Isoliquiritigenin suppresses microglial activation and neuroinflammation in primary microglia

摘要

OBJECTIVE Microglial activation contributes to neuroinflammation and neuronal damages in neurodegenerative disorders including Alzheimer and Parkinson diseases.It has been suggested that neurodegenerative disorders may be improved if neuroinflammation can be controlled.Isoliquiritigenin(ISL)isolated from Glycyrrhiza glabra possess potent anti-inflammatory capability,we thus investigate the inhibitory effects of ISL on LPS-induced microglial activation and neuroinflammation and the roles of neuroprotection on neurodegenerative disorders.Moveover,we would explore the mechanism of itsassociated inflammatory signaling passway.METHODS By cultivating,isolating and purifying,we got the primary microglia of the rat.The form of those cells was observed under an optical microscope,the purity was identified by the CD11b immunefluorescence staining.Firstly,the effects of ISL on microglial viability were explored by the MTT assay.The microglial cells were pretreated by ISL(5μmol·L-1)and then stimulated by LPS(100 ng·mL-1),the production of NO was detected by Griess reagent,the change of microglial morphology was observed by immunefluorescence staining.The production of IL-1βand TNF-αin culture medium were tested by ELISA,and the m RNA expression of pro-inflammatory factors,including IL-1β,TNF-α,i NOS and COX-2 was detected by real time PCR.The protein expression of i NOS and COX-2was detected by Western blotting.RESULTS(1)After purifying,microglial showed the typical morphological characteristics.The cell body was small,protruding outstretched in the branch rod,spindle.After LPS stimulation,the shape was changed to amoeba-like,and the protrusions retracted,cell rounding.In the CD11b immunefluorescence staining,the purity of the microglial was over 98%,and showed powerful phagocytic activity.(2)The results show that ISL(0.16-20μmol·L-1)concentration has no effect on primary microglia′s vitality.And at the concentration of 5μmol·L-1ISL has the significantly inhibitory effect on NO production induced by LPS.(3)The results shows that ISL pretreatment can significantly inhibit LPS-induced microglial activation,and can significantly reduce the production of NO in a dose-dependent manner(P<0.05).When stimulated by LPS for12 h,the expression of TNF-αand IL-1βm RNA in the culture medium are reduced by ISL pre-treatment(P<0.05).After LPS stimulation for 24 h,the expression of COX2 and i NOS m RNA and protein can be reduced(P<0.05).After stimulated by LPS from 0.5 to 2 h,p-ERK expression has be decreased.CONCLUSION We successfully obtain primary microglia with high-purity by culturing in vitro.We find that ISL can significantly reduce the levels of proinflammatory facters,which indicate ISL can inhibit microglia activation and neuroinflammation by blocking ERK1/2 signal pathway.