Effects Of Resveratrol And Its Derivatives On The Inhibition Of Microglial Activation

Microglia are macrophage-like cells,resident in the brain,that have been implicated as mediators of inflammation in the central nervous system(CNS).Activated microglia produce inflammatory mediators including nitric oxide(NO),reactive oxygen species,and cytokines,such as tumor necrosis factor-α(TNF-α).These are associated with several neurodegenerative diseases including Alzheimer's disease(AD),Parkinson's disease(PD), multiple sclerosis(MS),AIDS-associated dementia,as well as other neurodegenerative diseases.Thus,inhibition of microglial activation is generally considered a useful strategy for treatment of many neurodegenerative diseases.Our previous studies have shown that resveratrol(Res)potently inhibits the microglial activation and protects neuron.The present dissertation evaluated the effects of a series of resveratrol derivatives on the NO production in LPS-activated primary rat microglia,as well as the mechanisms of action of the active compounds.In order to avoid the possible effects of reduced viability on NO release,the cytotoxic activity of resveratrol and derivatives(0.3-30μM)on microglial cells in the presence or absence of LPS(1μg/mL)was assessed using MTT assays.Co-treatment of either unstimulated or stimulated microglial cells with resveratrol or 37 derivatives did not affect the cell viability.Then,the effects of resveratrol and 37 derivatives on the NO production in LPS-activated microglial cells were assessed by Griess reaction.Their structure-activity relationships were studied.It was found,for the first time,that:(1)Certain resveratrol derivatives that have 3,5-dimethoxyl groups in the A-ring strongly inhibited NO production.(2)The 4'-hydroxyl group in the B-ring(RV32,pterostilbene)had more potent effects than those with a methoxy group at this site.(3)Substituting the B-ring of resveratrol with quinolyl,such as in RV01 and RV02,enhanced the activity.From the results of structure-activity relationships,Res, RV01,RV02 and RV32 were selected for further studies.RV01,RV02 are novel compounds.The effects of Res,RV01,RV02 and RV32 on morphological changes in activated microglial cells were observed under an inverted microscope.The result indicated that LPS-induced morphological changes in microglial cells were markedly suppressed by the four compounds(30μM).Moreover,TNF-αlevels in the culture supematants were examined by ELISA assay.The results showed that the four compounds potently suppressed TNF-αrelease by LPS-activated microglia,and the inhibitory effects of RV01,RV02 and RV32 were stronger than those of Res.In addition,the results of RT-PCR showed that Res, RV01 and RV02 could significantly inhibit LPS-induced expression of IL-1βand IL-6 mRNA in microglia,while RV32 could not.In order to evaluate whether the inhibitory effects of NO release were attributed to direct NO-scavenging activities,the NO-scavenging activities of the selected compounds were examined using SNP as a NO donor.The results showed that,unlike resveratrol,the three active derivatives,in concentrations up to 30μM,did not affect NO levels in SNP solutions. Then,the effects of Res and the three active derivatives on iNOS protein and mRNA expression were studied.The results demonstrated that the four compounds could reduce both protein and mRNA expression of iNOS in LPS-activated microglia.In addition,Res and the three active derivatives(RV01,RV02 and RV32)could inhibit phosphorylation and degradation of IκBαand phosphorylation of p38 MAPK in LPS-activated microglia.These effects were mediated,at least in part,by inhibiting the generation of ROS.In order to further investigate the protective effects on the neuronal cell of the tested compounds which inhibited the activation of microglia,in the present study,the effects of the four compounds on DNA damage of rat cortical neurons induced by condition medium of LPS-activated microglial cells(L-CM)was studied.The results showed that treatment of rat cortical neurons with L-CM for 24 h could cause DNA damage.The four compounds alone did not influence the DNA of rat cortical neurons;however,they suppressed the DNA damage of rat cortical neurons induced by L-CM.These results show that the four compounds may play important neuroprotective roles in protecting the neurons from the toxic influence by the activated microglia.In conclusion,the results suggested that Res and the three active derivatives(RV01,RV02 and RV32)could inhibit the activation of microglia,and have protective effect on the neurons from the toxic influence by the activated microglia.These compounds can inhibit inflammatory responses of microglia.They may have therapeutic potential in the treatment of neurodegenerative diseases accompanied by microglial activation.