Exploring Anti-Neuroinflammatory Mechanism Of Bromo-Derivative Of 1,6-O,O-Diacetylbritannilactone(OABL)

Neuroinflammation constitutes a fundamental process involved in the progression of several neurodegenerative disorders,such as Parkinson’s disease,Alzheimer’s disease,amyotrophic lateral sclerosis and multiple sclerosis.The activation of microglia is the main feature of neuroinflammation,promoting the release of pro-inflammatory cytokines and resulting in the progressive neuronal cell death.In response to this phenomenon,existing studies have shown that natural products and their derivatives have the potential to alleviate neurodegenerative diseases probably related to their ability in inhibiting neuroinflammation.Therefore,the discovery of natural products with anti-neuroinflammation activity and the investigation of their mechanisms are of great significance.In this study,we constructed a library of natural products（1407）and screened out the1,10-seco-eudesmane sesquiterpenoids with significant anti-neuroinflammation activity through an anti-neuroinflammation model.Compound 18,a brominated derivative of1,6-O,O-diacetyl crotonide lactone（OABL）,with the more prominent anti-neuroinflammation activity was found in the re-screening.We analyzed the cell localization and biological function of potential targets of 18 through chemoproteomics strategy based on the site level.We proposed the targets covalently binded with 18.The specific research results are as follows:（1）Nitric oxide（NO）is an endogenous inflammatory factor in microglia.We firstly evaluated the NO level of microglia and found compound 18,the bromo-derivatives of OABL,with IC₅₀ of anti-neuroinflammation is 0.5μM,and CC₅₀ of neurotoxicity is greater than 20μM.（2）Compound 18 could attenuate tumor necrosis factor-α（TNF-α）and prostaglandin E₂（PGE₂）productions in activated BV-2 cells.18 also could inhibit the expression of inducible nitric oxide synthase（i NOS）and cyclooxygenase-2（COX-2）in BV-2 cells activated by lipopolysaccharide（LPS）.（3）Through the quantitative thiol reactivity analysis（QTRP）strategy based on the site level,the proteome in BV-2 cells after 18 treatment was analyzed,and the degree of binding of proteins to compounds was quantitatively evaluated by the ratio of light and heavy isotope R_L/H.The GO analysis showed that these binding proteins(R_L/H<0.5)were significantly localized in the mitochondria and cell membranes,and were also involved in transport,NF-κB negative regulation and oxidative stress response.（4）Fluorescence confocal experiments verified the effect of 18 on alleviating the dysfunction of mitochondria.Immunoblotting experiments confirmed that 18 can induce the expression of antioxidant HO-1 in the oxidative stress pathway.These results were consistent with the previous proteomic analysis of 18 acting on mitochondria proteins that perhaps induce the oxidative stress.（5）The expression of some proteins in the TLR/NF-κB pathway was detected by immunoblotting,immunofluorescence and flow cytometry.18 could down-regulate the expression of CD14 and IRAK1,up-regulate the expression of TAK1,and suppress the translocation of NF-κB/p65 into nucleus.These results of 18 inhibiting the activation of the TLR/NF-κB pathway supported the analysis of 18 inhibited the TLR pathway and the downstream negative regulation of NF-κB.（6）Combined with the verification results and referred to the binding strength of 18to cysteine sites,we predicted 8 potential targets covalently bound to 18:Acod1,Nlrp3,Pbrm1,Sucla2,Atp13a2,Irgm1,Copb1 and Gcn1,among which cis-aconitic acid decarboxylase Acod1 and inflammatory body NLRP3 may be particularly prominent.In summary,in this dissertation,the compound 18 with more significant anti-neuroinflammation activity was screened out from the library of natural products.Chemoproteomics analysis revealed the possible mechanism of its anti-neuroinflammation activity.Subsequently,we proposed Acod1 and Nlrp3 as the potential covalently binding targets.These results provide key insights and necessary references for the further development of target research and drugs development.