川芎-红花药对最佳配比及其抗缺血性脑卒中的作用OA

Objective:This study aimed to investigate the optimal compatibility ratio of the Chuanxiong Rhizoma-Carthami Flos(CR-CF)couplet medicines in ischemic stroke(IS)therapy and its pharmacological action mechanism,providing a scientific basis for the clinical application of CR-CF couplet medicines in IS therapy.Methods:The chemical composition of CR-CF was analyzed using liquid chromatography mass spectrometry(LC-MS/MS).The contents of eight characteristic chemical components in aqueous extracts of CR-CF with common clinical compatibility ratios(1∶1,1∶2,1∶3,3∶2,2∶1)were determined by ultra-high performance liquid chromatography(UHPLC).An oxygen-glucose deprivation/reoxygenation(OGD/R)-induced mouse hippocampal neuron HT22 cell injury model was established,and cells were treated with different CR-CF compatibility ratios.The collaborative index(CI)was calculated by using CompuSyn software.A cerebral artery occlusion/reperfusion(MCAO/R)model of rats was induced by using the modified Longa suture method.The rats were divided into the sham group,model group,Chuanxiong Rhizoma(CR)group(1.3 g·kg-1),Carthami Flos(CF)group(3.9 g·kg-1),CR-CF group(5.2 g·kg-1),and edaravone group(5 mg·kg-1).Neuronal defect scores were assessed by the Longa scoring method.Cerebral infarction volume was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Neuronal damage was observed via hematoxylin-eosin(HE)staining.Neuronal apoptosis of rats was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)staining,and the expression of apoptosis-related proteins was analyzed by Western blot.Label-free proteomics was employed to screen differentially expressed proteins,and Western blot was used to examine the expression of phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway-related proteins.Finally,molecular docking was performed to predict the binding affinity of eight active constituents in CR-CF(1∶3)with PI3K.Results:When CR-CF was combined at a 1∶3 ratio,the total content of the eight active constituents in the extract was the highest,and the synergistic protective effect on OGD/R-injured HT22 cells was the strongest(CI=0.308).Animal experiments showed that compared with the sham group,the model group exhibited increased neuroecological score points(P＜0.01),larger cerebral infarction volumes(P＜0.01),aggravated brain tissue damage,elevated neuronal apoptosis(P＜0.01),and increased B-cell lymphoma-2(Bcl-2)-associated X protein(Bax)/Bcl-2 and cleaved Cysteinyl aspartate specific proteinase-3/Cysteinyl aspartate specific proteinase-3(cleaved Caspase-3/Caspase-3)ratios(P＜0.01).Compared with the model group,CR-CF(1∶3)significantly reduced neurological scores(P＜0.01),significantly decreased cerebral infarction volume(P＜0.01),alleviated brain tissue damage,inhibited neuronal apoptosis(P＜0.01),and significantly lowered the Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 ratios(P＜0.01).The therapeutic effect of CR-CF(1∶3)was superior to that of CR or CF alone.Proteomic analysis revealed that CR-CF(1∶3)activated the PI3K/Akt signaling pathway.Validation experiments demonstrated that compared with the sham group,the model group showed obviously reduced p-PI3K/PI3K and p-Akt/Akt(P＜0.05).Compared with the model group,p-PI3K/PI3K and p-Akt/Akt ratios(P＜0.05)obviously increased.Compared with the CR-CF group,the 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one LY294002 inhibitor+CR-CF group exhibited obviously decreased p-PI3K/PI3K and p-Akt/Akt(P＜0.05).Molecular docking results indicated that the active constituents of CR-CF(1∶3)had strong binding affinity with PI3K.Conclusion:The CR-CF couplet medicines at a 1∶3 ratio exhibit optimal synergistic effects,and their anti-IS mechanism is closely related to activation of the PI3K/Akt signaling pathway and inhibition of neuronal apoptosis.