Neuroprotective Effects And Its Mechanism Of STVNa On NF-κB Signaling Pathway

Cerebrovascular diseases,as a leading"killer"endangering human health,are one of the three major diseases causing the highest lethality and disability rates globally.Currently,most treatments for cerebral ischemia are focused on thrombolysis and neuroprotection,and clinical therapeutic drugs are fairly limited,among which thrombolytic r-tPA is used most widely.However,this drug is restricted to relatively narrow time window and potential bleeding injury risk,and thus,its target patients are relatively few.Hence,it is particularly urgent to develop neuroprotective agents meeting clinical needs.In recent years,it is found in studies on neuroprotective agents that some natural or synthetic substances provide good protection for ischemic brain issues.Isosteviol,a tetracyclic diterpene compound with beyerane skeleton,is obtained from the natural sweetener stevioside through acid hydrolysis and has very good protective effect on cerebral ischemia-reperfusion in rats.However,standards used to evaluate pharmaceutical protection roles of this substance are not sound due to its low solubility,poor bioavailability,preventive administration methods,and so on.After years of research and development in our laboratory,isosteviol has been converted into STVNa,a kind of injectable sodium salt,with pharmacological and physiological activity being retained,and this substance is more satisfactory to requirements of clinical administration.In this paper,C57BL/6 mice and N2a cells are selected as study objects,and the specific study contents and results are as follows:1.A study was conducted on the protective effect of STVNa on acute permanent focal cerebral ischemia injury in mice.A stable acute permanent focal ischemic model was built with young male adult C57BL/6 mice,with emphasis being placed on study of the dose-response relationship of STVNa’s protection to permanent cerebral ischemic injury in mice.According to results of the dose-response relationship experiment,in groups which received 15,30,45 and 60 mg/kg STVNa respectively through intravenous injection in the case of ischemia,the cerebral infarction volume and neuroethology,compared with those of the solvent group,demonstrated significant differences（P<0.05）.The comprehensive results of infarct volume and neuroethology reflected that 30 mg/kg STVNa had better effect.Histological analysis revealed that,compared with the solvent group,30 mg/kg STVNa could remarkably improve neuronal necrosis and astrogliosis caused by permanent ischemic injury.In addition,in the study on time window,it was demonstrated that intravenous injection of 30mg/kg STVNa 4 hours after ischemia took place was still protective,with significant decreases in infarct volume and neuroethology compared to those in the solvent group.Thus,in the follow-up experiments aimed at exploring the mechanism,in vivo experiments used 30mg/kg STVNa,with immediate intravenous injection being used for administration after ischemia.2.A study was conducted on neuroprotective effect of STVNa on cells in vitro by building a CoCl₂-induced hypoxia injury model with N2a mice neuroblastoma cell lines.First of all,for the stable chemically induced N2a cell injury model,CCK-8 kit was used to detect the corresponding cell viability.It was found that 300μM CoCl₂ could inhibit about 50%viability of N2a cells.It was comparatively suitable for evaluation of neuroprotective effect of cells in vitro.Through comparison of effects of different STVNa concentrations on N2a cell viability subject to hypoxia injury,it was revealed that 20μM STVNa had the most significant effect on alleviating hypoxia injury.Therefore,this dose was used in follow-up experiments to study the effect and mechanism.Meanwhile,the experimental study found that when 20μM STVNa was given,it exerted marked impact on improvement of apoptosis and it was also able to reduce the mitochondrial membrane potential injury caused by hypoxia,inhibit the increase of ROS level resulting from the same and NF-κB into the nucleus.3.As the astrocyte proliferation,mitochondrial membrane potential disorder,ROS increase and so on are directly related to NF-κB activation,in the following experiment,this signaling pathway was also studied,with effect of STVNa on midstream and downstream signaling pathways of NF-κB being studied on gene and protein levels by combining in vitro and in vivo approaches together,so as to understand the mechanism of action.According to the experimental study,for in vivo experiment,30 mg/kg STVNa could significantly reduce the mRNA and protein levels of IKK,NF-κB,TNF-α,IL-1β,Bax and Caspase-3 and raise the mRNA and protein expression levels of anti-apoptotic factor Bcl-2.For in vitro experiment,it was further verified that 20μM STVNa could also remarkably bring down the mRNA and protein levels of IKK,NF-κB,TNF-α,IL-1β,Bax and c-Caspase-3 and increase the mRNA and protein expression levels of anti-apoptotic factor Bcl-2.The experimental results indicate that one of the action mechanisms of STVNa is to inhibit the activation of NF-κB and further affect the expression of downstream inflammation and apoptosis-related factor.4.According to Part 3,it was revealed that STVNa exerted neuroprotection by inhibiting the activation of NF-κB.So,which is the specific upstream factor affecting this pathway?Through screening of NF-κB-related mi RNAs,it was discovered that miR-181b had an obviously downregulated expression level in brain tissues of mice with permanent cerebral ischemia and CoCl₂-induced N2a cell hypoxia.To explore the role of miR-181b in CoCl₂-induced N2a cell injury,48h after pre-NC,pre-miR-181b,si-NC,and si-miR-181b were used to transfect cells,QPCR results showed that pre-miR-181b and si-miR-181b could significantly increase and decrease the expression level of miR-181b in N2a cells respectively,while other groups were not impacted.After CoCl₂ induced N2a hypoxia,the results of CCK-8 and Tunel indicated that the overexpression of miR-181b could significantly increase the degree of N2a injury induced by CoCl₂,while the interference of miR-181b could enhance the cell viability and decrease the apoptosis after hypoxia of cells.This result verifies that miR-181b aggravates Co Cl₂-induced ischemic injury of N2a cells.Then,bioinformatics was applied to predict that miR-181b might regulate CYLD.The results of QPCR and Western blotting showed that with the overexpression of miR-181b,CYLD mRNA and protein expression levels were significantly decreased;after expression of miR-181b was inhibited,mRNA and protein expression levels were increased significantly,which proves that CYLD is the direct target of mi R-181b.Later,a study was conducted on the action mechanism of STVNa upstream on cellular level.With hypoxia injury of N2a cells being induced by CoCl₂,miR-181b was downregulated significantly and the gene and protein expression levels of CYLD were increased remarkably when 20μM STVNa was administered;the mediated downstream inflammatory and apoptotic factors of NF-κB were also inhibited substantially.Conclusions:STVNa had significant protective effect on acute permanent focal cerebral ischemia in mice and chemically-induced N2a cell hypoxia.Since CYLD is the direct target of miR-181b and miR-181b can regulate IKK directly,the specific action mechanism of STVNa may be to upregulate the expression level of CYLD and inhibit the activation of downstream NF-κB signaling pathway by downregulating miR-181b,and thus inhibit the decline of nerve cells,reduce hyperplasia of astrocyte proliferation,enhance cell viability,mitigate apoptosis,improve mitochondrial membrane potential disorders,and reduce the expression of ROS,with neuroprotective effect of anti-inflammation and anti-apoptosis being exerted.