Effect And Mechanisms Of Nrf2 And It's Agonist On The Activation Of Astrocyte

BackgroundAstrocytes account for about 50 percent of all cells in the central nervous system(CNS).There is a growing evidence that astrocytes are not only simple to support cells,but also play an important physiological role in the central nervous system,including the formation of blood-brain barrier,participation in synaptic activity,mediated inflammatory response.Under pathological state,astrocytes could be activated which usually called astrogliosis.Astrogliosis was mainly manifested as accelerated cell proliferation,hypertrophy of cell body and high expression of intermediate filaments,extracellular matrix and inflammatory factors.Astrogliosis is a common pathological manifestation of many CNS diseases,and now it is widely accepted that mild and early phase of astrogliosis helps to limit the spread of inflammation,whereas excessive astrogliosis may lead to the formation of glial scar in the latter stage.Furthermore,the formation of glial scar is always accompanied by the secretion of CSPGs which is a group of powerful axon growth retardation molecules.Glial scar and CSPGs forms the main obstacles to axonal regeneration in many CNS diseases especially CNS traumatic injury.How to reduce the excessive activation of astrocytes is one of the hotspots in neural regeneration research.A large number of studies have shown that excessive oxidative stress and inflammatory response are the major factor leading to the activation of astrocytes.Inhibition of excessive oxidative stress and inflammatory response is the main strategy to reduce the excessive activation of astrocytes.Nrf2 is the main endogenous antioxidant and anti-inflammatory transcription factor,in a variety of CNS diseases it plays a neuroprotective effect.Previous studies of Nrf2 in CNS disorders have focused more on the direct protective effect on neurons but paid less attention to their effects on nerve regeneration and formation of glial scar.Sulforaphane(SFN)is an active component extracted from natural plants that effectively activates Nrf2 to play a role in anti-inflammatory and anti-oxidative effects and is widely used as a specific agonist of Nrf2 in many studies and researches have confirmed that it can play a positive role in the inhibition of tumor cell proliferation.All of these features make it potentially valuable in CNS disease therapy.To sum up,we guess whether by regulating Nrf2 can effectively inhibit the activation of astrocytes? Can Nrf2 agonists be used in the CNS to control the excessive proliferation of astrocytes and reduce glial scarring? Based on the above conjecture,we performed the following two parts of the study via an in vitro method:Part I Effect of Nrf2 on the activation of astrocyte induced by LPS and study of potential mechanismsObjectiveAstrocytes were activated by LPS treatment in vitro and Nrf2 was regulated by its agonist and gene deletion to explore the role of Nrf2 in LPS-induced activation of astrocytes and possible molecular mechanisms.MethodsThe primary mice astrocytes were stimulated with different doses of LPS(0,0.2,0.4,0.8,1.0ug / ml).The nuclei and cytoplasmic Nrf2 were detected by immunofluorescence and WB to investigate the influence of LPS on the Nrf2 nuclear translocation in vitro.The expression of NQO1 and HO-1 which directly regulated by Nrf2 was detected by WB to indirectly reflect the activation of Nrf2.The effect of up-regulation of Nrf2 on the activation of astrocytes stimulated by LPS was investigated by using SFN to activate Nrf2 and to detect the expression of GFAP and Neurocan,markers of astrogliosis,by immunofluorescence and WB.The effect of Nrf2 inactivation on the activation of astrocytes induced by LPS was investigated by immunofluorescence and WB assay by using Nrf2 knockout astrocytes.Intracellular ROS was detected by DCFH-DA method and phosphorylation of P38 was detected by WB to reveal the possible mechanism involved in the effect of Nrf2 on astrogliosis.Result1.LPS can promote the Nuclear translocation of Nrf2 and the expression of NQO1 and HO-1 in vitro,and this phenomenon is concentration-dependent.2.Nrf2 agonist SFN can reduce the expression of GFAP and Neurocan in astrocytes stimulated by LPS.3.Nrf2 knockout astrocytes express more GFAP and Neurocan than wild-type cells after LPS stimulation.4.LPS increased the amount of ROS in astrocytes after 24 h of intervention,and SFN could inhibit this effect and Nrf2 knockout astrocytes produce more ROS than wild-type cells.SFN can inhibit the phosphorylation of P38 MAPK induced by LPS,and the phosphorylation of P38 MAPK in Nrf2 knockout astrocytes is higher than that of wild-type cells.ConclusionA certain concentration of LPS can promote the activation of the endogenous Nrf2 in astrocytes and increase the expression of antioxidant enzymes.Nrf2 can affect the activation levels of LPS-stimulated astrocytes and astrogliosis related signaling molecules such as ROS and P38 MAPK can be modulated by Nrf2 at the same time and this maybe one of the potential mechanisms.Part II Effects of Sulforaphane on LPS-induced proliferation of astrocytes in mice ObjectiveTo investigate the effect and mechanism of sulforaphane on the proliferation of mouse astrocytes induced by LPS in vitro.Method Primary astrocytes are derived from newborn mice cortex and cultured in vitro.First,the effect of different concentrations(0,10,15,20 u M)SFN treatment on the proliferation of astrocytes induced by LPS was measured by CCK8 method.In subsequent experiments 20 u M SFN was selected to treat the cells.The number of Ki67 positive cells was detected by Ki67 immunofluorescence staining.Cell cycle was detected by flow cytometry.The expression of PCNA,Cyclin D1 and P27kip1 were detected by Western blot.Result1.CCK8 results suggest that a certain concentration of SFN can inhibit the proliferation rate of astrocytes under LPS treatment,which 20 u M inhibition effect is more obvious.Ki67 immunofluorescence double labeling results also showed that 20 u M SFN could decrease the number of Ki67-positive astrocytes under the treatment of LPS.2.Cell circle analysis was performed by flow cytometry test,the result showed that 20 u M SFN could reduce the proportion of astrocytes in S phase and S + G2 / M phase cells under LPS treatment.3.The result of western blot analysis showed that 20 u M SFN could decreas e the expression of PCNA and Cyclin D1 and increase the expression of P27kip1.Conclusion 20 u M SFN could inhibit the proliferation of astrocytes induced by LPS in vitro,and its mechanism may be related to the regulation of cycle-related proteins such as P27kip1 and Cyclin D1 so that inhibit the cell cycle.