Activation Of ARE Pathway By Curcumin Protects Astrocytes

Oxidative stress is described as an imbalance between generation and elimination of reactive oxygen species (ROS) and reactive nitrogen Species (RNS). Growing data from experimental models and human brain studies suggest oxidative stress may involve in mediating neuronal death and play an important role in neurodegenerationin diseases such as Parkinson's disease(PD), Alzheimer's disease(AD) and amyotrophic lateral sclerosis(ALS). It is now recognized that redox regulation involving ROS, is key to the modulation of critical cellular functions, notably for neurons and astrocytes. A net increasein reactive oxygen species can produce damage to lipids, proteins, DNA and induce apoptosis. Recent studies have emphasized the involvement of astrocyte dysfunction in the pathogenesis of neurodegenerationin disease through different mechanisms. Reactive astrocytes may contribute to free radical formation and neuronal death;Production of nitric oxide or peroxynitrite by astrocytes induces damage to mitochondrial complexes and enhances NMDA-induced excitotoxicity. Excitatory amino acid transmission is dependent upon rapid clearance of released glutamate from the extracellular space by high affinity glutamate transporters located in the plasma membranes of presynaptic terminals and astrocytes. Reactive oxygen species induce oxidants and lipid peroxides inhibit the high affinity glutamate uptake in astrocytes by oxidation of critical sulfhydryl groups of the transporter protein and disrupt glutamate uptake in astrocytes. Activated astrocytes are potent producers of cytokines, makes neurons vulnerable to undergo apoptosis in response to oxidative stress. Some of the produced cytokines such as FasL, TNF-a and NGF are capable of activating death receptors expressed in the diseased CNS. It plays an important role in neuron loss in neurodegenerationin disease.Several mechanisms of astrocytes are implicated in the pathogenesis of neurodegenerationin disease. Although disturbances in each of these pathways may contribute to amplification or even initiation of motor neuron injury.These mechanisms are not mutually exclusive but are activated as a communal response that may be coordinated by oxidative stress. Increased oxidative stress appears to be an early and sustained event in association with neurodegenerationin disease. Oxidative stress has a prominent role in the initiation of neurodegenerationin disease and is capable of activating pathways that elicit additional oxidative stress and propagate disease.Endogenous phase II enzymes, e g, NQO1, HO1, GST play an important role in protecting cells against oxidative stress damage. In this model, we want to study neuroprotective effects by inducing endogenous antioxidant agents. The accumulation of reactive oxygen species has been implicated in the pathogenesis of many neurodegenerative diseases.The ability of neutralizing reactive intermediates is, in part, dependent on the activation of a cis-acting regulatory element termed the antioxidant response element (ARE). The ARE is located in the 5'-flanking region of many genes essential for both detoxification and antioxidant proteins. In many tissues of human, rat, mice and primary culture neurons and astrocytes, there are many ARE-drived genes including GCLC, HO1, NQO1, GST and so on.Based on the oxidative stress pathogenesis, incubation of primary astrocytes culture in presence of H2O2, a powerful oxidantant, causes oxidative stress damage of astrocytes. We established the model of oxidative stress.In this study, the model of oxidative stress was used to study the effect of Curcumin on astrocytes protection and mechanisms. The first part: we show that established the model of oxidative stress in astrocytes. The second part: we show that Curcumin protects H2O2-induced astrocytes damage. The effects of protection are corresponding to phase II enzymes alteration.The third part: the protection mechanism of increasing of phase II enzymes by Curcumin and effects on susceptivity of oxidative stress.PartⅠModel of astrocyte oxidative stressObjective: Astrocyte oxidative stress plays an important role in neurodegeneration disease, the model of astrocyte oxidative stress is benefit to further explore oxidative stress and look for antioxidant medicines.Methods: Primary astrocyte culture was prepared from less than 2-day-old Nrf2+/+ mice and digested by trpsin.Then it is purified and verified. 25μmol/L H2O2 was added into the cultures for 30 minutes after cultured seven days. Immunohistochemistry staining by GFAP antibody was taken then observed by confocal microscopy; Cell vitality was tested by MTT method; LDH and MDA of the medium and ROS content inside the cell was examed by probe DCFH-DA, comparing with different group.Results: Results showed that the numbers of GFAP+ in generationⅠwere above 85%, the numbers in generationⅡwere above 95%. Comparing to control, cell vitality descend,LDH and the MDA content of the medium and generation of ROS increased in H2O2 intervention group.Conclusions: This study succeed in estabolishing method of primary astrocyte culture and the model of oxidative stress by H2O2.PartⅡThe effect of Curcumin on astrocyte oxidative stress and the relationship between Curcumin and phase II enzymesObjective: To study effects of Curcumin on astrocyte oxidative stress and alteration of phase II enzymes.Methods: Pre-treated various concentrations of Curcumin (5μmol/L,10μmol/L 15μmol/L,20μmol/L,25μmol/L,30μmol/L)for 24h then 25μmol/L H2O2 were added into the cultures for 30 minutes. Cell vitality was tested by MTT method; LDH and MDA of the medium and ROS content inside the cell was examed by probe DCFH-DA. Protein expression were measured by Western blot. Immunohistochemistry staining of HO1,NQO1 were taken then observed by confocal, comparing with different group.Results: Curcumin of the concentrations of 5μmol/L,10μmol/L and 15μmol/L increased protein levels of NQO1,HO1,GCLC,effects of 10μmol/L were obvious. Comparing to control, cell vitality descended,MDA content of the medium and generation of ROS decreased after incubated with curcumin for 24 h.While obvious changes of the concentrations of 20,25μmol/L Curcumin have never been seen . The level of LDH increased, indicateing damages of cells.Conclusions: Curcumin protect astrocytes from oxidative stress damage and the effects relevant to the increasing of phase II enzymes.PartⅢMechanism of increasing phase II enzymes and effects on susceptibility of oxidative stress by CurcuminObjective: Activated Nrf2 trans-located to nucleus and combined with ARE, adjusting ARE target gene. This study investigated mechanism of increasing phase II enzymes and effect on susceptibility to oxidative stress by Curcumin.Methods: 1. Pre-treated 10μmol/L Curcumin for 24h in the Nrf2+/+ and Nrf2-/- cultures, protein expression of phase II enzymes were measured by Western blot. 2. Pre-treated 10μmol/L Curcumin for 24h then 25μmol/L H2O2 were added into the cultures for 30 minutes. Protein expression of phase II enzymes and Nrf2 were measured by Western blot. Cell vitality was tested by MTT method; LDH and MDA of the medium and ROS content inside the cell was examed by probe DCFH-DA. Immunohistochemistry staining by Nrf2 antibody was taken then observed by confocal microscopy. 3. Pre-treated 10μmol/L Curcumin for 24h then various concentrations of H2O2 (50,100,200,300,400,500μmol/L)were added into the cultures for 30 minutes, testing cell vitality by MTT method.Results: 1.Curcumin promoted Nrf2 trans-located to nucleus and increased nucleu protein levels of Nrf2. Comparing to control, Nrf2+/+ astrocytes of MDA content of the medium and generation of ROS decreased. Cll vitality and protein levels of HO1,NQO1,GCLC increased after treatment of Curcumin. While in Nrf2-/- astrocytes, obvious changes have never been seen after treatment of Curcumin. 2.Comparing to Nrf2+/+ astrocytes, cell vitality of Nrf2-/- astrocytes decreased by exposuring to various concentrations of H2O2 (50,100,200,300,400,500μmol/L). Cell vitality of Nrf2+/+ astrocytes increased after treatment of Curcumin, While Nrf2-/- astrocytes have never seen obvious changes.Conclusions: Curcumin activated Nrf2-ARE pathway and promoted expression of ARE target gene, increased protein levels of HO1,NQO1,GCLC .It also plays an important role in decreasing susceptibility to oxidative stress.