Mechanism Studies Of Cyanidin Against Neurotoxicity Through Regulation Of Oxidative Stress And Apoptosis

Part1. Mechanisms studies of cyanidin against cisplatin-induced neurotoxicity in PC12 cells through regulation of oxidative stress and apoptosisBackgroundCisplatin as a first-line chemotherapy drug was common used in clinical for treating a variety of human cancers. However, its application was seriously limited because of its neurotoxicity and other serious side effects. Oxidative stress plays an important role in the neurotoxicity induced by cisplatin. and its mechanism is not very clear.Many studies indicated that cisplatin induced peripheral and central nervous system toxicity mainly because of the drug caused-oxidative damage, which induced ROS scavenging barrier, generation and excess accumulation of ROS, then caused DNA damage, inflammation and mitochondrial dysfunction. Cyanidin as a natural flavonoid compound exhibits powerful antioxidant activity. Hence, we investigated the protective effects of cyanidin in PC12 cells against cisplatin-induced neurotoxicity and explored the underlying mechanisms.ObjectiveTo establish the PC 12 cell model of cisplatin-induced neurotoxicity, and then to explore the effect of cyanidin and its mechanism in the process.MethodsPC 12 cells were seeded into 96 well plates,2.5-40 ?M of cisplatin or 10-80 ?M of cyanidin was incubated for 24 h, and the treatment group with cyanidin was treated by cisplatin 3 hours later. Cell viability was detected by MTT method, cell apoptosis and cell cycle distribution were detected by flow cytometry. Cells apoptotic fluorescent images were collected by TUNEL-DAPI staining. Caspase-3 activity and ROS content were tested by multifunctional enzyme mark detecting instrument after adding the probe substrate. The protein content of PARP?caspase-3 P53 and Ser139-H2A was detected by Western blot analysisResultsPC12 cell death and morphological changes were significantly reduced after cyanidin pretreatment. TUNEL-DAPI nuclear staining showed that cyanidin pretreatment could inhibit cisplatin-induced PC12 cells apoptosis, cell division and chromosome condensation.Cyanidin reduced Caspase-3 activation and PARP clevage in PC12 cells induced by cisplatin, and significantly reduced the expression levels of Ser139-H2A and p53 by inhibiting ROS production.ConclusionsOur findings revealed that cyanidin as an apoptotic inhibitor effectively reduced the Sub-Gl peak, PARP clevage, and activation of Caspase-3 and blocked cisplatin-induced neurotoxicity through inhibition of ROS-mediated DNA damage and apoptosis, which predicating its therapeutic potential in prevention of chemotherapy-induced neurotoxicity.Part 2. Protective effects and mechanism of cyanidin against A?-induced neurotoxicity in PC12 cellsBackgroundA?-induced oxidative damage is believed to be the major pathological feature of Alzheimer's disease. Oxidative damage induced by A? caused ROS accumulation, then lead to DNA damage, which will result in cell apoptosis and neurological deficit. Inhibition of A?-induced oxidative damage is considered to be an effective treatment against AD. Drugs with anti-oxidative damage activity have great potential in the treatment of neurodegenerative diseases.Cyanidin is a natural flavonoid compound shows multiple pharmacological properities against oxidative damage-mediated neurodegenerative disorders. Studies have shown that cyanidin can remove excess ROS produced by the body, and antagonize ROS-induced DNA damage, which can effective inhibit oxidative damage mediated neurodegeneration. However, the specific mechanism of this pharmacological effect is not clear, so we studied the protective effect of cyanidin against A?-induced neurotoxicity of PC 12 and its possible mechanism.ObjectiveTo establish the A?-induced neurotoxicity model of PC 12 cells, and to explore the protective effect and mechanism of cyanidin in this process.MethodsPC 12 cells were seeded into 96-well plates, A?25-35 was dissolved with PBS and incubated at 37? for 3 days. The cells were treated with the cyanidin (20,40,80 ?M) for 24 h or/and (10 uM) for another 24 h. Cell viability was detected by MTT method, and TUNEL-DAPI staining was used to detect the DNA fragmentation. JC-1 probe were used for detecting of mitochondrial membrane potential (??m). Ac-DEVD-AMC substrate and DCFH-DA method were used to analysis the Caspase-3 activity and ROS accumulation. Bax, Bcl-2, Ser139-H2A, p53 protein level were detected by Western blot.ResultsThe results showed that cyanidin pretreatment significantly attenuated A?-induced cell mortality and morphological changes in PC 12 cells. TUNEL-DAPI nuclear staining showed that cyanidin pretreatment can reduce the cisplatin-induced PC 12 cells apoptosis, chromosome condensation and reduce the Caspase-3 activity. Cyanidin pretreatment significantly restored the mitochondrial membrane potential through regulation of Bcl-2 family expression. Moreover, cyanidin pretreatment significantly blocked Ap-induced ROS accumulation, and inhibited the phosphorylation level of Ser15-p53 and Ser139-H2A in ciaplatin-treated cells.ConclusionsThe results revealed the evidences that cyanidin can regulate the expression of Bcl-2 family protein, restore the mitochondrial membrane potential to effectively block the A?-induced apoptosis of PC 12 cells. Cyanidin also suppresses A?-induced cytotoxicity by inhibition mitochondria-mediated apoptosis through suppressing ROS-mediated oxidative damage, which validated the therapeutic potential of cyanidin in the prevention of oxidative stress-mediated neurotoxicity.Part3. Protective effects and mechanism of cyanidin against photochemical-induced focal cerebral ischemia in miceBackgroundOxidative stress plays an important role in ischemic stroke, and its mechanism is not very clear. Many studies confirmed that central nervous system injury caused by cerebral ischemia related to body stress caused oxidative damage after ROS scavenging barrier, generation and accumulation which induced DNA damage, inflammation and mitochondrial dysfunction, and finally resulted in cell apoptosis. Cyanidin as a natural flavonoid compound shows multiple pharmacological properities against oxidative damage. Therefore, we investigated the protective effects of cyanidin against the neurotoxicity induced by ischemic stroke and to explore the possible mechanisms.ObjectiveTo establish the model of focal cerebral ischemia induced by photochemical method in mice, and to explore the protective effect and mechanism of cyanidin in this process.MethodsThe regional cerebral blood flow was measured by Laser Doppler, neurological function and behavior score was also evaluated. The infarct volume was measured by TTC staining, apoptotic fluorescent images was examined by TUNEL-DAPI staining. Brain tissue oxidative stress indicators such as ROS, SOD, MDA activity assay were detected with a multifunctional enzyme marker. Intracellular PARP, Caspase-3, and HO-1?Nrf-2 protein content was detected by Western blot.ResultsCyanidin pretreatment significantly increased the local cerebral blood flow, improve neurological function and behavioral score, infarct size and cerebral edema. TUNEL-DAPI staining showed that cyanidin pretreatment can reduce the cell apoptosis in the cerebral ischemia mice. We also found the reduced activation of Caspase-3 and PARP cleavage. Moreover, cyanidin could also inhibit the generation of ROS, superoxide dismutase (SOD) expression and the content of MDA. The expression level of HO-1?Nrf-2 were found up-regulation.ConclusionsThe results revealed the evidences that cyanidin pretreatment has obvious protective effect on focal cerebral ischemia in mice. The main mechanisms may be related to that cyanidin could reduced ROS accumulation and lipid peroxidation, which enhanced the body's antioxidant capacity and reduce DNA damage and apoptosis. We can come to the conclusion that cyaniding as a natural antioxidant and inhibitor of apoptosis, it has great potential in the prevention and treatment of ischemic stroke.