Roles And Mechanisms Of ?7nAChR In Ischemic Stroke And Crush Syndrome

?7 nicotinic acetylcholine receptor??7nAChR?belongs to cys-loop receptors superfamily and is a ligand-gated cation channel.It is composed of five homometric?7subunits.Different from other nicotinic acetylcholine receptors,?7nAChR is widely distributed in central nervous system and immune system.However,its function is distinguishing within these two systems.Studies indicated that,distinguish from other nAChR,?7nAChR was specific with rapid activation and rapid desensitization in central nervous system.When agonist binds into ligand-binding domain,the central ion channel opens simultaneously,the whole process is within millisecond.And then,it is desensitized within 100 ms.?7nAChR has a high permeability to calcium and it is reported that the permeability ratios of P_Ca/P_Na as high as 15 to 20.Activation of?7nAChR can trigger intracellular signal transduction dependent or independent of calcium influx.In immune system,stimulation of the vagus nerve causes anti-inflammatory effects which is called cholinergic anti-inflammatory pathway.Studies showed that activation of?7nAChR in macrophage could cause anti-inflammatory responses and reduce the release of macrophage pro-inflammatory cytokines.Thus?7nAChR plays an important role in the cholinergic anti-inflammatory pathway.Ischemic stroke is necrosis of brain tissue caused by insufficient blood supply due to cerebral vascular stenosis or occlusion.Stroke has a high morbidity and mortality rates worldwide especially in developed countries.According to statistics from the World Health Organization,stroke becomes a major fatal disease and major disabling disease,placing a heavy burden on families and society.Investigating the pathophysiological changes following ischemic stroke and finding out new therapeutic target are of great importance.Previously,our lab found that combining use of anisodamine and neostigmine could treat ischemic stroke which was mainly mediated by?7nAChR.However,the direct effect of?7nAChR on ischemic stroke and mechanism are unclear.The first part of this project is to explore the effect and mechanisms of?7nAChR on ischemic stroke with selective agonist PNU282987.Crush syndrome is a series of clinical syndromes caused by long-term compression of muscle-riched parts and decompression.The necrosis of muscle release amount of harmful substances including potassium ion and myoglobin which could cause acute renal failure,hyperkalemia,hypovolemic shock and other symptoms.Crush syndrome is common in catastrophic events such as earthquakes,mudslides,landslides,and traffic accidents and trampling.Especially in medical rescues for non-war military operations,large number of such wounded often occur.How to effectively treat crush syndrome and reduce the mortality are important.As an important role in cholinergic anti-inflammatory pathway,whether?7nAChR can be a new target in treatment is a question.Anisodamine is muscarinic acetylcholine receptor antagonist and neostigmine is a cholinesterase inhibitor.Previous study in our lab showed that combination of anisodamine and neostigmine could increase binding of acetylcholine to?7nAChR.The second part of this project is to explore the effect and mechanisms of?7nAChR on crush syndrome.1.?7nAChR can protect against ischemic stroke1.1?7nAChR can protect against ischemic stroke in vivoWe constructed tMCAO model in wild-type and?7nAChR knockout mice.The results showed that?7nAChR knockout mice gained higher neurological deficit score and large infarct volume.1.2 The expression of?7nAChR decreases following ischemic strokeWe detected the expression of?7nAChR mRNA level and protein level by RT-PCR,Western Blot and immunofluorescence.The results showed that the expression of?7nAChR mRNA and protein decreased in penumbral zone at 24 h after ischemic stroke.1.3?7nAChR regulates neuronal autophagy following ischemic strokeWe constructed tMCAO model in WT mice and?7nAChR KO mice and detected autophagy by Western Blot and immunofluorescence.Results showed that tMCAO WT mice showed an increased LC3 level however tMCAO?7nAChR KO mice didn't show this phenomenon.1.4 The expression of?7nAChR in primary neuron decreases following OGD/R conditionWe detected expression of?7nAChR mRNA and protein by RT-PCR and Western Blot.The results showed that the expression of?7nAChR decreased in OGD/R condition.Besides,we stained neuronal marker MAP2 and?7nAChR by immunofluorescence.The result was consistence with the above.1.5 Activation of?7nAChR can protect neuron from injury in OGD/R conditionWe treated neuron with?7nAChR agonist PNU282987 and detected CCK8 and LDH release.The results showed that activation of?7nAChR could protect neuron from injury in OGD/R condition especially with the concentration of 100?M.1.6 Activation of?7nAChR enhances neuronal autophagyWe stained with LC3 and MAP2 on neurons.The results showed that OGD/R condition increased the positive expression of LC3,while activation with PNU282987further increased LC3 expression.Besides,we also used lentivirus harboring tandem monomeric mRFP-GFP-LC3 to detect autophagy influx.The results revealed that OGD/R increased both numbers of autophagosomes and autolysosomes,and also PNU282987 further enhanced this phenomenon.Autophagy related proteins were also detected by Western Blot.Results showed that PNU282987 could enhance neuronal autophagy following OGD/R.1.7 Autophagy protects primary neurons from injury in OGD/R conditionAutophagy can be induced by rapamycin.Thus we detect the influence of induced autophagy by rapamycin to neurons.Results revealed that autophagy increased cell viability and reduced LDH release of primary neurons in OGD/R condition.1.8 The protective effect of?7nAChR activation on neurons is mediated by autophagyWe blocked autophagy by 3-MA and found that the protective effect of?7nAChR activation on neurons vanished which indicated that this effect was mediated by autophagy.1.9 The effect of?7nAChR on neuroprotection is mediated by AMPK-mTOR-p70S6K signaling pathwayWe detected expression and phosphorylation of AMPK,mTOR and p70S6K after giving PNU282987 following OGD/R condition.The results showed that phosphorylation of AMPK further increased after giving PNU282987 in OGD/R condition.While the phosphorylation of mTOR and p70S6K further decreased after giving PNU282987 following OGD/R condition.We also used compound C to inhibit AMPK and found that after blocking AMPK the protective effect of?7nAChR activation disappeared.1.10 Activation of?7nAChR reduced inflammatory cytokines produced by BV2microglia stimulated by LPSWe stimulated BV2 microglia with LPS and activated?7nAChR by PNU282987.We detected mRNA level of inflammatory cytokines by RT-PCR and found that the activation of?7nAChR could reduce the inflammatory cytokines produced by BV2.1.11 Activation of?7nAChR promotes microglia polarization from M1 to M2phenotypeTo explore the effect of?7nAChR on microglia polarization,we detected markers of M1 and M2 phenotype by RT-PCR.M1 phenotype leads to inflammation while M2phenotype has anti-inflammatory effect.The results showed that LPS stimulation promoted microglia M1 phenotype polarization while the activation of?7nAChR enhanced M2 phenotype polarization.In addition,we also detected these markers by immunofluorescence and the results were consistent with the above.Conclusion:?7nAChR can protect against ischemic stroke,on one hand,it induces neuronal autophagy to protect neurons,on the other,it can promote microglia polarization to M2 phenotype.2.Activation of?7nAChR have therapeutic effect on crush syndrome2.1 Combination administration of anisodamine and neostigmine increases 24 h survival rate in rats with crush syndromeWe constructed crush syndrome model with rats and administrated anisodamine,neostigmine or the combination.The results showed that high dose of combination?20mg/kg anisodamine and 40?g/kg neostigmine?increased 24 h survival rate significantly.2.2 Combination administration of anisodamine and neostigmine inhibits oxidative stress caused by crush syndromeWe constructed crush syndrome model with rats and administrated anisodamine,neostigmine or the combination.Serum and muscles were collected to detect oxidative stress related substances like H₂O₂,MPO and total NO.The results showed that crush syndrome induced oxidative stress however the combination use of anisodamine and neostigmine could inhibit oxidative stress.2.3 The therapeutic effect of anisodamine and neostigmine on crush syndrome is mediated by?7nAChRWe constructed crush syndrome model using WT mice and?7nAChR KO mice and administrated anisodamine,neostigmine or the combination.The results showed that combination of anisodamine and neostigmine could increase 24 h survival rate in WT mice.However there was no significant effect of the combination on survival rate in?7nAChR KO mice.2.4 The combination of anisodamine and neostigmine inhibits oxidative stress and inflammation caused by crush syndrome through?7nAChRWe detected substances related to oxidative stress and inflammation in WT mice and?7nAChR KO mice with crush syndrome.The results revealed that the treatment of anisodamine and neostigmine combination had effect of anti-oxidative stress and anti-inflammation in WT mice.However,this effect was not found in?7nAChR KO mice.2.5 The activation of?7nAChR by anisodamine and neostigmine can active JAK2-STAT3 signaling pathwayWe detected the total and phosphorylation level of JAK2 and STAT3 by Western Blot in both C57BL/6 mice and WT and?7nAChR KO mice.We found that in C57BL/6mice crush injury decreased the levels of p-JAK2/JAK2 and p-STAT3/STAT3,however,the combination could inhibit the decrease.The effect of anisodamine and neostigmine can be blocked by MLA.In consistent with the results above,crush injury induced a decrease of the p-JAK2/JAK2 and p-STAT3/STAT3 in both WT and?7nAChR KO mice.However the combination inhibited the decrease of the p-JAK2/JAK2 and p-STAT3/STAT3 in WT mice while it couldn't be rescued in?7nAChR KO mice.Conclusion:Activation of?7nAChR by anisodamine and neostigmine has anti-oxidative effect and anti-inflammation effect through JAK2-STAT3 signaling pathway activation.And it can be a therapeutic target for crush syndrome.