Effects Of Total Flavones Of Rhododendra Simsii Planch On SOCE Pathway Regulated By STIM-Orai At Different Stages After Cerebral Ischemia/Reperfusion In Rats

Objective:To investigate the effect of total flavones of Rhododendra simsii Planch（TFR）on STIM-Orai-regulated store-operated calcium entry（SOCE）on brain tissue at different stages of focal cerebral ischemia-reperfusion injury in rats.Methods:1.In vivo experiment:Middle cerebral artery occlusion（MCAO）was used to construct a focal cerebral ischemia-reperfusion injury model.SD male rats were randomly divided into Sham group,MCAO group,TFR group,TFR+SOCE inhibitor2APB group,2APB group.The latter four groups were divided into 1,3,and 14 d（day）according to the different time points of material collection,a total of 13 groups with 8rats in each group.The Sham and MCAO groups were given normal saline（NS）by intragastric administration.The rats in the one-day group were treated with tail vein injection of TFR（100 mg/kg）.The rats in the 3 d and 14 d groups were given TFR（60mg/kg）by intragastric administration until the material was obtained.The rats in the SOCE channel inhibitor group were intraperitoneally injected with 2APB（2.5 mg/kg）after surgery.The activity of inflammatory factors IL-1,IL-6 and TNF-αin serum were measured with the enzyme-linked immunosorbent assay.HE staining was applied to detect pathological changes in rat ischemic brain tissue.TTC staining was used to observe rat brain tissue infarction.Western blot and RT-q PCR were utilized to detect the protein and mRNA expression levels of STIM1,STIM2,Orai1,Caspase-3 and PKB in brain tissue at various periods,respectively.Immunofluorescence was used to detect the growth of rat brain neurons in each group at 14 d of chronic recovery.2.In vitro experiment:PC12 cells were cultured in vitro to detect the changes in cell levels in the acute phase and randomly divided into 5 groups,Normal group,Hypoxia reoxygenation（Model）group,TFR group（0.3 mg/m L）,TFR+2APB group（0.3mg/m L+22.51 mg/m L）,2APB group（22.51 mg/m L）.CCK-8 was used to detect the cell viability at different concentrations of TFR.Laser confocal technology was applied to detect the effects of TFR and SOCE signaling pathway blockers on the Ca²⁺concentration of hypoxia-reoxygenation PC12 cells variety.Flow cytometry was utilized to detect TFR and SOCE signal pathway inhibitors on apoptosis of hypoxia-reoxygenation PC12 cells.Results:1.In vivo experiments:（1）Compared with Sham group,the neurological function scores of rats in the MCAO group were significantly increased in each period（P<0.01）.Compared with the MCAO group at the same period,the neurological function scores of the rats in the TFR group were significantly lower at each period（P<0.05）.After treatment with blocker 2APB for 1and 3 d,the neurological function scores of rats further were decreased（P<0.01）.The neurological function score of the TFR+2APB group was significantly higher than in TFR group for 14 d（P<0.05）.（2）The results showed that the contents of serum IL-1,IL-6 and TNF-αin MCAO group were significantly increased at each period as compared with the Sham group（P<0.01）.TFR obviously reduced the levels of IL-1,IL-6 and TNF-αat each period（P<0.05,P<0.01）.Compared with the TFR group at the same period,the contents of IL-1,IL-6,and TNF-αin TFR+2APB group were significantly lower for 1 and 3 d（P<0.05）,and the above indexes were significantly higher than in TFR group at 14 d（P<0.05）.The levels of IL-1,IL-6,and TNF-αin the TFR+2APB group at each period were significantly reduced as compared with the2APB group（P<0.05,P<0.01）.（3）The results of HE staining showed that the cells in the Sham group were arranged regularly and the nucleoli were clear,while the MCAO group in different time had irregular morphology,which was manifested by cell structure disorder,vacuole formation and cell necrosis.Compared with the MCAO group at the same period,the necrosis of brain tissue in the infarcted side of the TFR group was significantly reduced at each period.After treatment with inhibitor 2APB,the degree of brain injury was further significantly reduced at 1 and 3 d,but the brain injury was aggravated at 14 d.TTC staining showed that obvious cerebral infarction was occurred in the MCAO group at each period.Compared with the MCAO group at the same period,the focus of cerebral infarction and the rate of cerebral infarction were decreased significantly in the TFR group at each period,especially in 14 d.Compared with the simultaneous TFR group,the cerebral infarction area was significantly reduced in the 1 d and 3 d TFR+2APB group,and the cerebral infarction rate was significantly reduced（P<0.01）,while the 14 d group was significantly increased（P<0.01）.（4）According to the results of Western blot and RT-q PCR detection,the protein and mRNA expression of STIM1,STIM2,Orai1 and Caspase-3 in the brain tissue of MCAO rats for 1 and 3 d were significantly higher than those in the Sham group（P<0.01）,while the protein and mRNA expression of PKB were significantly decreased（P<0.01）.The expression of STIM1,STIM2,Orai1,PKB protein and mRNA were decreased significantly（P<0.05）,while the expression of Caspase-3 protein and mRNA were increased significantly in MCAO group for 14 d（P<0.05）.Compared with the MCAO group at the same point,the expression of STIM1,STIM2,Orai1,Caspase-3protein and mRNA in the brain tissue of rats were significantly reduced in TFR group for 1 and 3 d（P<0.05）,while the expression of PKB protein and mRNA were increased（P<0.05）.After TFR treatment for 14 d,the expression of STIM1,STIM2,Orai1,PKB protein and mRNA were increased significantly（P<0.05）,while the expression of Caspase-3 protein and mRNA were decreased（P<0.05）.Compared with the TFR group and 2APB group,the STIM1,STIM2,Orai1,Caspase-3 protein and mRNA expression on the 1 and 3 d TFR+2APB group were significantly decreased（P<0.05）,while the PKB protein and mRNA expression were increased（P<0.05）.On the 14 d,the protein and mRNA expression of STIM1,STIM2,Orai1,PKB in the TFR+2APB group were significantly lower than that of the TFR group but higher than that in 2APB group（P<0.05）,while the expression of Caspase-3 protein and mRNA were higher than that in TFR group and lower than 2APB group（P<0.05）.（5）Immunofluorescence results showed that there were no Brdu/MAP-2 double-labeled positive cells in Sham group,MCAO group and 2APB group,while a small number of Brdu/MAP-2 double-labeled positive cells were found in TFR group and TFR+2APB group.2.In vitro experiment:（6）The results of CCK-8 showed that the cell survival rate was decreased significantly when the concentration of TFR was more than 1.05mg/m L（P<0.01）.（7）The results of laser confocal detection showed that after hypoxia-reoxygenation treatment,the Ca²⁺fluorescence intensity of PC12 cells was significantly higher than that of the Normal group（P<0.01）.The average fluorescence intensity in TFR group was significantly lower than that in Model group（P<0.05）.The mean fluorescence intensity of Ca²⁺in TFR+2APB group was significantly lower than that in TFR group and 2APB group（P<0.01）.（8）The apoptosis rate of PC12 cells in Model group was significantly higher than that in Normal group（P<0.01）.Compared with the Model group,the apoptosis rate of PC12 cells in the TFR group was significantly reduced（P<0.05）.Compared with TFR group or 2APB group,the apoptosis rate of PC12 cells in TFR+2APB group was significantly reduced（P<0.05）.Conclusion:Our data demonstrated that TFR can improve brain injury in MCAO rats,and its mechanism may be related to regulate the activity of SOCE channel in different periods of ischemia-reperfusion.In the acute stage of ischemic stroke,it may inhibit the activation of SOCE pathway,decrease the contents of inflammatory factors,and protect the damaged brain tissue.In the later stage,TFR may upregulate the expression of SOCE channel related genes to promote neurogenesis around the ischemic area and reduce neurons apoptosis,thereby playing a protective role in brain injury.