The Effect And Mechanism Of Meisoindigo On Macrophage/microglia Polarization After Cerebral Ischemic And Reperfusion Injury

Stroke is the leading cause of death and is the primary disabling disease worldwide,with ischemic stroke accounting for the majority.To date,there has been only one FDAapproved direct treatment for severe ischemic stroke,recombinant tissue plasminogen activator(rt-PA),which recanalizes blood vessels and restores blood flow.Although the therapeutic effect of thrombolysis is clear,the treatment time window is narrow,from 3 to 4.5h.Application beyond the time window will not only reduce the efficacy,but also increase the risk of secondary hemorrhage.Only a small number of patients are eligible for rt-PA treatment.After ischemic stroke,local cerebral blood flow is interrupted,causing ischemic brain tissue damage and partial neurological deficit within a few minutes.Ischemic brain tissue can be divided into ischemic core zone and penumbra zone according to the degree of ischemia.After cerebral ischemia,energy depletion and loss of glucose and oxygen lead to a sharp drop in ATP production,resulting in lactic acidosis and ion homeostasis.At the same time,these reactions trigger downstream "waterfall" cascades involving multiple cell types,leading to excitotoxicity and induction of apoptosis.DAMPs released from necrotic neurons can activate a large number of immune cells in the brain,and recruit peripheral immune cells accompanied by the destruction of the blood-brain barrier,among them,peripheral blood-derived monocyte/macrophage and activated microglia play an important role in the inflammatory reaction process of stroke.Macrophages can be polarized under local inflammatory microenvironment conditions to pro-inflammation M1 type or anti-inflammatory M2 type in a broad sense.Reduction in the activation/infiltration of inflammatory cells in acute phase after stroke,inhibition in macrophage/microglia polarization to M1 and promoted polarization to M2 perform important functions in reduce neuronal necrosis,brain tissue edema and the inflammatory response after stroke.Meisoindigo is a derivative of indirubin,and the solubility and clinical efficacy of meisoindigo are better than that of indirubin,and the adverse reactions are relatively mild.In clinical practice,meisoindigo has gradually replaced indirubin and become a common used drug for chronic myeloid leukemia(CML)clinical treatment in China since the 1980 s.Meisoindigo can inhibit the proliferation of various leukemia cells and promote apoptosis at the ?M concentration level.The molecular mechanism in how meisoindigo plays a role is not fully understood,mainly to inhibit DNA biosynthesis and microtubule assembly in leukemia cells.Our previous in vivo studies have found that meisoindigo inhibits chemotactic migration of leukocytes to the lesion,thereby alleviating local inflammatory responses.However,whether this effect can inhibit the inflammatory reaction in the acute phase after cerebral ischemia-reperfusion injury,thereby improving the cerebral ischemia-reperfusion injury and the mechanism of action,whether it has potential as a neuroprotective drug,further exploration is needed.In this research,we studied the neuroprotective effect of meisoindigo on cerebral ischemia-reperfusion injury and the regulation of macrophage/microglia polarization in mice t MCAO model,and introduced MCC950 as a comparison to explore the mechanism of protective effect of meisoindigo on CIRI.Inhibition of inflammasome expression after ischemia-reperfusion injury,combined with the inhibitory effect of meisoindigo on inflammatory cell activation/infiltration in the ischemic penumbra,found macrophage/microglia polarization and activation of inflammatory cells/ Infiltration is associated with NLRP3 inflammatory bodies.This study validated the neuroprotective effect of meisoindigo on CIRI in mice,and explained that it may play a neuroprotective role by regulating microglia/macrophage polarization by inhibiting inflammasomes,and also on CNS inflammatory cells after CIRI.Activation/infiltration has a direct inhibitory effect.It reveals the new pharmacological effects of meisoindigo,suggesting that meisoindigo has potential for the treatment of diseases related to inflammation or macrophage/microglia polarization in addition to cerebral ischemiareperfusion injury.ObjectTo study the neuroprotective effects of meisoindigo on cerebral ischemia-reperfusion injury in mice and to explore its effect and mechanism on microglia/macrophage polarization.Method1)C57BL/6 mice weighing 25-30 g were randomly divided into 5 groups,namely Sham group,3 doses of meisoindigo group(4mg/kg,8mg/kg and 12mg/kg)and MCC950(50mg/kg)group.Transient Middle Cerebral Artery Occlusion(t MCAO)model was prepared by inserting a silicon-coated 6-0 suture into the left internal carotid artery and block the left middle cerebral artery,reperfusion after 1h ischemia,drugs were injected intraperitoneal immediately after reperfusion in mice,and continued to administered the drug once a day at fixed time.3 days later,the neurobehavioral scores of the mice were scored and the brain was sacrificed.TTC staining was used to measure the size of cerebral infarction,and the water content of brain tissue was measured to study the effective dose of meisoindigo in the treatment of ischemic stroke.2)8 mg/kg was selected as the experimental dose of meisoindigo,using MCC950(50 mg/kg,ip,qd,3d)as the comparison.Immunofluorescence(IF)was performed to observe whether the polarization of macrophage/microglia was regulated by meisoindgo,the effect of meisoindigo on the expression of NLRP3 inflammasome in the ischemic cortex,it's capability to inhibiting the activation/infiltration of inflammatory cells.Extracting the m RNAs and proteins of the brain tissue 3 days after I/R.Real-time quantitative polymerase chain reaction(RT-q PCR)for M1-related cytokine m RNA(TNF-?,IL-1?,i NOS,CD16/32)and M2-related cytokine m RNA(Ym1/2,CD206 Arg-1)was detected by Western blotting(WB).The TLR4/NF-?B signaling pathway molecule,NLRP3 inflammatory corpuscle complex and cerebral ischemia-related inflammatory factors were detected.The above three experimental methods were used to study the effects of meisoindigo on inflammatory response in ischemic areas of mice after cerebral ischemia-reperfusion injury,inhibition of NLRP3 inflammatory body expression and polarization of microglia/macrophages.Results 1)Application of meisoindigo(4 mg/kg,8 mg/kg,12 mg/kg)and MCC950 had neuroprotective effects on mice 3 days after cerebral ischemia-reperfusion.Both meisoindigo and MCC950 can significantly reduce the size of cerebral infarction zrea after t MCAO,reduce the degree of cerebral edema,and improve neurobehavioral scores,of which 8 mg/kg is the smaller and most effective dose,so 8 mg/kg will be administered in subsequent experiments.2)Meisoindigo and MCC950 can reduce the quantity of pro-inflammation cells in the ischemic penumbra after 3 days of cerebral ischemia-reperfusion in mice.Immunofluorescence staining showed that meisoindigo and MCC950 can significantly reduce the number of CD68,i NOS and MPO positive cells in the ischemic area.Both of them can significantly increase the number of Ym1/2 positive cells.3)Double labeled immunofluorescence showed that NLRP3 inflammatory bodies were mainly expressed in neurons and CD68 positive cells,and their expression levels were positively correlated with the number of CD68 positive cells.4)Immunofluorescence results showed that both meisoindigo and MCC950 could reduce the number of lost neurons in the CIRI region,and both inhibited the expression of NLRP3 inflammatory bodies.The number of neurons surviving was negatively correlated with the expression of NLRP3 inflammasome.5)Both meisoindigo and MCC950 can inhibit the expression levels of TLR4/NF-?B signaling moleculars,components of NLRP3 inflammasome complex and proinflammatory inflammatory factors.6)Both meisoindigo and MCC950 can inhibit the production of M1-type macrophage polarization markers(i NOS,CD16/32)and inflammatory factors(TNF-?,IL-1?)at the m RNAs level,they both can also promote the production of M2 type macrophage polarization markers(Ym1/2,Arg-1,CD206)m RNAs,suggesting that meisoindigo can inhibit the polarization of macrophage/microglia to M1 type,and promote the M2 polarization.Conclusion 1)Meisoindigo performe neuroprotective effects by regulating microglia/macrophage polarization and activation/infiltration of inflammatory cells following cerebral ischemia-reperfusion injury.2)Inhibition of NLRP3 inflammatory bodies may be a mechanism that regulates macrophage/microglia polarization.The main innovations of this paper are: 1)Meisoindigo showes neuroprotective effects by regulating microglia/macrophage polarization and activation/infiltration of inflammatory cells following cerebral ischemia-reperfusion injury.Meisoindigo can significantly reduce the extent of cerebral infarction after t MCAO in mice,reduce the degree of cerebral edema,and improve the neuromotor dysfunction and general state caused by CIRI in mice.It also inhibits the activation/infiltration of penumbra macrophages/microglia to M1-type polarization and macrophage/microglia and neutrophils after CIRI,while promoting M2-type polarization.Thereby performing neuroprotective effects.2)Inhibition of NLRP3 inflammatory bodies may be a mechanism that regulates macrophage/microglia polarization.MCC950 is a NLRP3 inflammatory body-specific inhibitor.By contrast,it can be found that meisoindigo regulates macrophage/microglia polarization by inhibiting NLRP3 inflammasome.Although the inhibitory effect of NLRP3 inflammasome is not as significant as that of MCC950,its protective effect on cerebral ischemia-reperfusion injury after t MCAO in mice seems better than that of MCC950,suggesting that macrophage/microglia polarization regulated by meisoindigo via inhibiting NLRP3 inflammasome may be just one of the mechanisms.