Study On The Mechanism Of Butylphthalide Mediating CXCR4 In Cerebral Ischemia-reperfusion Injury

Objective: The specific mechanism of dl-3-n-butylphthalide(dl-NBP)in the treatment of cerebral ischemia-reperfusion injury(CIRI)remains unclear.In this study,we investigated the anti-apoptotic and anti-ferroptosis death effects of dl-NBP through in vivo experiments,and the protective effect of dl-NBP on CIRI was further investigated by in vitro experiments to determine whether dl-NBP mediates chemokine receptor 4(CXCR4).To reveal the pharmacological effects and new targets of action of butylphthalide and provide new ideas in the treatment of cerebral ischemia-reperfusion injury.Methods: 1.Animal experiments: Adult male Sprague-Dawley(SD)rats,weighing 220-240 g,were used to establish transient middle cerebral artery occlusion(t MCAO)model to induce ischemic stroke(IS)and simulate CIRI in vivo.SD rats were divided into 3 groups randomly,which were Sham operation group(Sham),model group(t MCAO)and administration group(dl-NBP + t MCAO).The balance beam is 150 cm long,2.5 cm wide and 1m high from the ground.All rats underwent beam training twice a day for 3 days before modeling.The dosing group was injected intraperitoneally with 80 mg/kg dose of dl-NBP 2 hours after the successful establishment of the model for 7 days.After 7 days of administration,Modified neurological severity score(m NSS)and corner test were performed to assess the degree of neurological deficits in rats.After completing the above behavioral evaluation,the brains were removed intact,cut into 2-mm-thick brain slices,the cerebral infarction volume of rats in each group was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining method,and the changes of brain water content were detected by dry-wet weight method.Protein immunoblotting assay(Western blot,WB)was used to detect the expression of Bax,Bcl-2,and sheared Caspase-3(Cleaved Caspase-3),which are apoptosis marker proteins,in rat brain tissue,and ferroptosis marker proteins ACSl4,GPx4 and XCT was assessed by Western blot(WB),also.2.Cell experiments: Highly differentiated rat adrenal chromaffin cells(PC12)have sympathetic neuron characteristics and are catecholaminergic cells,so they are widely used in the in vitro study of nervous system diseases.In this study,PC12 cell line was selected to establish an in vitro oxygen glucose deprivation / reoxygenation(OGD / R)model to mimic CIRI.The cells were divided into 0 h group,2 h group,4 h group,6 h group,8 h group and 12 h group to determine the duration of glucose and oxygen deprivation.Cell viability was measured at the above different time points using a cell counting kit(Cell counting kit-8,CCK-8).The cells were then divided into Sham group,OGD/R group,0.1 u M dl-NBP+OGD/R group,1 u M dl-NBP+OGD/R group,10 u M dlNBP+OGD/R group and 100 u M dl-NBP+OGD/R group.Cell viability was detected by CCK-8 to determine the concentration of dl-NBP in vitro.To determine the in vitro effect of dl-NBP,cells were divided into Sham group,OGD/R group and dl-NBP+OGD/R group,and the percentage of apoptotic cells contained in each group and the expression level of apoptotic marker protein were detected by flow cytometry and immunoblotting assay.Lipid oxidation levels and iron content were evaluated by measuring Malondialdehyde(MDA)content and iron ions in cells in each group.Western blotting assay was used to determine the level of iron death marker protein in these groups.In order to investigate whether dl-NBP mediates CXCR4 to play a protective role in CIRI in vitro,cells were pretreated with AMD3100,a CXCR4 inhibitor at different concentrations(1 u M,5 u M,10 u M,20 u M)for 24 h,and the expression of CXCR4 in cells was detected by WB to determine the optimal drug concentration of AMD3100.The cells were divided into Sham group,OGD/R group,dl-NBP+OGD/R group and AMD3100 + dl-NBP + OGD/R group.The apoptosis level of each group was evaluated by flow cytometry,immunofluorescence and Western blot.The death level of iron was evaluated by MDA,iron ion and Western blot.Results: 1.Animal experiments: After modeling,the cerebral infarction volume,brain water content,m NSS score,and the percentage of right turn in the corner test were significantly increased.After 7 days of intraperitoneal injection of dl-NBP,the infarct volume and brain water content were significantly reduced,and the neurological deficits were significantly improved.The results of WB indicated that brain proapoptotic protein Cleaved Caspase-3,Bax,Bax/Bcl-2 and iron death marker ACSL4 were increased significantly in t MCAO group,while anti-apoptotic protein Bcl-2 and anti-iron death protein GPX4 and x CT were decreased significantly.After dl-NBP treatment,Increased Cleaved Caspase-3,Bax,and ACSL4 and decreased Bcl-2,GPX4,and x CT were significantly reversed.2.Cell experiment: CCK-8 results indicated that cell viability decreased by 50% at 6 h of OGD,and the concentration of dl-NBP was 10 um with the best therapeutic effect.After stimulation by OGD/R,Bcl-2 expression level was significantly downregulated,while in contrast to Bcl-2,Cleaved Caspase-3,Bax protein levels and Bax/Bcl-2 ratio were significantly increased.Flow cytometry results suggested that the percentage of early apoptotic and late apoptotic cells was significantly increased in PC12 cells after OGD/R.In contrast,dl-NBP intervention reduced Cleaved Caspase-3,Bax protein levels and Bax/Bcl-2 ratio,and significantly downregulated the proportion of early apoptotic cells.The OGD/R group had significantly increased MDA level,iron ion content and ACSL4 expression,and significantly decreased GPX4 and x CT expression compared to the Sham group.dl-NBP treatment significantly reduced MDA level,iron ion content and ACSL4 expression,and significantly increased GPX4 and x CT expression.Pretreatment of cells with the CXCR4 inhibitor AMD3100 at 20 u M concentration for 24 h resulted in a 50% decrease in CXCR4 levels in cells.Immunofluorescence,WB and flow cytometry results showed that the combination of CXCR4 inhibitor AMD3100 significantly increased the fluorescence intensity and protein expression of Cleaved Caspase-3,elevated Bax protein levels and Bax/Bcl-2 ratio,and significantly decreased Bcl-2 expression,as well as upregulated the proportion of apoptotic PC12 cells compared with dl-NBP administration alone.The results of MDA detection,iron ion content detection and WB experiment showed that dl-NBP significantly reduced the level of lipid oxidation,iron ion content and ACSL4 protein expression,and significantly increased the expression levels of GPX4 and x CT.When CXCR4 was inhibited,the above trends were significantly reversed.Conclusion: 1.dl-NBP protects against cerebral I/R injury through anti-apoptosis and ferroptosis in vivo.2.dl-NBP still exerted anti-apoptosis and anti-ferroptosis effects in vitro.3.The anti-apoptotic and anti-ferroptosis effects of dl-NBP were mediated by CXCR4.