly increased the expressions of MAFB and MSR1,while decreased the expression and extracellular distribution of Prx1 in tPA-infused t MCAO mice.Chapter 2.The mechanism of CD21 in reducing hemorrhagic transformation after ischemic stroke in miceMethods1.The nucleic acid samples were extracted from tails of newborn mice,and subjected to agarose gel electrophoresis after amplification.2.Suture method was used to induce transient cerebral ischemia(2 hours occlusion),and tPA(10 mg/kg)was given intravenously immediately after reperfusion to trigger cerebral HT in mice.Wild type(WT)mice were divided into Sham group,tPA group,and tPA+CD21 group,and MSR1 knockout(MSR1-KO)mice were divided into tPA group and tPA+CD21 group,a total of 5 groups.CD21(13.78 mg/kg)or vehicle was injected intravenously following tPA,and repeated for 3 days.On the third day after operation,the volume of cerebral hemorrhage was detected using Drabkin’s reagent.Evans blue leakage was used to evaluate the degree of BBB damage in the ipsilateral brain hemisphere of mice.The transcription of proinflammatory factors(TNF-α and IL-1β)and anti-inflammatory factors(Interleukin 10(IL-10)and arginase-1(Arg-1))in the ipsilateral brain hemisphere of mice were detected by quantitative real-time polymerase chain reaction.Western blot was used to detect the expression of Prx1 and TLR4 in the ipsilateral brain hemisphere of mice.Immunofluorescence staining was used to detect the expression of MSR1 in polarized microglia/macrophages around the injured area of brain in cerebral hemorrhagic mice.3.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to detect the cytotoxicity of CD21 and N-acetylcysteine(NAC)to cultured RAW264.7 cells.The effect of CD21 and NAC on MSR1 transcription and expression in RAW264.7 cells were detected by quantitative real-time polymerase chain reaction and western blot,respectively.The effect of CD21 and NAC on the clearance of extracellular Prx1 in cultured RAW264.7 cells were detected by fluorescent labeling.Results1.Results of gel imaging showed that MSR1-KO mice used in this study were homozygous.2.In WT mice,CD21 significantly reduced tPA-induced cerebral hemorrhage,penetration of Evans blue,m RNA levels of pro-inflammatory cytokines(TNF-α and IL-1β),and the expression of Prx1 and TLR4,while significantly increased the m RNA levels of anti-inflammatory cytokines(IL-10 and Arg-1)in the ipsilateral brain hemisphere after cerebral ischemia.However,CD21 could not significantly improve these pathological changes induced by tPA in MSR1-KO mice.3.CD21 significantly promoted M2 polarization and MSR1 expression in M2 type cells around the injured area after cerebral HT in WT mice.But,the effect of CD21 on promoting M2 polarization of microglia/macrophages was inhibited in MSR1-KO mice.4.In vitro,NAC(8 m M)significantly inhibited the transcription and expression of MSR1 induced by CD21(20 μM)in RAW264.7 cells without cytotoxicity.Also,the ability of CD21 promoting clearance of extracellular Prx1 in lysosomes was inhibited by NAC.Chapter 3.Regulatory effect and mechanism of CD21 on sterile inflammation in primary microgliaMethods1.Mixed glial cells were isolated from brains of C57BL/6 mice within 24 hours after birth,and primary microglia were isolated and purified by upright hand shaking method.The specificity and purity of primary microglia were identified by immunofluorescence staining with IBA-1.Cell counting kit-8(CCK8)was used to detect the effects of different concentrations of CD21 and Prx1 on the survival of primary microglia.The effects of different concentrations of CD21 and Prx1 on releasing nitric oxide(NO)from primary microglia were detected by one-step NO kit.2.WT primary Microglia were divided into Sham group,Prx1(30 n M)group,and Prx1(30 n M)+ CD21(40 μm)group,and MSR1-KO primary Microglia were divided into Prx1(30 n M)group and Prx1(30 n M)+ CD21(40 μm)group.The concentrations of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 in the medium were detected by enzyme linked immunosorbent assay(ELISA)after 24 hours of group processing.The expression of TLR4,the nuclear translocation of NF-κB p65,and the expression of MSR1 in polarized primary microglia were detected by immunofluorescence staining.Hoechst/lysotracker labeling living cells was used to detect the lysosomal clearance of extracellular Prx1.Results1.The results of immunofluorescence staining showed that IBA-1 labeled microglia cytoskeleton was almost completely coincident with DAPI labeled nucleus,indicating that high purity primary microglia could be obtained by this method.2.CCK8 and NO results showed that Prx1(0-50 n M)had no significant cytotoxicity on primary microglia,and Prx1(30 n M)could stimulate inflammation in primary microglia.CD21(0-40 μM)had no significant cytotoxicity on primary microglia,and CD21(30-40 μM)concentration-dependently inhibited Prx1(30 n M)-triggered inflammation.3.Prx1(30 n M)induced the release of pro-inflammatory cytokines(NO and TNF-α)and the activation of TLR4/NF-κB signaling pathway in primary microglia.After co-cultured with Prx1(30 n M)and CD21(40 μM),the release of pro-inflammatory cytokines(NO,TNF-α)and the activation of TLR4/NF-κB signaling pathway were significantly decreased,while the release of anti-inflammatory cytokines(IL-10)and the lysosomal phagocytosis of Prx1 were significantly increased in WT primary microglia.In MSR1-KO primary microglia,the release of NO,TNF-α and IL-10,the activation of TLR4/NF-κB signaling pathway,and the clearance of Prx1 had no significant differences between Pxr1-cultured and Prx1+CD21-co-cultured group.4.CD21 inhibited M1 polarization induced by Prx1,and promoted M2 polarization as well as MSR1 expression in M2 type cells in WT primary microglia.However,CD21 had little effect on cell polarization in MSR1-KO primary microglia.Chapter 4.Evaluation of other medicinal properties of CD21Methods1.High performance liquid chromatography(HPLC)method was used to obtain the chromatogram of CD21 and NBP,and to establish appropriate analytical methods.2.The pharmacokinetic parameters(drug concentration-time curve)and the bloodbrain distribution of CD21 and NBP after intravenous administration were determined by high performance liquid chromatography-triple quadrupole mass spectrometry(HPLC-Qq Q-MS)method,and the atrioventricular models were established.3.The safety of CD21 was evaluated by acute toxicity test.Results1.The chromatographic peaks of CD21 and NBP were close to but completely separate from the chromatographic peaks of standard samples,respectively.2.The concentration change pattern of CD21 was similar to that of NBP,but CD21 had the characteristics of shorter peak time,higher peak concentration,and easier to cross through BBB.3.One-time intravenous injection of 20 times of pre-set clinical dose of CD21 did not cause death in mice.ConclusionsThis study demonstrated the neuroprotective effect of CD21,a novel neuroprotectant,on stroke mice for the first time.CD21 alleviated ischemic brain injury and inhibited cerebral HT induced by tPA.The mechanism was related to the reduction of neuroinflammation after stroke.CD21 upregulated the expression of MSR1 mainly in M2 microglia/macrophages and accelerated the clearance of extracellular DAMP(Prx1),thereby reducing the neuroinflammation induced by Prx1/TLR4/NF-κB signaling pathway,and finally alleviated BBB breakdown and HT in tPA-infused cerebral ischemic mice.Compared with NBP,a phthalide drug on the market,CD21 has the characteristics of faster onset of action,stronger BBB permeability and higher security.Therefore,CD21 might be a potential drug for clinical cerebral ischemic treatment,reducing the side effects such as cerebral HT combined with tPA.