Formononetin Sodium Sulfonate Protects Cerebral Ischemic Injury By Inhibiting Oxidative Stress And Apoptosis

Cerebral ischemia is a cerebrovascular disease that seriously threatens the health of human life.Ischemia and hypoxia may lead to a series of injuries such as inflammatory cascade,release of free radicals,induction of neuronal apoptosis and necrosis,and cerebral edema.In severe cases,limbs may be paralyzed or life-threatening.A variety of factors regulate neuroprotection after cerebral ischemia,thus interventions for cerebral ischemia rehabilitation are also characterized by multiple targets,multiple levels,and multiple channels.Studies have shown that cell death can be reduced by inhibiting oxidative stress and inflammatory responses,and through the inhibition of these processes,the effects of cerebral ischemia-related symptoms can be treated.A number of basic and clinical studies have confirmed that estrogen may have neuroprotective effects on cerebral ischemia by inhibiting the release of excitatory amino acids,anti-inflammatory and anti-apoptosis.Formononetin is an isoflavone phytoestrogens with anti-tumor,hypolipidemic,anti-lipid peroxidation,anti-inflammatory,estrogen and other physiological functions.Sodium formononetin-3'-sulfonate is its sulfonated derivative which greatly improves water solubility.Studies have shown that Sul-F has neuroprotective effects on cerebral ischemia,but its specific mechanism is still unclear.Therefore,we speculated that Sul-F may exert protective effects through anti-inflammatory,anti-oxidation and anti-apoptosis pathways.This study explored the protective effects of Sul-F on cerebral ischemic injury and related molecular mechanisms from both in vivo and in vitro studies.Firstly,a permanent focal cerebral ischemia model was established in C57BL/6 mice.The neuroprotective effect of Sul-F on cerebral ischemic injury was confirmed by neurological function score,cerebral infarction volume and activation of microglia.Secondly,a hypoxia model of PC 12 cells was established,and the molecular mechanism of Sul-F protecting cerebral ischemic injury through anti-oxidative stress and apoptosis was revealed by immunofluorescence and Western blotting.Results:1.Sul-F significantly attenuated focal cerebral ischemic injury in mice.The C57BL/6 male mice were intraperitoneally injected with Sul-F at a dose of 10 mg/kg for seven consecutive days.The pMCAO model was prepared and the saline group was used as a control.Neurological function,cerebral infarct volume and activation of microglia in the SGZ region of the ischemic side of the hippocampus were evaluated.The results showed that Sul-F significantly reduced the neurological function score and cerebral infarction volume after cerebral ischemia,and inhibiting the activation of microglia.2.Sul-F significantly inhibited the decrease of PC 12 cell viability caused by hypoxia.The normal cultured PC 12 cells were pretreated with 0.1-1280?M of Sul-F for 24h,and the cytotoxicity was examined by MTT assay.The hypoxia injury model was established by PC 12 cells to screen the optimal hypoxia injury time point.It was found that when the concentration of Sul-F was more than 160?M,it was toxic to cells;when the cells were hypoxic for 48h,the survival rate was about 50%,which is suitable for subsequent experiments as a model.PC 12 cells were pretreated with 0.1-160?M Sul-F for 24h,and hypoxic for 48h.Normal cultured cells were used as control group,and cell viability was detected by MTT assay.The results showed that Sul-F significantly increased the survival rate of cells after hypoxia At the concentrations of 20 and 40?M,the survival rate was the highest.Therefore,20?M was selected as the dosing concentration of the subsequent experiments.And the release of LDH indicated that Sul-F significantly inhibited cell damage caused by hypoxia.3.Sul-F significantly inhibits oxidative stress caused by hypoxia.Normally cultured PC 12 cells were pretreated with 20?M Sul-F for 24h,and ROS levels were measured after hypoxia for 12h,24h and 48h.The normal cultured PC 12 cells were pretreated with 20pM Sul-F for 24h,and the levels of MDA and MMP were detected after 48h of hypoxia.The results of ROS,MDA and MMP showed that Sul-F could significantly decrease the level of ROS after hypoxia for 12h,and the level of MDA decreased significantly after 48h of hypoxia,and the mitochondrial membrane potential increased significantly.After hypoxia,the ROS content in the cells increased,the MDA production increased,and the mitochondrial membrane potential decreased.Sul-F treatment significantly inhibited the production of ROS and MDA and the decrease of mitochondrial membrane potential induced by hypoxia.4.Sul-F significantly inhibits apoptosis induced by hypoxia.The normal cultured PC 12 cells were pretreated with 20?M Sul-F for 24h.After 48h hypoxia,Hoechst/PI staining was used to detect the cell death level,and Western blot was used to detect the expression of apoptosis-related proteins and the changes of AKT/ERK signaling pathway.It was found that Sul-F significantly reduced the cell death caused by hypoxia and the expression of Cleaved Caspase-3,Bax and Cleaved PARP.At the same time,it was found that Sul-F significantly inhibited the decrease of Akt/Erk phosphorylation caused by hypoxia.Conclusion:1.Sul-F exerts significant neuroprotective effects on cerebral ischemic injury in mice,which is characterized by the decrease in neurological function score and cerebral infarction volume and the inhibition of activated microglia.2.Sul-F reduced the level of oxidative stress by regulating the AKT/ERK signaling pathway,further inhibiting the occurrence of apoptosis,thereby achieving neuroprotection.