Protection Of C-p On Hypoxia/reoxygenation Injured Astrocytes And Its Mechanism

BackgroundLyophilized Powder of catalpol and puerarin(C-P) is a compound injection developed by our team, composed of Chinese herbal monomer catalpol and puerarin. It aims to be used in the treatment of ischemic stroke. C-P was found to have neuroprotection on the model of ischemic stroke in our previous experiments in vivo and in vitro. But the underlying mechanism was still unclear. In this study, astrocytes isolated from cerebral cortex in SD rats were suffered from hypoxia/reoxygenation injury to simulate ischemic stroke. The neuroprotection and its molecular mechanism of C-P on the damaged astrocytes were evaluated. This work was supported by the National Natural Science Foundation of China(31402237, 81473549), the Ministry of Education Doctoral Program Special Fund(20110182110012), the Key Projects of Chinese Medicine Research of Chongqing Municipal Health Bureau(2010[60] 2010-1-4), the Fundamental and Front Research Funds of Chongqing(CSTC2014jcyjA80023), and the Fundamental Research Funds for Central Universities(XDJK2014C058).ObjectiveTo evaluate the neuroprotection of C-P on astrocytes isolated from cerebral cortex in SD rats which suffered from hypoxia/reoxygenation, and clarify its underlying molecular mechanism, so that providing experimental basis for the development of C-P as a new medicine.Methods1. The primary cultivation and identification of astrocytes isolated from cerebral cortex of SD ratsThe cerebral cortex was isolated from SD neonatal rats(2-3 d) in a sterilized condition. After the digestion and filtration, the cell suspension was transferred to a sterilized bottle to cultivate the astrocytes with DMEM-F12 medium containing 10% FBS. When it grew to fusion, the cell was oscillated at a speed of 220 rpm for 18 h in a shaker. Using FITC labelled GFAP and DAPI, the astrocytes were identified by immunofluorescence. The purity of cell was analyzed with Image-Pro Plus 6.0.2. Establishment of hypoxia/reoxygenation injury model of astrocytes and the protection of C-PThe experiment was divided into normal group, model group, excipients group(β-CD) and three groups of C-P(12.25 μg·mL-1, 24.50 μg·mL-1, 49.00 μg·m L-1). Each group set up 6 repeated holes. The establishment of hypoxia/reoxygenation injury model of astrocytes was conducted according to the previous method, and hypoxia 6 h/reoxygenation 12 h was chosen as damage condition. At the beginning of hypoxia and reoxygenation, excipients group and C-P groups were administrated for once respectively. The inverted microscope was used to observe the morphologic change of cell morphology before and after the damage. After the reoxygenation, MTT and LDH leakage assays were used to test the survival rate and the damage degree of cell membrane respectively. Meanwhile, TUNEL fluorescent staining combining with DAPI were used to detect the apoptosis rate of astrocytes. Western blot was used to determine the protein expression level of caspase-9, caspase-3, and PARP-1.3. The research of the inhibition of C-P on inflammatory response of the hypoxia/reoxygenation injured astrocytes and its mechanismAt the end of the reoxygenation, the released amount of NO in medium of each group were measured with the method of Nitrate reductive enzymatic. Whereas the amount of TNF-α, IL-1β, and PGE2 released in medium were tested by ELISA. Western blot was used to determine the expressions of iNOS, COX-2, NF-κB p65, p-NF-κB p65, IκB-α, pIκB-α and p-IKKα/β in astrocytes.4. The research of the inhibition of C-P on oxidative stress of the hypoxia/reoxygenation injured astrocytes and its mechanismAfter reoxygenation, the content of SOD, MDA, and GSH in astrocytes were tested with the method of xanthine oxidase, Glucosinolates barbituric acid chromogenic, and Disulfide generation 2 nitro benzoic acid chromogenic respectively. The fluorescence intensity of DCFH-DA was examined with microplate system to reflect the accumulation of ROS in the cell. Meanwhile, the protein expression of Nrf-2 and HO-1 in astrocytes were determined by western blot.Result1. The primary cultured astrocytes was isolated from cerebral cortex of SD rats successfullyThe planted astrocytes completed adherence within 24 h. 2 days later, the cells proliferated gradually and had the fastest proliferation speed at the 3-4th d. At the 5th d, the cell number further increased, and the cells covered the bottom of bottle at the 6th d. After that, the cells were shaken for purification. The result of immunofluorescence identification using FITC labelled GFAP combining with DAPI verified that the cells as astrocytes. Cell purity was more than 97%.2. C-P showed a protection on the hypoxia/reoxygenation injured astrocytes Compared with the normal group, the cell survival rate significantly decreased, while the LDH leakage rate markedly increased in the model group(P<0.01). The soma of cell shrinked. Part of the cells fell down from the wall and the number of the cell decreased. The cell damaged seriously and the glossiness was poor. Meanwhile, the apoptosis rate as well as the expression level of caspase-9 and caspase-3 were all increased notably, and the content of PARP-1 was decreased notably in the model group(P<0.01). Compared with the model group, the cell survival rate increased, while the LDH leakage lowered significantly in groups of C-P 12.25 μg·m L-1, 24.50 μg·m L-1 and 49.00 μg·m L-1(P<0.05). Meanwhile, the cell morphology had a obvious improvement. The cell soma stretched relatively. The density of cell increased markedly, and the glossiness also changed better. Meanwhile, the apoptosis rate as well as the expression level of caspase-9 and caspase-3 were all decreased significantly, and the PARP-1expression had a significantly increase in 12.25 μg·m L-1, 24.50 μg·m L-1 and 49.00 μg·m L-1 groups(P<0.05).3. C-P could efficaciously inhibit the inflammatory response in hypoxia/reoxygenation injured astrocytesCompared with the normal group, the amount of TNF-α, IL-1β, NO and PGE2 released in medium and the expression level of i NOS and COX-2 in the model group were all increased significantly(P<0.01). Compared with the model group, the amount of IL-1β, NO, and PGE2 released in medium and the expression level of i NOS in groups of C-P 12.25 μg·m L-1, 24.50 μg·m L-1 and 49.00 μg·m L-1 decreased markedly(P<0.05). And the amount of TNF-α released in medium and the expression level of COX-2 in C-P 24.50 and 49.00 μg·m L-1 groups also decreased dramatically(P<0.05).Compared with the normal group, the phosphorylation level of IKKα/β, IκB-α and NF-κB p65 in cells of model group had a notably increase(P<0.01). Compared with the model group, the phosphorylation level of NF-κB p65 in cells of C-P 12.25 μg·m L-1, 24.50 μg·m L-1 and 49.00 μg·m L-1 groups were lowered significantly(P<0.05). Meanwhile, the phosphorylation level of IKKα/β and IκB-α in cells of C-P 24.50 and 49.00 μg·m L-1 groups also decreased significantly(P<0.05).4. The C-P could significantly inhibit the oxidative stress induced by the hypoxia/reoxygenation in astrocytesCompared with the normal group, the content of ROS and MDA increased notably, and the activity of SOD and the content of GSH were all lowered markedly in the model group(P<0.01). Compared with the model group, the content of MDA and ROS markedly decreased, and the content of GSH significantly increased in C-P 12.25 μg·m L-1, 24.50 μg·m L-1 and 49.00 μg·m L-1 groups(P<0.05). And the activity of SOD also had a significant increase in C-P 24.50 μg·m L-1 and 49.00 μg·m L-1 groups(P<0.05).Compared with the normal group, the expression level of Nrf-2 and HO-1 was markedly increased(P<0.01). Compared with the model group, the expression level of Nrf-2 and HO-1 in 12.25 μg·m L-1, 24.50 μg·m L-1 and 49.00 μg·m L-1 groups was further raised significantly(P<0.05).Conclusions1. Primary astrocytes were successfully separated and cultivated from cerebral cortex of SD rats, and its purity was more than 97%, which conformed to the requirements of the experiments. 2. C-P showed a protection on hypoxia/reoxygenation injured astrocytes. It could significantly improve the morphological characters, increase the cell survival, lower the LDH leakage of cell, and inhibit the apoptosis rate of astrocytes. 3. C-P could inhibit the occurrence of inflammatory response in hypoxia/reoxygenation injured astrocytes effectively. Through inhibiting the activation of NF-κB signaling pathway, C-P could significantly decreased the release amount of inflammatory cytokine from cell. 4. C-P showed an obvious inhibition on the occurrence of oxidative stress induced by hypoxia/reoxygenation injury in astrocytes. It could effectively suppress the generation of oxygen radical, and promote the production of Peroxiredoxins at the same time. The mechanism may related to that the durg could activate of Nrf-2/HO-1 signaling pathway.