Resolvin D1 Alleviates PC12 Neuronal Cytotoxicity Mediated By Stimulated Microglia Cells And Its Mechanism

Background and ObjectiveAcute and chronic neurodegenerative diseases,(such as cerebral ischemia, brain injury, Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, etc.) are complex diseases which featured of neuronal loss or degeneration. The diseases are incurable and developing over time that can lead to impaired cognitive and sensorial functions and problems in movement. Studies show that inflammation in the central nervous system is an important component of many neurodegenerative diseases. Although the basic mechanism of neuronal loss is not fully understood, it is believed that the activation of microglia plays an important role. Microglia are resident immune cells in the central nervous system whose function is similar to macrophages. They can release inflammatory mediators including pro-inflammatory cytokines to clear the pathogen and the infected cells and restore homeostasis to external stimuli. But below pathology circumstance, too much inflammatory mediators including proinflammatory cytokines can cause chronic inflammation of the nerve and promote a variety of neurodegenerative diseases which eventually lead to the death of nerve cells. Resolvin D1( RvD1) is a new type of pro-resolving lipid mediators, which can play an effective role in many inflammatory diseases.The study selected supernatant of BV-2 microglia cells which had been treated by drugs to culture the neuronal PC12 cells, in order to simulate the interaction between microglia cells and neuronal cells. The study was aimed to observe whether RvD1 can alleviate PC12 neuronal cytotoxicity mediated by stimulated microglia cells and its mechanism. Materials and Methods Cell culture and groupingBV-2 microglia cell lines were seeded on the culture liquid. Cells in Logarithmic growth phase was used in the experiment. Experimental groups: BV-2 cells were divided into control group, LPS group, RvD1+LPS group and RvD1 group. The control group cells were added with 0.038% anhydrous ethanol; The LPS group cells were added with 0.038% anhydrous ethanol,and then added with 100 ng / ml LPS after 30 minutes; RvD1+LPS group cells were firstly pretreated with 100 n M RvD1 30 min and then added with 100 ng / ml LPS; RvD1 group cells were added with 100 n M RvD1. Methods1 BV-2 cells were treated with medicine according to the experimental group, then we collected the supernatant after 24 h.The PC12 cells were respectively cultured with the supernatant of each group for 24 h. We use DAPI staining to observe the apoptosis of PC12 cells.2.CCK-8 assay was used to detect the survival rate of PC12 cells in each group.3 Apoptosis rate of PC12 cells was measured by flow cytometry(V-FITC/PI Annexin Kit).4 We used Enzyme linked immunosorbent assay(ELISA) to detect the content of IL-1β, IL-6, TNF-α in the supernatant of BV-2 cells in each group.5 BV-2 nuclear translocation of nuclear factor kappa B p65(NF- ΚB p65) was detected by immunofluorescence assay in each group.6 Western blot method was used to detect the expression of NF- ΚB p65 protein in the cytoplasm and nucleus of BV-2 cells in each group. Statistical analysisAll data was analyzed with statistical software SPSS 17.0. Measurement data was expressed in mean ± standard deviation(x-± s). Differences among groups were compared with one-way analysis of variance, the two-two comparisons were done by LSD method, andα=0.05 was the significant test level. Results1. DAPI staining showed, compared to the control group, PC12 cells in LPS group showed obvious morphologic characteristics of apoptosis: staining cytoplasm condensed, highly condensed and marginalized; RvD1+LPS group cells also have some cells showed apoptotic morphology, but the number was significantly lower than that of LPS group; There were no statistically significant differences compared RvD1 group with control group.2. CCK-8 results showed that, compared with the control group, we observed decreased cell survival rate in LPS group, the difference was statistically significant(P< 0.05); RvD1+LPS group showed that the cell survival rate was significantly higher than that of LPS group, the difference was statistically significant(P< 0.05); Compared RvD1 group with control group, we did not found significant difference(P> 0.05).3 Harvested PC12 cells were analyzed by flow cytometry after V-FITC/PI Annexin staining. The apoptosis rate in the LPS group was increased than the control group, the difference was statistically significant(P< 0.05), and RvD1+LPS group showed that the cell apoptosis rate was significantly lower than that of LPS group(P< 0.05); Compared RvD1 group with control group, we did not found significant difference(P > 0.05).4.After LPS stimulated BV-2 cells for 24 h, the cell culture supernatant of IL-1, IL-6, TNF-a concentrations in LPS group cells were significantly higher than the control group(P < 0.05); The cell culture supernatant of IL-1, IL-6, TNF-a concentrations in RvD1+LPS group were significantly lower than the LPS group(P < 0.05); Compared RvD1 group with control group, we did not found significant difference(P > 0.05).5. Immunofluorescence images showed that, compared with the control group, the NF-κB p65 entering the nuclears in LPS group was increased significantly; The NF-κB p65 entering the nuclears in RvD1+LPS group were significantly lower than that in the LPS group, There were no significant differences compared RvD1 group with control group.6. Western blot results showed that, compared with the control group, the NF-κB p65 entering the nuclears in LPS group was increased significantly(P < 0.05); The NF-κB p65 entering the nuclears in RvD1+LPS group were significantly lower than that in the LPS group(P < 0.05), Compared RvD1 group with control group, we did not found significant difference(P > 0.05). ConclusionRvD1 can relieve the toxic effect of LPS activated BV-2 microglia to neural PC12 cells, and the possible mechanism is that RvD1 inhibits the activation of microglia and nuclear translocation of NF-κB p65 in microglia.