Objective To investigate the effects of Buyang Huanwu Decoction on nerve regeneration after cerebral ischemia in mice based on Cav-1/Notch1/Hes1 signaling pathway.Methods Wild(WT)and Cav-1-/-(KO)male mice were randomly divided into sham-operation group,model group and Buyang Huanwu Decoction group,with 10 mice in each group.The middle cerebral artery occlusion method was used to construct the mouse cerebral ischemia model.After modeling,5-ethynyl-2'-deoxyuridine(EdU)was injected intraperitoneally every 7 days for 3 times(with an interval of 8 hours).Buyang Huanwu Decoction group was given 18.5 g/kg Buyang Huanwu Decoction by gavage daily,while the sham-operation group and model group were given distilled water of equal volume by gavage for 21 consecutive days.The neurological function of mice was evaluated using modified neurological severity score,brain tissue morphology was observed through HE staining,nerve regeneration in the ischemic side injury area was detected using dual immunofluorescence staining,Western blot was used to detect the expressions of Notch1 and Hes1 protein in ischemic cortical area.Results Compared with the same genotype sham-operation group,the neurological function score of the mice in the WT and KO model groups significantly increased(P<0.01),the arrangement of neurons in the ischemic cortical area was disordered,with nucleoli shrinking,marginalization,and even rupture,widened intercellular spaces,intracellular edema and vacuolar changes,the co-localization of EdU/Nestin,EdU/DCX and EdU/NeuN in the ischemic side injury area significantly increased(P<0.01),and the expression of Notch1 and Hes1 protein in ischemic cortical area significantly increased(P<0.01).Compared with the same genotype model group,the neurological function score of the WT and KO Buyang Huanwu Decoction group significantly decreased(P<0.01),the neurons in the ischemic cortical area were arranged neatly and structurally intact,with reduced intercellular space and clear nucleoli,the co-localization of EdU/Nestin,EdU/DCX and EdU/NeuN in the ischemic side injury area was significantly increased(P<0.01),and the expression of Notch1 and Hes1 protein in ischemic cortical area were significantly decreased(P<0.01).Compared with the corresponding WT group,the neurological function scores of mice in the KO model group and KO Buyang Huanwu Decoction group significantly increased(P<0.01),the neurons in the ischemic cortical area had more severe nucleolar condensation,marginalization and rupture,with more vacuolar changes and ischemic injury,the co-localization of EdU/Nestin and EdU/NeuN in the ischemic side injury area was significantly decreased(P<0.01),and the expression of Notch1 and Hes1 proteins in ischemic cortical area significantly increased(P<0.01).Conclusion Buyang Huanwu Decoction can promote nerve regeneration after cerebral ischemia,which may be related to the regulation of Cav-1 thereby inhibition Notch1//Hes1 signaling pathway activity.

Objective To investigate the effects of Buyang Huanwu Decoction on nerve regeneration after cerebral ischemia in mice based on Cav-1/Notch1/Hes1 signaling pathway.Methods Wild(WT)and Cav-1-/-(KO)male mice were randomly divided into sham-operation group,model group and Buyang Huanwu Decoction group,with 10 mice in each group.The middle cerebral artery occlusion method was used to construct the mouse cerebral ischemia model.After modeling,5-ethynyl-2'-deoxyuridine(EdU)was injected intraperitoneally every 7 days for 3 times(with an interval of 8 hours).Buyang Huanwu Decoction group was given 18.5 g/kg Buyang Huanwu Decoction by gavage daily,while the sham-operation group and model group were given distilled water of equal volume by gavage for 21 consecutive days.The neurological function of mice was evaluated using modified neurological severity score,brain tissue morphology was observed through HE staining,nerve regeneration in the ischemic side injury area was detected using dual immunofluorescence staining,Western blot was used to detect the expressions of Notch1 and Hes1 protein in ischemic cortical area.Results Compared with the same genotype sham-operation group,the neurological function score of the mice in the WT and KO model groups significantly increased(P<0.01),the arrangement of neurons in the ischemic cortical area was disordered,with nucleoli shrinking,marginalization,and even rupture,widened intercellular spaces,intracellular edema and vacuolar changes,the co-localization of EdU/Nestin,EdU/DCX and EdU/NeuN in the ischemic side injury area significantly increased(P<0.01),and the expression of Notch1 and Hes1 protein in ischemic cortical area significantly increased(P<0.01).Compared with the same genotype model group,the neurological function score of the WT and KO Buyang Huanwu Decoction group significantly decreased(P<0.01),the neurons in the ischemic cortical area were arranged neatly and structurally intact,with reduced intercellular space and clear nucleoli,the co-localization of EdU/Nestin,EdU/DCX and EdU/NeuN in the ischemic side injury area was significantly increased(P<0.01),and the expression of Notch1 and Hes1 protein in ischemic cortical area were significantly decreased(P<0.01).Compared with the corresponding WT group,the neurological function scores of mice in the KO model group and KO Buyang Huanwu Decoction group significantly increased(P<0.01),the neurons in the ischemic cortical area had more severe nucleolar condensation,marginalization and rupture,with more vacuolar changes and ischemic injury,the co-localization of EdU/Nestin and EdU/NeuN in the ischemic side injury area was significantly decreased(P<0.01),and the expression of Notch1 and Hes1 proteins in ischemic cortical area significantly increased(P<0.01).Conclusion Buyang Huanwu Decoction can promote nerve regeneration after cerebral ischemia,which may be related to the regulation of Cav-1 thereby inhibition Notch1//Hes1 signaling pathway activity.

Objective To explore the effects of Buyang Huanwu Decoction on neuronal cell apoptosis after cerebral ischemia based on mediating Cav1 in regulating Wnt pathway.Methods Male wild-type(WT)and Cav1-/-(KO)C57BL/6 mice were randomly divided into sham-operation group,model group and Buyang Huanwu Decoction group(18.5 g/kg).Cerebral ischemia model was prepared using middle cerebral artery occlusion method,and drug intervention was given for 14 days.Neurobehavioral score was performed,HE staining was used to observe the morphology of ischemic cortical area of brain tissue,TUNEL staining was used to detect neuronal apoptosis in ischemic cortical area,immunohistochemistry was used to detect the expressions of apoptosis related proteins and Wnt1,glycogen synthase kinase 3β(GSK3β)and β-catenin protein in ischemic cortical area.Results Compared with the same genotype sham-operation group,the neurobehavioral score of the model group mice significantly increased,neuronal cells in the ischemic cortical area showed vacuolar changes,with nuclear condensation and widened intercellular spaces,the apoptosis rate of nerve cells significantly increased,with increased expressions of Bax,GSK3β and decreased expressions of Bcl-2,Wnt1 and β-catenin(P<0.01).Compared with the same genotype model group,the neurobehavioral score of mice in Buyang Huanwu Decoction group were significantly decreased,the pathological damage of the ischemic cortical area improved,the apoptosis rate of nerve cells decreased,the expressions of Bax and GSK3β decreased,and the expressions of Bcl-2,Wnt1 and β-catenin increased(P<0.01).Compared with the WT model group,the KO model group showed an increase in neurobehavioral score,aggravated damage in ischemic cortical area,significantly increased neuronal apoptosis rate,and increased expression of GSK3β(P<0.05).Compared with the WT Buyang Huanwu Decoction group,the KO Buyang Huanwu Decoction group showed an increase in neurobehavioral score,aggravated damage in ischemic cortical area,significantly increased neuronal apoptosis rate,increased expressions of Bax and GSK3β,and decreased expressions of Bcl-2,Wnt1 and β-catenin(P<0.01).Conclusion Buyang Huanwu Decoction can inhibit neuronal cell apoptosis after cerebral ischemia,and its mechanism may be related to regulating the expressions of apoptosis-related proteins by mediating Cav1 to regulate the Wnt signaling pathway.

Objective To explore the mechanism of Buyang Huanwu Decoction against cerebral ischemia-reperfusion injury in rats by regulating lipid metabolism through the cAMP/PKA/PPARγ pathway.Methods 60 rats were randomly divided into sham operation group(Sham),model group(Model),Buyang Huanwu Decoction low-dose group(BHD-L),Buyang Huanwu Decoction medium-dose group(BHD-M),Buyang Huanwu Decoction high-dose group(BHD-H)and Butylphthalide group(NBP).The cerebral ischemia-reperfusion model was prepared by transient middle cerebral artery embolization.The BHD low-,medium-and high-groups were given different doses of Buyang Huanwu Decoction(6.413,12.825,25.65 g·kg-1)by intragastric administration.The NBP group was administered with Butylphthalide(54 mg·kg-1).The sham operation group and the model group were administered with an equal volume of distilled water,all given for 14 days.The rats were subjected to neurobehavioral scoring.HE staining was used to observe brain pathological changes,and the kit was used to detect the levels of phosphocholine(PC),phosphatidylethanolamine(PE),diacylglycerol(DAG),and free fatty acid(FFA)on the ischemic side.RT-qPCR and Western Blot were applied to detect the mRNA and protein expressions of cyclic adenosine monophosphate(cAMP),protein kinase A(PKA),and peroxisome proliferator-activated receptor γ(PPARγ).Results Compared with the sham group,the neurological deficit score was significantly increased(P＜0.01),pathomorphological damage in ischemic cortex was found,the contents of PC and PE were reduced,the contents of DAG and FFA were increased(P＜0.01),and cAMP mRNA expression increased(P＜0.05)in the model group.Compared with the model group,the neurological deficit score of the BHD-L group was decreased(P＜0.05),and the neurological deficit score of the BHD-M,BHD-H and NBP groups was significantly decreased(P＜0.01),the cells in each treatment group were regularly arranged,the intercellular spaces were reduced,and the normal cells were increased.PC and PE were significantly increased,DAG and FFA were significantly decreased(P＜0.01)in the BHD-M,BHD-H and NBP groups.PC was increased,FFA and DAG were decreased in the BHD-L group(P＜0.05,P＜0.01).The mRNA level of PPARγ was increased in the BHD-L group(P＜0.05),and the mRNA and protein levels of cAMP,PKA,and PPARγ were increased in the other treatment groups(P＜0.05,P＜0.01).Conclusion Buyang Huanwu Decoction has a neuroprotective effect on cerebral ischemia-reperfusion injury rats,and its mechanism may be related to regulating the expression of key factors in the cAMP/PKA/PPARγ signaling pathway and lipid metabolism.