Protective Effects Of TRPC1 Channel Against OGD/R-induced Neuronal Injury Via SOCE-regulated Endoplasmic Reticulum Stress

Ischemic stroke?IS?is a neurologic disorder resulting from loss of blood supply to local brain tissue caused by various reasons.It may cause permanent damage of the central nervous system and even threaten the victim's life.Studies have shown that the degree of cerebral ischemia-reperfusion?I/R?injury is closely related to its prognosis.Therefore,in-depth study on the detailed mechanism and strategies for prevention and treatment of cerebral ischemia-reperfusion injury is of great clinical significance for improving the prognosis of patients with ischemic stroke.The transient receptor potential canonical 1?TRPC1?channel is one of the members of TRPC channel family?TRPC1^TRPC7?,which is widely expressed in the central nervous system.TRPC channel is a family of nonselective cation channels,which participates numerous pathophysiological processes of the body.It has been known that some members of this family,such as TRPC3 and TRPC6,have neuroprotective effects on hypoxic/ischemic neurons.It has also been reported that TRPC1may reduce hypoxia-induced injury in the salivary gland cells and SHSY-5Y cells.However,much less is known about the protective effects of TRPC1 on oxygen-glucose deprivation/reoxygenation?OGD/R?neurons.TRPC channel,which is highly permeable to calcium,is an important type of ion channels to regulate intracellular calcium homeostasis.Intracellular Ca²⁺is an essential second messenger that mediates multiple intracellular signal transduction pathways and has a vital role in the regulation of diverse cellular processes such as proliferation,survival,and apoptosis.The regulation of intracellular Ca²⁺concentration mainly depends on the release of intracellular calcium store and the influx of extracellular calcium.Store-operated calcium entry?SOCE?,which is mediated by store-operated calcium channel?SOC?,is the main form of calcium influx in neurons.A decrease in calcium influx mediated by SOC could lead to decreased ER calcium store refilling and calcium deprivation,and then disrupt intracellular calcium homeostasis resulting in endoplasmic reticulum stress?ERS?.Unfolded protein response?UPR?is a process to re-establish ER homeostasis by promoting correct folding of secreted proteins or enhancing removal of misfolded proteins from the ER.It is a self-protection mechanism against ERS.However,prolonged or irremediable activation of UPR triggers apoptotic signaling pathways,committing the cells to injury or death.Previous studies have suggested that ERS contributes to neuron injury after cerebral ischemia and reperfusion and ERS inhibition alleviates I/R-induced brain damage.TRPC1,an important molecular component of SOC in the nervous system,constitutes SOC together with the stromal interaction molecule 1?STIM1?and Orail proteins.However,it is unclear that the influence of TRPC1-modulated SOCE on OGD/R-induced neuronal injury and its relationship with ERS.ObjectiveThis study focused on TRPC1 channel,SOCE and ERS,using primary cortical neurons subjected to oxygen-glucose deprivation/reoxygenation,to investigate the protective effect of TRPC1 channel against ischemic brain injury and its underlying mechanism.Methods1.ModelCortical neurons extracted from fetuses of Sprague Dawley?SD?rats were cultured in vitro for 5⁷ days.The primary cortical neurons were subjected to oxygen glucose deprivation 1.5h followed by reoxygenation 24h to establish OGD/R model.2.Groups?1?Controlgroup,OGD/Rgroup,OGD/R+4-PBAgroup,OGD/R+DMSO group.?2?Control group,OGD/R group,OGD/R+SKF96365 group,OGD/R+DMSO group.?3?Control group,OGD/R group,OGD/R+si-TRPC1 group,OGD/R+Scramble group.3.Detection Indicator?1?The cell viability of neurons was detected by MTT assay;the integrity of cell membrane was used by LDH leakage;and apoptosis was tested by Hochest33342 staining and TUNEL staining.?2?Western blot was used to detect the expressions of diverse proteins.?3?The interactions between TRPC1 and STIM1 or Orai1 were tested by the Co-Immunoprecipitation.?4?Flou-4AM staining and laser confocal microscopy were used to detected the calcium concentration in endoplasmic reticulum and SOCE after OGD/R.Results1.An ERS inhibitor,4-PBA,could significantly increase the cell viability,decrease the rate of LDH leakage and apoptosis in OGD/R neurons as well as depress the expressions of ERS marker protein GRP78 and IRE1signaling pathway-associated proteins.2.The TRPC channel blocker SKF96365 or siRNA-TRPC1 could significantly reduce the cell viability,and increased the leakage rate of LDH and the rate of neuronal apoptosis after OGD/R.3.SKF96365 or siRNA-TRPC1 up-regulated the expression of GRP78 in OGD/R neurons,while the phosphorylation levels of IRE1 and downstream JNK were increased significantly,and the expression of anti-apoptotic protein Bcl-2 was down-regulated.4.Blockage of TRPC1 channels or down-regulation of TRPC1 expression decreased the ER calcium concentration and SOCE in OGD/R neurons significantly.5.Down-regulation of TRPC1 expression reduced STIM1 or Oria1binding with TRPC1 after OGD/R.ConclusionTRPC1 channel plays a protective role on OGD/R neurons.The underlying mechanism may be that TRPC1 channel could restore ER calcium homeostasis through regulating SOC-mediated calcium influx in OGD/R neurons,and then inhibit ERS-associated signaling pathways and sequential apoptosis of neurons.