Roles Of TRPC1&4 In Early Brain Injury After Experimental Subarachnoid Hemorrhage In Rats

PartⅠ: Research on the impact of Transient receptor potential channel on the early brain injury after experimental subarachnoid hemorrhageObjective: To investigate the effects and mechanisms of Transient receptor potential channel after experimental subarachnoid hemorrhage(SAH).Methods: 70 adult SD rats were divided into four groups: normal group,SAH group,SAH+DMSO group,and SAH+SFK96365 group.The rat SAH model was induced by injection of 0.3 ml fresh arterial,nonheparinized blood into the prechiasmatic cistern in 20 s.In SAH+SFK group,SFK was administered i.p.at 1.0 mg/kg 30 minutes before and each day after the induction of SAH.Part of rats in each group were killed on 72 hr after SAH.The brain sample was removed and keeped in 10% formalin solution.We use the western blot to find out the time cost of the expression of TRPC1&4 after SAH,then we kill the rats on the peak of expression of TRPC1&4.TUNEL and FJB staining show the apoptosis and degeneration rate of neurons,western blot results shows the albumin to determine the damage of blood brain barrier.In vitro experiment: Prepare the dish 1 day in advance with sterile 12-well plates,take the brain of 16th–18th days fetal rats,digest and wash,treated with 200 mesh filter.Changing the neurol basil medium every 2 days,each time for half the liquid.Treat with siRNA and over-expression mRNA(Ribobio,Guangzhou,China)and hemoglobin after 14 days.Extract the extracellular medium,proteins and coverslips at the certain time after treatment according to the experimental design.Cells cultured for 14 days-18 days,the cells were divided into 4 groups: normal group,oxyHb group,oxyHb +TRPC down-regulated group,oxyHb +TRPC up-regulated group.Western blot results shows the outcomes of the regulation of TRPC1&4 expression,TUNEL staining show the apoptosis rate of neurons,LDH assay shows the degeneration rate of neurons.Results: 1.During the EBI after SAH,the expressions of TRPC1&4 continue increasing.The 72 hour is the peak of expression during EBI.2.Compared with normal control group,SAH group neuronal apoptosis rate increased significantly(p <0.01),apoptosis rate of SAH+SFK96365 treated group higher than the SAH + DMSO treated group(p <0.05),and SAH+DMSO treated SD rats showed no differences between SAH group SD rats(p>0.05).3.Compared with normal group,the degeneration rate of SAH group were significantly increased(p <0.01),degeneration rate of SAH+SFK96365 treated group was higher than that in SAH+DMSO treated ones(p <0.05),and SAH+DMSO treated SD rats showed no differences between SAH group SD rats(p>0.05).4.Blood brain barrier was damaged after SAH(p<0.01),but SAH+SFK96365 treated group was worse comparing with SAH group(p<0.001).5.In the in vitro experiment,OxyHb group shows higher apoptosis rate(p<0.01)and degeneration rate(p<0.01);the down-regulated TRPC1&4 results in higher apoptosis rate(p<0.01)and degeneration rate(p<0.01)compared to OxyHb group,the up-regulated TRPC1&4 results in lower apoptosis rate(p<0.01)and degeneration rate(p<0.05)compared to OxyHb group.Conclusion: Early brain injury caused by SAH showed an acute rise of apoptotic and degeneration rate,blood-brain barrier is destroyed after subarachnoid hemorrhage.While intracellular TRPC1&4 expression increased,it plays a neuroprotective role,with the downward of TRPC1&4,the neuronal apoptosis and degeneration is more severe.On the contrary,when expression of TRPC1&4 increases,the apoptosis and degeneration reduced.Part Ⅱ: Research on the mechanisms of the protective effects of TRPC1&4 in EBI due to SAHObjective: To establish subarachnoid hemorrhage model and observe the expression changes of TRPC1&4 cause calcineurin-mediated N-methyl-D-aspartate receptor changes.Methods: Rats experiment:90 adult SD rats were divided into four groups: normal group,SAH group,SAH + DMSO treated group,SAH + TRPC antagonist(SFK96365)treatment group,SAH + CN antagonist(FK506)treatment group and SAH + CN agonist(CHA)treatment group.The rat SAH model was induced by injection of 0.3 ml fresh arterial,nonheparinized blood into the prechiasmatic cistern in 20 s.In TRPC antagonist / CN antagonist / CN agonist treatment group,half an hour before the model,daily intraperitoneal injection of 1.0mg per kg of body weight SFK96365,FK506,CHA solution(concentration 10 umol / L dissolved in DMSO after model solution).In the DMSO group,the same amount of liquid and the same time by intraperitoneal injection of DMSO.The rats were killed on the peak of expression,western blot is used to detecting TRPC1&4 expression,NMDAR phosphorylation,degeneration is detected measuring FJB staining.In vitro experiment: Prepare the dish 1 day in advance with sterile 12-well plates,take the brain of 16th–18th days fetal rats,digest and wash,treated with 200 mesh filter.Changing the neurol basil medium every 2 days,each time for half the liquid.Treat with si RNA and over-expression m RNA(Ribobio,Guangzhou,China)and hemoglobin after 14 days.Extract the extracellular medium,proteins and coverslips at the certain time after treatment according to the experimental design.Cells cultured for 14 days-18 days,the cells were divided into six groups: normal group,oxy Hb group,oxy Hb +TRPC down group,oxy Hb +TRPC regulation group,oxy Hb +CN exciting group,oxy Hb +CN antagonistic groups.Western blot results shows the outcomes of the regulation of TRPC1&4 expression and the phosphorylation of NMDAR,TUNEL staining show the apoptosis rate of neurons,LDH assay shows the degeneration rate of neurons,CN assay shows the activity of CN.Results: 1.During EBI after SAH,up-regulated TRPC1&4 positively regulated the activity of CN(p<0.01),down-regulated TRPC1&4 negatively regulated the activity of CN(p<0.05).2.up-regulated TRPC1&4 negatively regulated the phosphorylation of NMDAR(p<0.05),down-regulated TRPC1&4 positively regulated the phosphorylation of NMDAR(p<0.05).3.When TRPC1&4 are down-regulated,acitivated CN is shown to restore the phosphorylation of NMDAR(p<0.01);when TRPC1&4 are up-regulated,depressed CN is shown to promote the phosphorylation of NMDAR(p<0.01).4.Acitivated CN can promote the intranuclear NFAT expression(p<0.05),which can up-regulate the expression of TRPC1&4(p<0.01),depressed CN can reduce the intranuclear NFAT expression(p<0.05),which can down-regulate the expression of TRPC1&4(p<0.01).Conclusion: TRPC1&4 positively regulated the activity of CN.Up-regulated TRPC1&4 promoted the dephosphorylation of NMDAR via CN,that may subsequently lead to a lowering neuron toxicity,thus shows the neuron protective effect of TRPC1&4 in EBI after SAH.