The Therapeutic Effects And Mechanisms Of Poly-IC Treatment Against Cerebral Ischemic/Reperfusion Injury

Background and objective: Ischemic stroke, a set of diseases resulted from cerebralblood circulation disorder characterized by local nerve function deficiency, is one of maindiseases which severely threat human health, and is the most common cause of severedisability and the second most common cause of death in the world. After research ofseveral decades, recombinant tissue plasminogen activator (rt-PA) thrombolytic therapy3-4.5h after stroke onset supported by evidence-base medical evidence is currently the onlyeffective treatment for ischemic stroke. However, it was limited by short treatment timewindow and poor medical condition, patients with ischemic stroke were rarely treated withrt-PA. Therefore, it is important to explore the effective treatment for stroke, which hasimportant clinical significance. This paucity of treatment options related to the fact that thepathophysiological mechanisms of ischemic injury have not been clearly elucidated. Recentresearch has revealed that inflammation plays an important role in secondary brain insultfollowing cerebral ischemia.The innate immune system is the major contributor to acute inflammation induced bymicrobial infection or tissue damage. The TLR family is one of the best-characterized PRRfamilies and is responsible for sensing invading pathogens outside of the cell andintracellular endosomes and lysosomes. They induce inflammation by recognizingstructures conserved among microbial species, named（PAMPs). Recent evidence suggeststhat TLRs are also responsible for recognizing endogenous molecules released fromdamaged cells, termed damage-associated molecular patterns (DAMPs). TLRs arecharacterized by N-terminal leucine-rich repeats (LRRs) and a transmembrane regionfollowed by a cytoplasmic Toll/IL-1R homology (TIR) domain. Eleven TLRs have beenidentified in humans and13in mice. Different TLRs recognize the different molecularpatterns of microorganisms and self-components. TLR2senses lipoproteins of bacteria andmycoplasma. TLR2recognizes its ligands by forming a heterodimer with either TLR1or TLR6. TLR3detects viral double-stranded RNA in the endolysosome. TLR3is involved inthe recognition of polyinosinic polycytidylic acid (poly-IC), a synthetic dsRNA analog.Mouse TLR7and human TLR7/8recognize single-stranded RNAs from RNA viruses,however TLR9senses unmethy-lated DNA with CpG motifs derived from bacteria andviruses. Toll-like receptors are expressed on various immune cells, such as neutrophils,macrophages, dendritic cells, B cells, and microglial cells, as well as on nonimmune cells,such as fibroblasts, epithelial cells, keratinocytes, myocytes, skeletal muscle, and neurons.TLRs recognize DAMPs and PAMPs, and subsequently activate TLRs pathway and induceinflammatory reaction.Toll-like receptors (TLRs) have been demonstrated to play critical roles in theinduction of immune and inflammatory responses. DAMPs activate TLRs and induce theexpression of NF-κB-dependent pro-inflammatory cytokines and/or type I interferon, andthese molecules participate in pathophysiological processes. Recent study has shown thatTLR4mRNA were upregulated in ischemic cerebral tissue after mice MCAO, and DAMPsreleased from ischemic neurons can activate TLR4/MyD88/NF-κB signaling pathway in theneighboring microglia and induce the release of pro-inflammatory cytokines, which givesrise to the degeneration and apoptosis of neurons. Our group and others havedemonstrated that cerebral infarction volume is decreased, brain water content is alleviatedand the content of TNF-α is reduced in peripheral blood in TLR4-deficient mice after focalcerebral I/R injury and total cerebral ischemia. Recent studies have shown that modificationof the TLR-mediated NF-κB signaling pathway significantly attenuates ischemic injuries tothe organs, and preconditioning with TLR2,4,7, and9ligands can increase tolerance tosubsequent ischemia; furthermore, significant reductions in neurological impairments havealso been reported. More importantly, recent report have indicated that TLR2ligand,Pam3csk4, treatment after cerebral ischemia also decreases cerebral ischemic reperfusioninjury in focal MCAO mice.Upon activation by double-stranded RNA viruses or poly-IC, TLR3recruits theregulatory protein TIR-domain-containing adapter-inducing interferon (TRIF), and TRIFassociates with TRAF3and TRAF6through TRAF-binding motifs present in its N-terminalportion, which further activate interferon regulatory factors (IRFs) and NF-κB via aTRIF-dependent signaling pathway. TLR3-deficient mice do not exhibit reduced cerebral infarct volumes after cerebral I/R compared to wild-type (WT) mice. But recent reportshave shown that polyinosinic-polycytidylic acid (poly-IC) preconditioning protects againstcerebral I/R injury. However, it is unclear whether poly-IC treatment after cerebral I/Rinjury is also effective. Additionally, if treatment is effective, the protective mechanismsremain unknown.It has been demonstrated that poly-IC attenuates lipopolysaccharide (LPS)-inducedliver injury by downregulating TLR4expression in macrophages and reducing the levels oftumor necrosis factor alpha (TNF-α). Therefore, we proposed that post-ischemia treatmentwith the TLR3ligand poly-IC would also have protective effects against cerebral I/R injury,which could be mediated by activation of TLR3and subsequent downregulation of theTLR4signaling pathway.We utilized the mouse/rat middle cerebral artery occlusion (MCAO) and celloxygen-glucose deprivation (OGD) models to evaluate the therapeutic effects andmechanisms of post-ischemia poly-IC treatment.Methods:PartⅠPoly-IC reduced focal cerebral I/R injury1, The MACO model in mice was founded, and the protective effects treated withpoly-IC before and after cerebral ischemia against cerebral ischemic reperfusion injury wereevaluated by TTC staining; meanwhile, the brain water content and the neurologicalfunction score were detected.2, Following48h in mice MCAO, the damage degree of hippocampal neuron andmitochondria were examined by Nissle staining and transmission electron microscopy, andthe levels of TNF-α, IL-1β and IFN-β were examind in ischemic brain tissues, cerebralspinal fluid (CSF) and peripheral blood by ELISA.3, Following48h in mice MCAO, the expression of Bcl-2, Bax, Hsp27and Hsp70inischemic brain tissues were detected by Western blot, and the TUNEL and FJB positivecells were examined by immunofluorescence.PartⅡ Poly-IC conferred protection against cerebral I/R injury through thedownregulation of TLR4signaling via TLR31, Following48h in TLR3-/-mice MCAO, the effects of poly-IC treated after ischemiawere observed against cerebral I/R injury, and the TUNEL and FIB positive cells were examined by immunofluorescence.2, Following48h in mice MCAO, the effects of activated TLR3signal molecule onTLR4signaling treated with poly-IC were examined by Western blot and EMSA, and thelevels of TNF-α and IL-1β treated with poly-IC were detected in LPS stimulated mice.3, Following48h in TLR4-/-mice MCAO, the protective effects treated with poly-ICwas observed by TTC staining and neurological function score. In mice MCAO model, theprotective effects and TLR4signaling treated with intracerebral ventricular injection ofrmIFN-β were observed by TTC staining, Western blot and EMSA.Part Ⅲ Poly-IC conferred the protective effects against OGD hippocampal neuronthrough the downregulation of TLR4signaling via TLR31, Cell OGD model was established, the levels of TNF-α and IL-1β were examined byELISA in microglia treated with poly-IC, and the hippocampal neuron survival rate wasdetected by immunofluorescence after treated with poly-IC. The expression of Bcl-2andBax in mixed neuronal-glial cell cultures were examined by Western blot.2, Following24h in OGD microglia, the effects of activated TLR3signal molecule onTLR4signaling treated with poly-IC were examined by Western blot and EMSA, and thelevels of TNF-α and IL-1β treated with poly-IC were detected in LPS stimulated microglia.3, Following24h in OGD microglia, the downregulated TLR4signaling by poly-ICwas detected after blocking TLR3, and the effect of TLR4signaling treated with IFN-β wasexamined.Part Ⅳ Poly-IC exerted the long-term protective effects after cerebral I/R injury inmice1, The MACO model in mice was established, the long-term protective effects ofpoly-IC after cerebral I/R injury in mice were examined by TTC staining and neurologicalfunction score.2, The persistent cerebral ischemic MCAO model in rats was established, theprotective effects treated with poly-IC against cerebral I/R injury in rats were examined byTTC staining and neurological function score.Results:Part ⅠPoly-IC reduced focal cerebral I/R injury1, Poly-IC pre-treatment significantly reduced the cerebral infarct volume, brain water content and improved neurological function score compared with the untreated I/R group inmice MCAO model. Importantly, poly-IC treatment after brain ischemia has therapeuticeffects against cerebral I/R injury.2, Nissl staining showed neuronal damage in the cornu ammonis1(CA1) and dentategyrus (DG) fields of hippocampal formation characterized by shrunken cell bodiesaccompanied by shrunken and pyknotic nuclei in the I/R mice for48h. However,hippocampal neurons damage was significantly reduced in the poly-IC treatment group.Transmission electron microscopy demonstrated mitochondrial membranes were severelydamaged, resulting in irregularly shaped organelles with poor membrane integrity in I/Rgroup; in contrast, in poly-IC group, mitochondria showed evidence of edema with matrix,but with intact surrounding membranes. ELISA results indicated that compared with I/Rgroup, TNF-α and IL-1β levels were markedly decreased, whereas IFN-β levels weregreatly increased in the ischemic brain tissues, CSF, and peripheral blood in poly-ICtreatment group.3, Western blot results showed that compared with I/R group, the levels of Hsp27,Hsp70and Bcl2were significantly increased, and the level of Bax was decreased in theischemic brain tissues. The numbers of TUNEL-positive and FJB-positive cells also weredecreased significantly in the ischemic brain tissues by TUNEL and FJB staining.PartⅡ Poly-IC conferred protection against cerebral I/R injury through the downregulationof TLR4signaling via TLR31, In TLR3-/-mice MCAO model, the protective effects of poly-IC was abolished,immunofluorescence results showed that the TUNEL-and FJB-positive cell counts in theischemic cerebral tissues with poly-IC treatment were not decreased compared with I/Rgroup.2, Western blot and EMSA results indicated that TLR3signaling molecule activated bypoly-IC downregulated the TLR4signaling and NF-κB activity. Moreover, poly-ICtreatment attenuated the levels of TNF-α and IL-1β in serum and CSF of mice stimulated bythe LPS.3, In TLR4-/-mice MCAO model, poly-IC treatment did not reduce cerebral infarctionvolumes or brain water content and did not improve neurological scores compared with theI/R group. Mice treated with intracerebral ventricular injection of rmIFN-β showed a significant reduction in infarct volume, the expressions of TLR4, MyD88, NF-κB p65, andNF-κB activity.Part Ⅲ Poly-IC conferred the protective effects against OGD hippocampal neuronthrough the downregulation of TLR4signaling via TLR3.1, In cell OGD model, ELISA results indicated that compared with OGD group, TNF-αand IL-1β levels were markedly decreased, whereas IFN-β levels were greatly increased inmicroglia treated with poly-IC. In mixed neuronal-glial OGD model, Western blot resultsshowed that the level of Bcl2were significantly increased, and the level of Bax wasdecreased in the poly-IC treatment group. The immunofluorescence result indicated thatpoly-IC postconditioning increased the survival rate of hippocampal neuron after cell OGD.2, Western blot and EMSA results indicated that TLR3signaling molecule activated bypoly-IC downregulated the TLR4signaling and NF-κB activity in OGD microglia.Moreover, poly-IC treatment attenuated the levels of TNF-α and IL-1β in microgliastimulated by the LPS.3, In microglia OGD model, the downregulation of TLR4signaling by poly-IC wereabolished after blocking TLR3, and exogenous IFN-β significantly downregulated theexpression levels of TLR4, MyD88, NF-κB p65and NF-κB activity.Part Ⅳ Poly-IC exerted the long-term protective effects after cerebral I/R injury inmice.1, In mice I/R model, the neurological scores of the poly-IC treatment group weresignificantly higher than those of the I/R group at1,3,10, and14days after ischemia andthe cerebral infarct volumes were significantly reduced at14days.2, In the persistent cerebral ischemic MCAO model of rats, cerebral infarct volumesand the severity of neurological deficits were significantly reduced in the poly-IC treatmentgroup at48h after cerebral ischemia.Conclusions:1, Poly-IC pre-treatment significantly reduced focal cerebral I/R injury. Importantly,poly-IC treatment after brain ischemia has therapeutic effects against cerebral I/R injury; inaddition, TNF-α and IL-1β levels were markedly decreased, whereas IFN-β level wasgreatly increased in the ischemic brain tissues, CSF, and serum in poly-IC treatment group,suggesting that the decreased levels of TNF-α and IL-1β and the increased IFN-β content played an important role in the protective effects of poly-IC against cerebral I/R injury.2, Poly-IC increased the expression of Bcl2, Hsp27, and Hsp70in ischemic braintissues, whereas decreased Bax expression, and reduced cellular degeneration and apoptosis,suggesting that the therapeutic effects of poly-IC against cerebral I/R injury were related tothe antiapoptosis and the protective effects of Hsp27and Hsp70.3, Poly-IC decreased TNF-α and IL-1β expression in microglia, wheras increased theexpression of IFN-β, and increased the hippocampal neuron survival rate, suggesting thatpoly-IC attenuated the inflammation response and conferred the protective effects ofhippocampal neuron.4, After TLR3knockout, the protective effects of poly-IC against cerebral I/R injurywere abolished, suggesting that poly-IC protected against cerebral ischemia via TLR3.5, Poly-IC conferred protection against cerebral I/R injury through the downregulationof TLR4signaling via TLR36, Poly-IC exerted the long-term protective effects after mice cerebral I/R injury andprotective effects against permanent cerebral ischemia in rat.