| Objectives:Buyang Huanwu decoction(BHD)has been widely used to treat ischemic stroke(IS)-induced brain injury.Our research group previously screened the potential bioactive ingredients of BHD using cell membrane affinity chromatography with brain microvascular endothelial cells,PC12 cells,platelets and red blood cells as stationary phase.The results showed that calycosin-7-O-β-D-glucoside(CG)and paeoniflorin(PF)had good anti-ischemic effect on these cell models of oxygen glucose deprivation/reperfusion(OGD/R).However,it is not clear whether CG and PF have anti-IS effects in vivo.Based on this,this study intends to explore the protective effect of CG combined with PF on IS,and to explore the mechanism of promoting recovery after IS based on axon regeneration,which may help to verify the bioactive compounds of BHD.Methods:1.Dosage ratio determination of CG and PFA high-performance liquid chromatography(HPLC)method was established to determine the content at ion of CG and PF in BHD.The chromatographic conditions were:Column:Uranus C18 Column(250×4.6 mm,5 μm);Mobile phase:acetonitrile(A)-water containing 0.2%formic acid(B);Gradient elution:0~20 min,20%A→0%A,20~23 min,40%A~20%A,23~30 min,20%A;Detection wavelength:230 nm;Flow rate:1 mL/min;Column temperature:25℃;Injection volume:10μL.Three batches of Buyang Huanwu decoction solutions were extracted and analyzed.The peak areas of CG and PF were recorded,and the concentration and content of which were calculated.Each set of experiment was performed three times and the average content was taken as the final content.2.Pharmacodynamic study on the combination of CG and PF to promote IS recoveryA model of middle cerebral artery occlusion(MCAO)in rats was established and randomLy divided into eight groups with six animals in each group:sham group,model group,CG group,PF group,CG+PF low-dose group,CG+PF meddle-dose group,CG+ PF high-dose group and nimodipine group.A modified nerve function score experiment and a balance beam experiment were performed every day after CG+PF administration to observe the neurological recovery of MCAO rats.Seven days after the administration,rat brain tissues were removed,and TTC staining was used to observe cerebral infarction;Brain water content was measured to observe cerebral edema;HE staining and Nissl staining were performed to observe the brain tissue morphology and neuronal damage.3.Study on the underlying mechanism of CG combined with PF to promote axonal regenerationMCAO model in rats was established and randomLy divided into six groups with eight animals in each group:sham group,model group,CG+PF low-dose group,CG+PF middle-dose group,CG+PF high-dose group and fasudil group.Seven days after the administration,brain tissues were obtained.Axonal degenaration was observed by Bielschowsky’s silver staining.The expression level of NF-200,MAP2,GAP43,GFAP and CSPGs was observed by immunofluorescence staining.The mRNA and protein expression of RGMa,Rho,ROCK2,MLC2 and CRMP2 related to Rho/ROCK signaling pathway were detected by western blot and real-time quantitative PCR.The regeneration of myelin sheath was observed by Luxol fast blue staining.Results:1.Dosage ratio determination of CG and PFIn the established HPLC method,the standard curve of CG was Y=34722 X+7296.8,and its detection range was 10.1~151.5 μg/mL;the standard curve of PF was Y=116065 X+758568,and the detection range was 10~200μg/mL.The reproducibility,precision,stability,accuracy and specificity all met the content determination requirements.The average content of CG in the three batches of Buyang Huanwu decoction sample was 20.68 mg/111g herbs,and PF was 32.59 mg/111g herbs.The content ratio of the two compounds was about 1:2.2.Pharmacodynamic study on the combination of CG and PF to promote IS recoveryCompared with the model group,after seven days intragastric administration of CG combined with PF,low-dose,middle-dose and high-dose significantly reduced the cerebral infarction volume and brain water content of MCAO rats(P<0.05),while the effect of middle-dose group was the most obvious(P<0.05).The pathological results showed that the cortex and hippocampal pathological morphology were improved and the number of neurons necrotic were reduced after middle-dose and high-dose administration(P<0.05).Also,neurologic function of the MCAO rats was improved(P<0.05),and the effects became more obvious with longer administration time,while lose-dose group could not promote neural function of the rats.Besides,CG and PF administration for 7 days,respectively,could not reduce the cerebral infarction rate and brain water content of MCAO rats,nor could it improve neural function and pathological damage.3.Study on the underlying mechanism of CG combined with PF to promote axonal regenerationCompared with the model group,combined administration of CG and PF of middle-dose and high-dose significantly reduced axonal degeneration of MCAO rats,and increased the expression of axon growth-related factors:NF-200,MAP2 and GAP43(P<0.05),while lose-dose group could only upregulated the expression of NF-200.Middle-dose and high-dose administration group could inhibite the expression of GFAP and CSPGs.Further research results showed that CG combined with PF inhibited the mRNA and protein expression of RGMa,Rho,ROCK(P<0.05),and reduced the phosphorylation level of MLC2 and CRMP2(P<0.05).LFB staining results showed that CG combined with PF could also promote myelin regeneration after brain injury,and the effect of middle-dose and high-dose were betterConclusions:Combined administration of CG and PF is capable of reducing cerebral infarction and cerebral edema,as well as the morphological damage and neuronal apoptosis of brain tissue caused by ischemia.In addition,combined administration of CG and PF can reduce axonal regenaration,promote axon regeneration,and inhibite astrocyte proliferation and secretion of axon inhibitor,thereby improving neural function after IS,which may be related to the inhibition of Rho/ROCK signaling pathway. |
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