Apigenin Mediated Protection Of OGD-evoked Neuron-like Injury In Differentiated PC12 Cells

ObjectiveIschemic stroke has been confirmed to cause neuronal disorganization injury and cerebral infarcts due to its insufficient supply of glucose and oxygen then reperfusion to brain tissue. With the deep development in "Toxin impaired brain meridian" theory of Modern Traditional Chinese Medicine (MTCM) and oxidative stress pathogenic mechanismon ischemic stroke of West, there was the same or similar realization in the toxicity effect during oxidative stress and the endogenous toxic materialpathopoiesia of MTCM. Moreover, finding the efficient, low toxicity antioxidant from the natural plants is current tendency to treat ischemic cerebrovascular disease via inhibition of oxidative stress.Apigenin (4',5,7-trihydroxyflavone), a common plant dietary flavonoid found at high levels in Apium graved ens L. var. dulce DC, especially with the presence of 2,3-double bond on the C ring and free phenolic hydroxyl groups (OH groups), exhibits a lower oxidation potential, a lower dissociation enthalpy and a higher radical scavenging capacity. Recent studies found that apigenin has enormous potential in central nervous system protection, based on these observations, we hypothesized that apigenin may protect neuronal cells from ischemic injury. This compound has been discovered to protect neurons from the injury in middle cerebral artery occlusion in rat. However, mechanisms responsible for the protective effects of apigenin on ischemia-induced neuronal damage have been little understood, so it's necessary to study in deep.Oxygen and glucose deprivation (OGD)/reperfusion (R) is a widely used in vitro model for mimicing stroke, this study was to investigate the potential mechanisms underlying the neural protection of apigenin on OGD/R induced neuronal injury in differentiated PC12 cells, and provide a new clue and evidence for developing natural antioxidant in stroke treatment.Methods1 The effect of apigenin on OGD/R induced PC12 cells neuron-like injury.PC12 cells were differentiated into neuron-like cells by nerve growth factor (NGF) treatment, and then exposedto OGD/R to establish the mimetic ischemic stroke model in vitro. After the cells were pretreated with apigenin in appropriate time, we obversed and evaluated the protective effect of apigenin and its optimal concentration by morphological observation, MTT and LDH methods.2 Apigenin influenced the biochemical criterion in the OGD/R-treated PC12 cells.We analyzed the effect of apigenin on apoptosis induced by OGD/R via Hoechst staining and AnnexinV-FITC/PIstaining in flow cytometry. Moreover, the apigenin potential radical elimination on ROS level, which is the representation for oxidative stress, was examined by DCFH-DA fluorescent probe, while the Mitochondrial Membrane Potential (MMP), which is the representation for mitochondrial health, was detected by JC-1 fluorescent probe.3 The antioxidative mechanism about apigenin protecting the OGD/R injured PC12 cells.In order to explore the probable antioxidative mechanism of this compound further in deep, we inspected the transcription and translation level of transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2)and its downstream targetsSOD, GSH-Px, CAT, NQ01 and HO-1 in mRNA levels.4 The antiapoptotic pathways about apigenin protecting the OGD/R injured PC12 cells.To indicate the underlying antiapoptotic mechanism of apigenin, we also observed its effect on mRNA and protein expression of P53 tumor suppressor, BAX, BCL-2 and gene transcription of the P53 downstream targets P21, PUMA, BCL-2, BAX.ResultsAfter being differentiated, PC12 cells became into polygonal shape and the sympathetic neuron-like synapses increase to form a network in a stable status. The results showed that oxygen and glucose deprivation/reperfusion (OGD/R) signi fi cantly decreased cell viability, mitochondrial membrane potential, mRNA levels of antioxidant and detoxifying enzymes and Nrf2 protein, mRNA expression, while elevated the release of LDH, cell apoptosis, intracellular ROS level, P53 protein and gene expression and upregulated its downstream genes in PC12 cells. However, apigenin in 1?10?20? Meffectively inhibited these undesirable changes induced by OGD/R.ConelusionIn summary, the experimental results indicate that apigenin exerts its significant activities against OGD/R-induced neuronal injury in NGF-differentiated PC12 cells. Furthermore, the antioxidative effect of apigenin are probably related to its oxygen radical absorbance capacity, which may be essential in increasing Nrf2 activation, enhancing expression of Nrf2-regulated cellular protective genes to maintain mitochondrial redox state and the function of cells free radical defense system. However, the results also imply that the anti-apoptotic effect of apigenin are related to its capability of regulating P53 expression and its downstream targets. With the complicated relationship between Nrf2 pathway and P53 pathway, it suggests that apigenin maybe regulate the P53 signal by activating to Nrf2 pathway, stabilizing mitochondrial function to fight against the neuronal apoptosis in OGD 12 h/R 24 h.The results are benefit to demonstratethe pharmacodynamic material basis and mechanism of apigenin, finding thatit maybe has the potential to be developed as a neuroprotective assistant agent to provide a good therapeutic strategy for the cerebral ischemia. In brief, this study is helpful to provide new insight into development and application of new type natural antioxidants.