| Ischemic injury has attracted attention in domestic and international research. It has been suggested that some herbal medicines, or their products, may have the functions of improving microcirculation in the brain, protecting against ischemic injury and inhibiting neuron apoptosis. However, they cannot be widely applied in clinical practice due to the ambiguous pharmacological components and the uncertain mechanism. It may explain the growing interest in discovering drugs from traditional Chinese medicine. As one of the major effective ingredients of Rhizoma Chuanxiong, ligustrazine (TMP) has been widely used in the treatment of ischemic injury. To further improve the neuroprotective property of TMP, a novel series of TMP derivatives were synthesized based on traditional Chinese medicine theory and the composition principle. Compound 247 showed protective effect against CoCl2-induced neurotoxicity in differentiated PC12 cells. Therefore, this study aims to further study the neuroprotective effect of compound 247 and the possible mechanism related to apoptosis.Objective:This study aims to explore the effect of compound 247 on differentiated PC12 cells and the possible mechanism related to apoptosis by observing the expression of Bax, Bcl-2, Cyt-C, Caspase-3 and Caspase-9.Methods:1. Effect of compound 247 on cell viability after CoCl2-induced neurotoxicity in differentiated PC 12 cells:Differentiated PC 12 cells were divided into normal group, model group and compound 247 group. PC 12 cells were pretreated with compound 247 at various concentrations (3.75,7.5,15,30,60?M) and then exposed to CoCl2 insult. MTT assay was used to evaluate the effect of compound 247 on PC 12 cells.2. Effect of compound 247 on apoptosis after CoCl2-induced neurotoxicity in differentiated PC 12 cells:AO/EB fluorescent staining was used to observe the apoptosis of PC 12 cells after CoCl2-induced neurotoxicity. Further studies on mitochondrial membrane potential and free calcium level were detected by laser scanning confocal microscope.3. Effect of compound 247 on morphological changes after CoCl2-induced neurotoxicity in differentiated PC12 cells and its mechanism:Cellular morphology was observed with hematoxylin and eosin (HE) staining. The expression of Bax and Bcl-2 was detected by immuno-histochemical analysis.4. Effect of compound 247 on the expression of apoptosis protein after CoCl2-induced neurotoxicity in differentiated PC 12 cells:Western blot was applied to evaluate the expression of Cyt-C and spectrophotometry was used to detect the expression of Caspase-3 and Caspase-9.Results:1. Based on the optical density (OD) values of MTT assay, compound 247 could increase PC12 cell viability after CoCl2-induced neurotoxicity in a dose-dependent manner (EC50=13.02?M).2. Effect of compound 247 (7.5?M,15?M,30?M) on apoptosis after CoCl2-induced neurotoxicity in differentiated PC12 cells:?Compared with model group, cells with the orange fluorescence decreasing in compound 247 groups which indicated that compound 247 inhibited cell necrosis and apoptosis. ?The mean fluorescence intensity of mitochondrial membrane potential in compound 247 group (0.1384±0.0032,0.1458±0.0069 and 0.1553±0.0009) increased significantly compare with the model group (0.0765±0.0093) (P<0.01).?Compared with model group(0.1456±0.0063), the mean fluorescence intensity of free calcium in compound 247 group (0.1108±0.0142,0.1059±0.0113 and 0.0855±0.0155) decreased significantly (P<0.01).3. Observation of morphological changes were as follows: ?Cells in normal group had round cell bodies with fine dendritic networks and intact cell edges, however, apoptosis with nuclear chromatin condensation and cell shrinkage as well as the disappearance of synapse were observed in model group. Cells in compound 247 groups had long synapse and the amount of neurite-bearing cells increased compared with model group.?The immunohistochemical results showed that compound 247 could increase the expression of Bcl-2 and decrease Bax expression compared with model group,4. Effect of compound 247 on the expression of apoptosis protein after CoCl2-induced neurotoxicity in differentiated PC 12 cells: ?Compound 247 decreased the expression of Cyt-C, and the gray value in high dose group of compound 247 reduced significantly compared with model group (P<0.01).?The activity of Caspase-3 in compound 247 groups (1.8921±0.0691,1.5543±0.2311 and 1.1919±0.0741) decreased significantly compared with model group (2.7064±0.1215) (P<0.01). Compound 247 decreased the activitiy of Caspase-9 (3.3011±0.1097,2.8167±0.0822 and 2.6842±0.2279) significantly compared with model group (3.5860±0.1383) (P<0.05).Conclusion:The protective effects of compound 247 against CoCl2-induced neurotoxicity in differentiated PC12 cells are as follows:compound 247 could increase mitochondrial membrane potential by promoting the expression of Bcl-2, reducing Bax expression and decreasing free calcium, so as to inhibit apoptosis. Moreover, compound 247 could decrease the expression of Cyt-C, inhibit the activities of Caspase-3 and Caspase-9, and helps to prevent ischemic injury by blocking mitochondrial apoptotic pathway... |
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