TRPV4 Channels Stimulate Ca²⁺-induced Ca²⁺ Release In Neurons Of Mouse And Trigger Endoplasmic Reticulum Stress After Intracerebral Hemorrhage

Intracerebral hemorrhage(ICH)is a subtype of stroke with high disability and mortality.Many factors,such as peripheral blood circulation disorders caused by mechanical compression of the hematoma,inflammatory reactions,and changes in permeability of the blood-brain barrier,metabolites of hematoma catabolism,can lead to secondary brain injury and severe neurological dysfunction.Although some drugs or molecular targets with anti-oxidation,anti-inflammatory or neuroprotective effects have shown positive therapeutic effects in animal experiments,they did not exert obvious therapeutic effects in clinic after intracerebral hemorrhage.The TRPV4 channels,one of the TRP family members,are widely distributed in the central nervous system.It is located on chromosome 12 in humans.Hypoosmotic stimulation,cell swelling,temperature stimuli,mechanical stimuli,arachidonic acid metabolites and other signals can lead to its activation.Therefore,TRPV4 channels are considered as the important sensors in the internal environment.At the same time,the activated TRPV4 channel mediate Ca2+ influx,and is involved in a variety of regulatory activities under pathophysiological conditions,for example neuronal apoptosis caused by ischemia and hypoxia,inflammatory reaction in chemical acute lung injury,brain edema caused by traumatic brain injury,and so on.However,its role in cerebral hemorrhage has not been confirmed.It has been showed that Ca2+ influx induced by TRPV4 channel-mediated Ca2+ influx(CICR)cause intracellular calcium oscillations,which are involved in the formation of calcium oscillations,and plays an important role in maintaining intracellular calcium homeostasis.When various pathological stimuli result in endoplasmic reticulum protein error synthesis,folding disorders,or emptying of calcium stores,endoplasmic reticulum stress(ERS)is induced.ERS triggers the unfolded protein response(UPR)to restore protein homeostasis.If chronically emptying ERS calcium pool or overloading of unfolded proteins,which will lead to apoptosis.Whether intracerebral hemorrhage may lead to the activation of TRPV4 channel and induce the emptying of endoplasmic reticulum calcium reservoir in the form of CICR,and then induce endoplasmic reticulum stress response and lead to neuronal apoptosis.it will be the focus of our researches.In summary,this study intends to investigate whether TRPV4 channels are protective or injurious after ICH,and then demonstrate whether TRPV4 channels trigger neuronal apoptosis through the CICR trigger endoplasmic reticulum stress pathway.Part One Role of TRPV4 Channel in Cerebral Hemorrhage in Mice and Its Effect on Neuronal ApoptosisObjective To investigate whether TRPV4 channels play a role in cereb=ral hemorrhageMethods 1.Group Sham group,vehicle group,GSK1016790A+ICH group,HC-067047+ICH group 2.Detection Indicator(1)The protein expression of TRPV4 channels around the hematoma after intracerebral hemorrhage was detected by western blot.(2)Different doses of TRPV4 channel agonists or blockers were administered immediately after intracerebral hemorrhage(within 15 minutes)to the lateral ventricles,and then neurological function tests(neurological function score,grasping rope test)were performed.(3)TRPV4 blockers were administrated at different time points after intracerebral hemorrhage and neurological function changes were observed.(4)When excited or blocked TRPV4 channel,it was measured the changes in brain edema(dry/wet method).(5)When excited or blocked TRPV4 channel,it was measured the apoptosis of neurons after ICH(TUNEL method,nissl staining).Results 1.The expression of TRPV4 channels was up-regulated in mice after intracerebral hemorrhage.TRPV4 protein expression gradually increased in the ipsilateral brain tissue at 6 h after intracerebral hemorrhage,reached the highest level at 24 hours,and gradually decreased after 48 hours.At the same time,the administration of the agonist/blocker group had the same degree of upregulation of TRPV4 expression compared with the vehicle group.This shows that pathological changes after ICH can stimulate the expression of TRPV4 upregulation.2.Blocking TRPV4 channels after ICH improve the neurobehavioral performance of mice.When administrating different doses of TRPV4 agonist/blocker after intracerebral hemorrhage,that was observed dose-dependent neurobehavioral improvement.In addition,we also observed the administration of TRPV4 antagonist before 12 h after intracerebral hemorrhage could improve the behavior,and the improvement effect was not obvious after ICH 12 hours.3.Blocking TRPV4 channel to improve the degree of cerebral edema after cerebral hemorrhage.The swelling of cells around the hematoma was significant after cerebral hemorrhage,which was aggravated by the GSK1016790 A,and relieved by the HC-067047.4.Blocking TRPV4 channel to alleviate neuronal apoptosis after intracerebral hemorrhage Administration of TRPV4 agonists aggravated brain injury and neuronal apoptosis.Blocking TRPV4 channels alleviated neuronal apoptosis.This shows that TRPV4 channels participate in neuronal apoptosis after intracerebral hemorrhage.In conclusion TRPV4 channels that are up-regulated after intracerebral hemorrhage,and activated by various pathological stimuli,which in turn leads to neuronal apoptosis.Blocking TRPV4 channels can improve brain damage and animal behavioral changes.Part two TRPV4 channels stimulate Ca2+-induced Ca2+ release in neurons of mouse and trigger endoplasmic reticulum stress after intracerebral hemorrhageObjective To investigate whether TRPV4 triggers the emptying of the endoplasmic reticulum calcium pool through CICR and whether neuronal apoptosis is induced by PERK-CHOP-Bcl-2 and/or IRE1-JNK pathways after endoplasmic reticulum stress.Methods 1.Group 1.1 primary cultured neurons Control group,GSK1016790 A group,Ca2+-free extracellular fluid + GSK1016790 A group,Ca2+-free extracellular fluid + GSK1016790 A + Ca Cl2 group,thapsigargin +GSK1016790A group 1.2 mouse cerebral hemorrhage model Sham group,vehicle group,GSK1016790A+ICH group,HC-067047+ICH group,2-APB+ICH group,GSK1016790A+2-APB+ICH group,HC-067047 group,2-APB+ICH group 2.Detection Indicator(1)To detect whether GSK1016790 A exert a excitotoxicity function by MTT and TUNEL.(2)To detect CICR response in TRPV4 channels by calcium imaging.(3)To detect the combination of TRPV4 and IP3 R after ICH by co-immunoprecipitation.(4)To detect the changes of PERK-CHOP-Bcl-2 and IRE1-JNK pathways after ICH by Western blot.(5)After blocking IP3 R,to detect the changes PERK-CHOP-Bcl-2 pathway.(6)After blocking IP3 R,to detect the apoptosis of neurons in the ipsilateral brain tissues.Results 1.Low concentrations of GSK1016790 A did not damage primary cultured neurons.The result of MTT and TUNEL assays showed that GSK1016790 A concentration from 0.1μmol/L to 10μmol/L,cell survival rate was almost unchanged,while 30μmol/L GSK1016790 A can affect cell survival rate,which may be related to the toxic effects of drugs at high concentrations.2.Activation of the TRPV4 channel on neurons induces a CICR response.In normal extracellular fluids,GSK1016790 A induced two forms of changes in intracellular calcium elevation.In the calcium-free extracellular fluid,GSK1016790 A could not induce the increase of intracellular Ca2+ and increased again when added Ca2+ into extracellular fluid;if emptying ERS ahead,GSK1016790 A could not induce the increase of intracellular calcium.These results indicated that TRPV4 channels stimulate Ca2+-induced Ca2+ release in neurons of mouse.3.Intracerebral hemorrhage recruits IP3 Rs into TRPV4 protein complex.The results showed IP3 Rs was physically associated with TRPV4 receptors,intracerebral hemorrhage promoted the IP3 Rs combining with TRPV4 receptors.Under physiological conditions,TRPV4 positively regulates IP3 R.After cerebral hemorrhage,IP3 R may trigger the continuous opening of the TRPV4 channel and the chronic and sustained release of calcium ions from the endoplasmic reticulum calcium pool,thereby inducing endoplasmic reticulum stress.4.Neuronal apoptosis induced by mouse cerebral hemorrhage was associated with activation of PERK-CHOP-Bcl-2 pathway.When activation/inhibition TRPV4 channel after ICH,PERK-CHOP-Bcl-2 pathway changed,but no change of p-JNK expression in IRE1-JNK pathway,which indicated that TRPV4 channel was mainly via PERK-CHOP-Bcl-2 pathway to lead to neuronal apoptosis.5.IP3 Rs inhibition relieved endoplasmic reticulum stress and improved hemorrhagic brain injury After blocking IR3 R,apoptosis-related proteins in the ERS pathway decreased,and neurons apoptosis relieved,which showed that IP3 R was an important link in TRPV4-induced endoplasmic reticulum stress and neuronal apoptosis pathways.In conclusion TRPV4 induced the emptying of the endoplasmic reticulum calcium reservoir by CICR,which led to the activation of PERK-CHOP-Bcl-2 and involved in the apoptosis of neurons after intracerebral hemorrhage in mice.