PROG Protect The Neurons Against Impairment Induced By The Aβ Activated Astrocytes

Objective: Alzheimer disease (AD) is the most common age-relatedneurodegenerative disorder, the most prevalent cause of dementia in humans.Numerous pathological changes have been described in the postmortem brainsof AD patients, including deposits of β-amyloid (Aβ) in senile plaques,neurofibrillary tangles, synapse loss, neuronal death and the activation of glialcells. Astrocytes play double edged role in AD. Activated astrocytes wasusually found to be associated with the amyloid plaques. Activation ofastrocytes leads to release neurotrophic factors, uptake and clearance ofamyloid peptide. However, excessive activation of astrocytes leads toproduction and release of inflammatory cytokines and other neuron toxicproducts which contribute to neuronal dysfunction and cell death. However,little is known about the regulation of these cells in the AD brain.The neuroactive steroid family in CNS includes steroids hormones andtheir metabolites directly synthesized from cholesterol by glial or neuronscells. And they always act as important endogenous modulators in multipleneurodegenerative disorders. Recent studies have shown that the levels ofneuroactive steroids can be affected by pathology or injury, as demonstratedin cerebrospinal fluid of AD patient, especially the significantly decrease ofPROG and AP. The previous studies in our laboratory have shown the effectsof Aβ on the production of neurosteroids in primary rat cortical neurons, andthe neuroprotective activity of PROG in AD models. But the changes ofneurosteroids synthesis in Aβ treated astrocytes and the effect of neurosteroidson astrocytes and interaction with neurons was still unknown. In this study,we used Aβ1-42treated the rat cortical astrocytes, investigate the underlyingmolecular mechanism of astrocyte activation in response to Aβ1-42, researchthe changes of neurosteroids synthesis and Metabolism in Aβ1-42activated astrocytes; explain the effects of neurosteroids on activated astrocytes, theinteraction of activated astrocytes and neurons. The purpose of this paper is toadd something valuble for AD prevention and medication.Material and Methods:1Cell cultures1.1Primary cultures of rat cortical astrocytesPrimary astroglial cell cultures were obtained from cerebral cortices of1-day neonatal Sprague-Dawley rats, which were minced and subsequentlydigested for15min at37C0.125%trypsin and mechanically dissociated tosingle cells. Cells were standing and then resuspended in DMEM containing10%fetal bovine serum (FBS). Cultures were incubated at37C in a5%CO2incubator, first renewal the medium at24h，then medium renewaled every2-3days until confluence. Cultures were purified from contaminating microgliaand oligodendrocytes by gentle shaking，200rpm. The astrocyte purity wasover95%, determined by GFAP-staining.1.2Primary culture of rat cortical neuronsBriefly, cortical neurons were obtained from new born SD rats by milddigestion and mechanical dissection. The cells were seeded on the cultureplates pre-coated with poly-L-lysine in the DMEM containing10%fetalbovine serum at the density of1.0×109·L-1. The neurobasal medium werechanged24hours after culture and followed by changing half medium every2days. Cultures were maintained at37oC, saturation humidity in an atmospherecontaining5%CO2for8to10days prior to experimentation. Invertedmicroscope was used to observe morphological changes of cultured neurons.1.3Co-culture of astrocytes and neuronsThe astrocytes were treated with or without Aβ and PROG, then take theglasses coverd of prepared neurons into the treated astrocytes, renewal theDMEM co-culture for48h.2Grouping and Drug treatment2.1Aβ1-42treatment on rat cortical astrocytesThe cultures were treated with or without Aβ1-42(0,1μM,5μM, or10 μM) for24h, used to the research of morphology and cytokine production;treated with or without cholesterol and Aβ1-42, ivided into3groups: Blank,Control (cholesterol Substance) and Aβ (Substance plus Aβ1-42), for theresearch of neurosteroids.2.2PROG trentment on Aβ1-42activated astrocytesAstrocytes were treated with PROG (0,0.01μM,0.1μM,1μM) and Aβ(5μM) for24h, for the research of cytokine production and co-culture ofastrocytes and neurons.3The GFAP immunofluorescence observe the morphology change ofastrocytes.Immunofluorescent analysis of GFAP (an astrocyte-specific intermediatefilament component) was performed to observe the morphological activationof astrocytes. After various treatments, astrocytes were fixed with4%paraformaldehyde for30min and incubated with a rabbit antibody againstGFAP (1:1000) at4C overnight. After being washed, the cells were treatedwith the appropriate secondary antibodies for1h at room temperature. Cellimages were captured with a fluorescence microscope.4Determination the leavls of IL-1β and TNFα in culture media of astrocytesby ELISAThe culture media were collected, centrifugate5minutes at3000r·min-1.The supematant was determinated using Enzyme-Linked ImmunosorbentAssay (ELISA).5Determination of neurosteroids’ concentrations in culture media ofastrocytes using HPLC-MSOne milliliter of cultured media samples were mixed withmethyltestosterone (MT), the internal standard, and methanol, then isolatedusing ethylacetate/hexane. Thereafter, samples were derivatized with2-nitro-4-trfluo-romethylphenylhydrazine (2NFPH) and determinated by highperformance liquid chromatography-mass spectrometry (HPLC-MS).6Determination the activaty of NF-κB6.1Immunofluorescence Immunofluorescent analysis of p65(an functional subunit of NF-κB) wasperformed to observe the activation of NF-κB. Astrocytes were fixed with4%paraformaldehyde for30min and incubated with a rabbit antibody against p65(1:100) at4C overnight.Then treated with secondary antibodies. Cell imageswere captured with a fluorescence microscope.6.2Western blotEqual amounts of protein (40μg) were separated by10%SDS-PAGE,and electrotransferred to a PVDF membrane (Millipore, Billerica, MA, USA).Membranes were blocked with5%BSA for2h at room temperature, andincubated with the primary antibodies,1:1000rabbit anti-p65, and1:3000rabbit anti-laminA overnight, respectively, and then with secondary antibodyfor2h. ECL luminescenced and exposure.7Cell viability assayThe MTT assay was used to evaluate the viability of the neurons culturedin the glass. Briefly,20μL MTT was added to the culture media with a finalconcentration of3.6mmol·L-1. After4hours of culture, the culture media wasremoved and200μL DMSO was added to dissolve the crystals. Theabsorbance of the solution was read at570nm on a spectrophotometer and thecell viability was calculated by compare to controls.Results:1Effect of Aβ1-42on morphological change of astrocytesGFAP staining revealed that astrocytes treated with Aβ1-42(1-10μM)exhibited morphological signs of activation as compared with untreatedastrocytes. Astrocytes were flat in the absence of Aβ1-42, and activated withextension of their processes, appeared stellate-shaped in the presence of Aβ (5μM,10μM).2Effect of Aβ1-42treatment on the release of IL-1β and TNFα in media ofcultured rat cortical astrocytesCompared with control group, the level of IL-1β was significantlyincreased in media of AβM、AβHtreated rat cortical astrocytes (P<0.01). Thelevel of TNFα was dose dependently increased in media of AβL, AβMand AβH treated rat cortical astrocytes (P<0.05).3Effect of Aβ1-42on the production of neurosteroids in rat cortical astrocytesCompared with control group, the PROG level were decreased by43.8%,greatly varied (P<0.05) whit Aβ1-42treatment; no change in PREG and DHEAlevels were observed after treated with Aβ1-42(P>0.05).4Effect of PROG treatment on the release of IL-1β and TNFα in media ofactivated astrocytesCompared with control group, the levels of IL-1β and TNFα weresignificantly increased in media of Aβ1-42treated rat cortical astrocytes(P<0.01), and PROG (0.1μM,1μM) treatments significantly decreased thelevels of IL-1β and TNFα in Aβ treated rat cortical astrocytes (P<0.05).5Effect of PROG treatment on the activity of NF-κB in activated astrocytesImmunofluorescence showed that p65was mainly distributed incytoplasm in the control group, translocated from the cytoplasm to the nucleusin Aβ1-42treated cells. This translocation was significantly inhibited by PROGtreatment.Western blot showed that the p65expression in nuclear protein extractswas significantly enhanced in the presence of Aβ1-42compared with thecontrol group, PROG (0.1μM,1μM) treatments significantly decreased thep65expression in nuclear protein extracts. Furthermore, low-dose PROG(0.01μM) treatment did not alter the expression of p65in nuclear proteinextracts.6PROG protected against the neurons impairment from activated astrocytesThe MTT assay showed that compared with control group, the cellviability of Aβ1-42group significantly decreased to70.1%(P<0.05);meanwhile, PROG protected against the neurons impairment from activatedastrocytes dose dependently. PROG (0.1μM) and PROG (1μM) elevated thecell viability to115.9%and129.6%(P<0.05），compared with Aβ group.Conclusions:1The Aβ1-42treatment induced cell morphology changes, inflammatorycytokines release and the activition of NF-κB cell signal of astrocytes, suggesting Aβ1-42indeed activate the astrocytes.2The level of PROG was significantly decreased in astrocytes after Aβ1-42treatment, indicating that Aβ1-42treatment effect the levels ofneurosteroids.3Further studies demonstrated that PROG can effectively inhibit theactivity of NF-κB cell signal, and then decrease the release ofinflammatory cytokines in a dose dependent manner.4PROG could efficaciously prevent the rat cortical neurons impairmentinduced by activated astrocytes in a dose dependent manner, throughinhibiting the activation of astrocytes.