The Interventions Of Flavonoids From Scutellaria Barbata On Astrocytes Damage Induced By Aβ25-35

Senile dementia is divided into Alzheimer’s disease (AD), vasculardementia and mixed dementia, and the AD is the most well known to all of us.A serial of researches demonstrated that the characteristic pathologicalchanges of AD are senile plaque formation from aggregation of β-amyloidprotein and neurofibrillary tangles from hyperphosphorylation of tau protein.Then, the abnormal changes of structure and function of neuroglial cell hasclosed relationship to AD physiopathological development.It is well known that there is a mount of neuroglial cells in brain exceptneurons, in which the astrocytes are the most and closely associated withmany neurodegenerative diseases. On the one hand, the atrocities sustain andnourish neurons and on the other hand, they are abnormal hyperplasia andaccompanied by unusual expression of cell some factors and proteins in AD. Itis reported that Aβ is composed of39-43amino acid residues, Aβ25-35isone toxic fragment and harmful to neurons in vitro and in vivo. With thedevelopment of the study in AD, the influence of neuroglial cells has beenmore and more important, and the study of astrocytes becames a new target.Then, any improvement in astrocytes abnormal changes is beneficial for thetreatment of neurodegenerative diseases.SBF is a group of flavonoid which is extracted and separated fromScutellaria barbata. Modern pharmacology found that flavonoid group has avariety of pharmacological effects on anti-tumor, anti-oxidation and improvememory impairment, which results from the strong reductibility of flavonoids’polyhydroxy structure. Our previous studies have proved that SBF has aprotective effect in neuron, but its influence on astrocytes has been notreported. In the present study, we established an in vitro model by usingAβ25-35to damage astrocytes, and to explore the effects of Scutellaria barbata flavonoids and its possible effective mechanism in astrocytes.Objective:To study the intervention SBF on astrocytes damaged by Aβ25-35invitro.Methods:The cerebral cortex cells of1-3days newborn rats were cultured in vitro.Through differential velocity adherent technique removed fibroblast cells andneurons. GFAP is a specific substance that can be used to identifing astrocyteby immunohistochemical measurement. The astrocytes were divided into5groups following the third passaged. The three treatment groups were givenSBF at dose of17.5μg/ml,35μg/ml and70μg/ml for24h, and then the modelgroup and three doses of SBF groups were exposed to Aβ25-35100μM for24h. The cell survival rate was detected by MTT, the lactate dehydrogenase(LDH) release、the level of interleukin-1β(IL-1β) and interleukin-6(IL-6) inculture supernatant were determined by spectrophotometry and enzyme linkedimmunosorbent assay (ELISA) respectively, the expression of LDH、neuronalnitric oxide synthase (nNOS), inducible nitric oxide synthase(iNOS)、endothelial nitric oxide synthase(eNOS) of cultured cells were assayed byimmunohistochemical method, the expression of heat shock protein70(HSP70)was estimated by western blotting and the mRNA expression ofapolipoprotein E(apoE) was assessed with reverse transcription-polymerasechain reaction(RT-PCR).Results:1. Culture and identification of astrocytes from rats cerebral cortex in vitroThe astrocytes showed typical morphological features and grew well,account for more than95%in third generation cells. Specific antigen GFAPappeared positive expression and two-layer cells. The upper layer cells arefibrous astrocytes and the sublayer cells are protoplasmic astrocytes. Themorphological status is difference of two kinds of the cells. The fibrousastrocyte is a small body、 nucleus and long neuritis, the protoplasmicastrocytet is big body and nucleus, flat neurites, and less branches. 2. Determination of the cell survival rate and expression of LDH in astrocytesThe cell survival rate was detected by MTT; the LDH release wasdetermined with spectrophotometry; the expression of LDH was observed byimmunohistochemical method. Compared with the control group, the cellsurvival rate(P<0.01)、 the LDH release(P<0.01) and the expression ofLDH(P<0.05) were decreased in model astrocytes. Compared with the modelgroup, SBF (17.5,35and70μg/ml) can differently increase the cell survivalrate (P<0.05), the LDH release (P<0.01) and the protein expression ofLDH(P<0.05).3. Determination of the protein expression of nNOS、eNOS and iNOS inastrocytesThe protein expression of nNOS, eNOS and iNOS in astrocytes wereassayed by immunohistochemical method. There was no expression of nNOSin all group astrocytes. Compared with the control group, the proteinexpression of eNOS and iNOS was significantly decreased and increased(P<0.01) in model group, respectively. However, these abnormal changes ofmodel astrocytes were marked improved following treatment with SBF (17.5,35and70μg/ml). The protein expression of eNOS was increased（P<0.05），and the iNOS was decreased(P<0.01) in astrocytes by SBF.4．Determination of the levels of IL-1β and IL-6in astrocytesThe levels of IL-1β and IL-6were assayed with ELISA kit. Comparedwith control group, the level of IL-1β and IL-6in supernate were markeddecreased in model astrocytes(P<0.01). However, SBF(17.5,35and70μg/ml)can reverse the disorders of IL-1βand IL-6in supernate.5．Determination of the expression of HSP70and apoE mRNA in astrocytesThe protein expression of HSP70was assayed by western blotting andthe mRNA expression of apoE was determined by RT-PCR. The resultsshowed that the protein expression of HSP70and mRNA expression of apoEwere notablely increased (P<0.01) in model group, respectively. However,these disturbances can be attenuated by SBF at dose of17.5,35and70μg/ml(P<0.01). Conclusion:1. We establish a culture method for rat cerebral cortical astrocytes.2. SBF can increase the cell survival rate and improve the abnormal changesin LDH when exposed to Aβ25-35.3. SBF can increase protein expression of eNOS and decrease proteinexpression of iNOS.4. SBF can increase the level of IL-1β and IL-6of supernate in damagedastrocytes induced by Aβ25-35.5. SBF can decrease the the expression of HSP70and apoE mRNA indamaged astrocytes induced by Aβ25-35.6. SBF has obvious protection on astrocytes damage induced by Aβ25-35.which provides that these influences mechanisms of SBF may be fromincreasing the cell survival rate、LDH protein expression of cell, regulatingthe NOS activities、IL-1β and IL-6levels of supernate, decreasing the the ofHSP70and apoE expression. The results suggest that the influences of SBFon astrocytes may be helpful to the treatmenf of AD.