Mechanism Of Hyperoside Regulating Cerebrovascular TRPV4 Through IP₃/PKC Signaling Pathway To Ameliorate Ischemic Cerebral Apoplexy

Objective:To investigate whether Hyperoside?Hyp?affects TRPV4 and its downstream channels IKca and SKca in cerebral basilar artery after ischemic stroke through IP₃/PKC signaling pathway and explore its possible mechanisms.Methods:104 male Sprague-Dawley rats,2 months old,weighing 250±20 g,free diet and drinking water.After a week of adaptive feeding,a global cerebral ischemia-reperfusion?CIR?model was established by four-vessel occlusion.Rats were randomly assigned about Sham group?NS?,CIR group?NS?,Hyp group?50mg/kg?,Hyp+TRPV4 blocker HC-067047 group?50 mg/kg+10 mg/kg?,HC-067047group?10 mg/kg?,Hyp+IKca blocker TRAM-34 group?50 mg/kg+0.5 mg/kg?,Hyp+SKca blocker Apamin group?50 mg/kg+0.3 mg/kg?,Hyp+IP₃ blocker 2APB group?50 mg/kg+2 mg/kg?,2APB group?2 mg/kg?,Hyp+PKC blocker BisI group?50 mg/kg+2.5 mg/kg?,BisI group?2.5 mg/kg?,Hyp+2APB+BisI group?50 mg/kg+2mg/kg+2.5 mg/kg?,2APB+BisI group?2 mg/kg+2.5 mg/kg??n=8 for each group?.Except Sham group,the rats in other groups were injected intraveously the above drugs 30 minutes before the bilateral common carotid arteries were clamped,and the rats were killed 24 hours after the completion of the modeling.Brain electroencephalogram?EEG?was recorderd before ischemia,20 min after ischemia and 2 h after reperfusion in rats by brain wave recorder.The body weight of rats was measured accurately before the establishment of the model,and the weight of the brain was accurately weighed after the completion of the model,the difference of organ coefficient of the brain tissue in each group was compared.Blood samples were takens from abdominal aorta of rats and serum was separated,the activity of LDH,the contents of MDA,NSE and S100?in serum were detected by ELISA.Pathological changes of hippocampal region in rats were observed by HE staining and Nissl staining,respectively.The changes of relaxation and contraction of basilar artery?CBA?produced by Hyp in CIR rats were observed by arterial pressure perfusion,and the effect of TRPV4,IKca and SKca channel blockers on the changes were observed and recorded.Western blot immunoassay?Western blot,WB?was used to observe the effects of Hyp and IP₃/PKC signaling pathway blockers on the expression of TRPV4,SKca and IKca channel proteins in CBA endothelial cells of CIR rats.Effects of Hyp and IP₃/PKC signaling Pathway blockers on Ca²⁺concentration in CBA smooth muscle cells of CIR Rats detected by Laser Confocal technique.Flow cytometry was used to detect the effect of Hyp on the apoptosis of CBA endothelial cells in CIR rats and the effect of IP₃/PKC signal pathway blocker on the degree of apoptosis.Results:?1?Compared with Sham group,there was no obvious change in the brain wave of model group before ischemia,but the fluctuation of the brain wave slowed down and the amplitude decreased at 20 min of ischemia,and the waveform slowly recovered to the state close to that of the pre-ischemia after reperfusion.?2?Compared with sham group,the organ coefficient of brain tissue in the model group was significantly higher than that in the model group,and compared with CIR group,the organ coefficient of brain tissue in Hyp group and Hyp combined with each channel blocker group decreased in varying degrees?P<0.01?.Compared with Hyp group,the organ coefficient of brain tissue in Hyp combined with HC-067047 group was significantly up-regulated?P<0.05?,while Hyp combined Apamin group and TRAM-34 group increased slightly,but the difference was not statistically significant.?P>0.05?.?3?HE staining showed that a large number of cells in hippocampal CA1region of model rats were deformed and necrosed,arranged disorderly,nerve fibers disintegrated and nucleus concentrated staining pyknosis,Nissl staining showed that the arrangement of neurons in hippocampal region of model group was scattered and loose.The pathological state of hippocampal region in Hyp group and Hyp combined with various blockers group was improved to a certain extent.?4?The results of ELISA showed that compared with Sham group,the activity of LDH,the content of MDA,NSE and S100?in serum of the model group showed a obvious upward trend?P<0.01?.Compared with the model group,LDH activity,MDA,NSE and S100?content in Hyp group and Hyp combined with each channel blocker group were significantly decreased?P<0.05,P<0.01?;while LDH activity,MDA,NSE and S100?content in Hyp combined with each blocker group were increased to a certain extent compared with Hyp group?P<0.05,P<0.01?.?5?According to the method of arterial pressure perfusion,it was found that Hyp could relax the basilar artery in a dose-dependent manner in the model group,but after pretreatment with TRPV4,SKca and IKca channel blockers,compared with the Hyp group,the basilar artery showed different degrees of vasoconstriction.?6?According to the results of western blot detection,compared with sham group,the expression of IP₃,PKC,TRPV4,SKca and IKca protein in rat basilar artery endothelial cells was significantly down-regulated in the model group?P<0.01?.The expression levels of IP₃,PKC,TRPV4,SKca and IKca protein in basilar artery endothelial cells in hyp group were significantly up-regulated,as compared with the CIR group,while IP₃,PKC,TRPV4,SKca and IKca protein in basilar artery endothelial cells in combination with various channel blockers were up-regulated?P<0.05,P<0.01?.The expression of IP₃,PKC,TRPV4,SKca and IKca protein in basilar artery endothelial cells was significantly down-regulated in each channel blocker group.?P<0.05,P<0.01?.Compared with Hyp group,the expression levels of IP₃,PKC,TRPV4,SKca and IKca protein in basilar artery endothelial cells were down-regulated to a certain extent in each combination of channel blockers?P<0.05?.?7?The results of laser confocal technique suggested that the fluorescence intensity of Ca²⁺in the smooth muscle cells of basilar artery in the model group was obviously higher than that in the sham-operate group.In comparision with CIR group,the fluorescence intensity of Ca²⁺in Hyp group and Hyp combined with each blocker group showed a decreasing trend in varying degrees,and in comparision with Hyp group,the Ca²⁺fluorescence intensity of Hyp combined with each blocker group was obviously rised.?8?The results of flow cytometry showed that the apoptosis rate of CBA endothelial cells in CIR group was significantly higher than that in Sham group?P<0.01?.and the apoptosis rate of CBA endothelial cells in Hyp group and Hyp combined with blockers group was decreased to some extent compared with CIR group?P<0.05,P<0.01?.And compared with Hyp group,the apoptosis rate of CBA endothelial cells in Hyp combined with each blocker group was obviously rised.Conclusion:Hyp has a significant improvement on the brain injury of rats with global cerebral ischemia and reperfusion.The mechanism may be that Hyp acts on the cerebral basilar artery,which activates the expression and activity of TRPV4,through IP₃/PKC signaling pathway,and then activates SKca and IKca channels,ultimately promote the increase of EDHF,resulting in endothelium-dependent hyperpolarization of smooth muscle cells,blocking the Ca²⁺influx,contributes to the relaxation of the cerebral basilar artery.