Ferulic Acid Attenuates Brain Microvascular Endothelial Cells Damage Caused By Oxygen-Glucose Deprivation Via The Punctate-mitochondria-dependentmitophagy

BackgroundVascular dementia (VD) is a clinical syndrome characterized by dementia performance cuased by cerebral vascular disease, which accounts for at least 20% of dementia and it is second only to Alzheimer’s disease (AD). With the increasing of VD prevalence year by year, it has a profound significance to study the pathogenesis of VD, create a new drug, prevent the occurrence of VD and improve patient quality of life.Ferulic acid (FA)is one of the effective traditional Chinese medicine effective components, such as angelica sinensis, rhizoma chuanxiong and ferulic, and it owns the role in scavenging free radicals, antithrombotic, inhibiting tumor, antibacterial, anti-inflammatory and so on. Now, a variety of pharmacological effects in ferulic acid for reducing the neurodegenerative disease has attracted more and more attention.ObjectiveThis study is to investigate both protective effect and its possible mechanism of ferulic acid on oxidative injury of brain microvascular endothelial cells (BMEC) induced by oxygen-glucose deprivation (OGD). The OGD was established by BMEC based on low perfusion injury in vitro model.Methods1. The FA protective effects of BMEC cell damage induced by OGD:Cell viability of BMEC was determined by MTT assay. Reactive oxygen species (ROS) generation was assessed by flow cytometry. SOD and MDA generation was measured by kit. Apoptosis-related protein (caspase3 and Bcl-2/Bax) was evaluated by Western blotting.2. The FA effects of BMEC mitochondrial damage in the cell induced by OGD:Antioxidants (NAC) was applicated as a positive medicine to inspect the production changes of mitochondrial ATP, MMP was determined by flow cytometry. Drpl, p-Drpl protein was evaluated by Western blotting. Mitochondria morphology was observed by transmission electron microscope.3. The influence of regulating autophagy of FA:Based on autophagy agonist rapamycin and autophagy antagonist 3-methyladenine and chloroquine, mitochondria morphology was observed by transmission electron microscope, autophagy related protein expression of LC3-Ⅱ was investigated, the changes in MMP was detected, the changes of ROS generation and the content of ATP, SOD and MDA were detected.4. The regulation of mitochondrial fission and fusion can have an impact on autophagy. To study the cell viability and MMP level in OGD, the mitochondrial fission inhibitor mdivi-1, and Drpl siRNA were uesed to investegate mitochondrial fission protein (Drpl, p-Drpl) and autophagy related protein (LC3-Ⅱ).Results1. FA increases cell activity and significantly reversed the oxidative stress state of OGD-treated BMEC.FA significantly reversed the oxidative stress state of OGD-treated BMEC, such as increased viability, decreased levels of ROS and MDA(p<0.01), and increased SOD activity(p< 0.05). In addition, the expressions of apoptosis-related proteins showed no alterations(p>0.05).2. FA protects and recoverys mitochondria function under the condition of OGD.FA significantly reversed the mitochondrial dysfunction of OGD-treated BMEC, such as increased levels of ATP and MMP (p<0.01), declined the Drpl, p-Drpl protein expression. Tubular mitochondria was transferred into swell, roundout and punctate ones under OGD condition, which was showed by transmission electron microscopy (TEM). FA can effectively improve mitochondrial morphology.3. FA protects the damaged cells and mitochondria under the condition of OGD by raising autophagy level.Western blotting showed that FA upregulated the expression of LC3-Ⅱ and mitophagy was also observed by TEM. The effects of FA on mitochondrial damages were enhanced by autophagy agonist rapamycin while inhibited by autophagy antagonist 3-MA and chloroquine, aggravated the mitochondrial dysfunction.4. Tubular mitochondria transferred into punctate one under OGD are the essential condition for FA inducing autophagy occurs.Mitochondrial fission inhibitor mdivi-1 can increased cell viability and MMP level (p<0.01), decreased the expression of Drpl, P-drpl at the same time(p <0.05, P<0.01). Compare with the control group and siRNA control group, Drpl siRNA significantly reduced Drpl,p-Drpl expression(p<0.01). OGD-induced injury resulted in a slight accumulation of LC3-II in BEMC. Administration of FA significantly enhanced LC3-II accumulation in OGD-induced injury. Compare with OGD group, OGD+FA group can significantly increased LC3-II expression (p< 0.01); Compare with OGD+FA group, OGD+FA+Mdivi-1 and OGd+FA+Drpl siRNA groups can significantly reduced LC3-II expression (p< 0.01).Conclusion1.FA can obviously reduce the BMEC damage induced by OGD; 2. FA can obviously improve the BMEC mitochondria injury induced by OGD; 3. Ferulic acid attenuates BMEC damage caused by OGD via the punctate-mitochondria-dependent mitophagy; 4. Mitochondrial fission protein Drpl mediated tubular mitochondria transferred into punctate ones that term of mitochondrial dynamics, which is the necessary conditions of mitochondrial autophagy occurs.