The Mechanism Of Jujuboside A On Neuroinflammation In Astrocyte C6 Cells Induce By LPS

Neuroinflammation is an innate immune response to the central nervous system and mainly mediated by glia cells of the brain.When glia cells are damaged by various pathological factors,they can be changed from rested state to activated state and removed the fragments of denatured nerve tissue to protect neurons.When glial cells are be over-activated to cause neuroinflammation and release amounts of inflammatory cytokines,such as tumor necrosis factor(TNF-?),reactiveoxygen species(ROS),interleukin(IL)and nitric oxide(NO),which lead to neuron damage.Although the mechanism of neuroinflammation is not fully understood,some studies have shown that Oxidative stress(OS)plays a key role in neuroinflammation.In this study,Jujuboside A was used as a research object.With reference to relevant research reports,the protective effect of Jujuboside A and the related mechanisms were investigated from the neuroinflammation.Using LPS to build the vitro model of neuroinflammation in astrocytes to study that Jujuboside A could inhibit the release of intracellular inflammatory mediators and the intervention of LPS-induced oxidative stress in C6 cells,and further study of Jujuboside A on the regulation of NF-?B/Nrf2 signaling pathway to play a possible theoretical basis for inhibiting the occurrence of neuroinflammation.Objective: To study the protective effect of Jujuboside A against LPS-induced neuroinflammation in astrocytic C6 and explore its mechanisms.Methods: MTT assay to detect cell viability rate after treatment with LPS and Jujuboside A for different times and concentrations and Griess assay to measure the level of NO in cell supernatant to establish model of neuroinflammation;Western blot detect the expression of GFAP;ELISA assay to detect the content of TNF-?,IL-1?and PGE2 in the cells;the expression of iNOS and COX-2 were detected by Western blot;Reverse transcription PCR dected the expression of TNF-?,IL-1?,iNOS and COX-2 mRNA;the DCFH-DA and Rhodamine-123 to analyzed the production of ROS and the level of MMP by flow cytometry;the level of malondialdehyde(MDA)and glutathione(GSH)and the activity of superoxide dismutase(SOD)were measured by the kits;separate the cellular proteins and the location of NF-?B/p65 and Nrf2 were observed by immunofluorescence and Western blot;the expression of HO-1,NQO1 and GCLC proteins which downstream of Nrf2 signaling pathway were detected by Western blot.Results:1.The protective effect of Jujuboside A on LPS induced C6 cells(1)The results of MTT assay showed that LPS and Jujuboside A had a significant inhibition on the viability of C6 cells at a certain time and concentration.When treated with LPS for 24 h and the concentration of 1 ?g/mL,which was the best viability of cells,While Jujuboside A had no significant effect on the cell viability in the treatment time of 24 h and the concentration of 0.125-50 ?M.(2)The results of Griess assay showed that the intracellular of NO level was increased in a time-dependent in C6 cells at the concentration of 1 ?g/mL LPS(P<0.01).(3)Treating with LPS and compared with the control group the expression of GFAP protein was significantly increased and the astrocytes was activated(P <0.05),Jujuboside A pretreated with LPS and the protein expression of GFAP was significantly reduced(P <0.01).2.The effect of Jujuboside A on LPS induced oxidative stress and inflammatory in C6 cells.(1)The result of microplate reader showed that the content of NO in C6 cells increased remarkably after treatment of LPS compared with that of control group(P<0.05),Jujuboside A pretreated with LPS and the content of NO was significantly reduced(P <0.01).(2)Elisa experimental results showed that the expression of TNF-?,IL-1? and PGE2 were significantly increased after treat with LPS(P<0.05),Jujuboside A significantly inhibited the level of these inflammatory cytokines(P<0.01),in a significant concentration dependent manner.(3)The result of Western blot showed that compared with the control group,after treat with LPS the expression of iNOS and COX-2 were significantly increased(P<0.05),Jujuboside A pretreated with LPS and the protein expression of iNOS and COX-2 were reduced remarkably(P <0.01).(4)The result of reverse transcription PCR also proved that Jujuboside A significantly inhibited the increase of the level of these inflammatory cytokines induced by LPS.(5)Flow cytometry detected DCFH-DA fluorescence staining found Jujuboside A effectively reduce the production of ROS induced by LPS(P <0.01).(6)Jujuboside A inhibited dissipation of the mitochondrial membrane potential monitored using the Rh-123 in C6 cells induced by LPS.(7)The result of treat with LPS was significantly increased the content of MDA and also decreased the activities of GSH and SOD(P <0.05),when C6 cells were treated with Jujuboside A of 12.5-50 ?M,the content of MDA were reduced significantly,and the activities of GSH and SOD were increased significantly(P <0.01).3.The mechanism of Jujuboside A inhibits the neuroinflammatory response induced by LPS in C6 cells(1)Immunofluorescence to observed the location of NF-?B/p65,LPS could significantly increase the expression of NF-?B/p65 in nuclear(P <0.05),Jujuboside A pretreated with LPS and the expression of NF-?B/p65 in nuclear was significantly reduced(P <0.01),in a significant concentration dependent manner.(2)Western blot had the same result to prove that Jujuboside A could inhibit the protein expression of NF-?B/p65 in nuclear induced by LPS,thus inhibiting the release of inflammatory cytokines.(3)Immunofluorescence analysis indicated that Jujuboside A stimulate a translocation of Nrf2 protein from the cytosol to the nucleus(P <0.01).(4)Western blot had the same result to prove that Nrf2 could induce antioxidant enzymes to protect cells against oxidative damage.(5)Western blot analysis indicated that the expression of HO-1,NQO1 and GCLCwere significantly increased after treat with LPS(P<0.05),while 12.5-50 ??Jujuboside A could increase the expression of these protiens(P <0.01),in a significant concentration dependent manner.Conclusion: Taken together,LPS could induce astrocyte C6 activation,upregulate the expression of GFAP and stimulate the content of ROS,MMP and MDA,while decrease the activity of GSH and SOD.Also induce the release of some inflammatory factors leading to the occurrence of neuroinflammation.Jujuboside A could stimulate a translocation of Nrf2 in nuclear and significantly increase the expression of HO-1,NQO1 and GCLC and decrease the content of NF-?B/p65 in nuclear to protect astrocytes in anti-inflammatory and antioxidant.