Improvement Effects And Underlying Molecular Mechanisms Of Chicoric Acid On Neuroinflammation And Cognition Defects

With continued elevation of the standard of living and the degree of population ageing worse off continuously,researches on the exploration of natural food components with brain nutrition and healthy function and their underlying mechanisms have become the hotspot in the field of food science.Chicoric acid（CA）,a natural phenolic acid extracted from vegetables such as chicory,lettuce and plants like Echinacea purpurea,dandelion,possesses powerful antioxidative,anti-HIV virus and anti-obesity activities.However,it has been rarely reported the neuroprotective effects of CA.Based on the above,the present study was designed to examine the metabolism and antioxidative properties of CA,and to investigate the intervention effects of CA on neuroinflammation and the underlying mechanisms in vivo and in vitro.The main investigations and results are as follows:（1）In order to investigate the metabolism of CA and its antioxidative abilities,liquid tandem mass spectrometry（HPLC-MS/MS）was applied to identify the metabolites of CA in rat liver microsomes and free radical scavenging capacities,biomolecules oxidative damage induced by free radicals,microglial inflammation triggered by lipopolysaccharide（LPS）in BV2 cells were constructed to compare the bioactivities of CA and its metabolites.Results showed that most CA exist in its prototype,while a little bit of CA was metabolized to caffeic acid（CFA）and caftaric acid（CTA）on cytochrome P450s in rat liver microsomes.Besides,the free radical scavenging abilities and the inhibition effects of CA on protein,lipid,DNA oxidative damage were stronger than its metabolites within a certain concentration range.Furthermore,CA significantly suppressed LPS-induced the production of reactive oxygen species（ROS）,which was more powerful than its metabolites.（2）To uncover the effects of CA on inflammation triggered cognitive defects and its underlying mechanisms,dietary CA supplementation on an intraperitoneal injection of LPS-stimulated C57BL/6J systemic inflammation mouse model was constructed.Results of animal behavior tests showed that supplementation of CA significantly decreased the escape latency and increased the crossings of the platform location,which implied the improvement in learning and cognitive abilities of LPS-induced mouse.Besides,CA obviously alleviated neuron impairment,promoted neuron proliferation,suppressed the excessive activation of glial cells and consequently reduced the levels of inflammatory mediators such as iNOS、COX-2、IL-6、IL-1βand TNF?.In addition,CA prevented LPS-induced expressions of amyloid precursor protein（APP）and neuronalβ-secretase 1（BACE1）,inhibiting the synthesis and accumulation ofβ-amyloid 1-42(Aβ_1-42).Furthermore,CA normalized the unbalanced cholinergic system by elevating acetylcholine（Ach）level and enhancing choline acetyl transferase（ChAT）activity,and suppressing acetylcholin esterase（AChE）activity.The results demonstrated that CA down regulated the phosphorylation of ERK、JNK、p38 MAPK and NFκB in LPS-induced mouse brain.On the other hand,the levels of nuclear transcription factor Nrf2,Keap-1 and their downstream II phase detoxification enzymes HO-1,NQO-1 and antioxidant enzymes including glutathione peroxidase-1（GPX-1）,manganese superoxide dismutase（MnSOD）and catalase（CAT）were enhanced in CA supplementation group,which contributed to the alleviation of CA in memory impairment and amyloidogenesis triggered by systemic inflammation.（3）In order to study the effects of CA on neuroinflammation,intervention of CA on LPS-stimulated BV2 microglia inflammation response and the underlying molecular mechanisms were investigated.Results demonstrated that CA significantly reversed LPS-elicited cell viability decrease,morphological changes,and reduced the levels of inflammatory mediators such as iNOS,COX-2,IL-1βand TNF-α.Besides,CA significantly restored LPS-induced defective mitochondrial membrane potential,promoted the protein expressions of mitochondrial respiratory chain complex I,IV,and elevated the levels of ATP and second messenger cAMP and Ca²⁺,which improving mitochondrial function.In addition,molecular modeling study demonstrated that six hydrogen bonds and twoπ-πinteractions were formed in the docking structure of CA with Keap-1,which contributing to the enhanced Nrf2 expression in the nucleus and downstream HO-1,NQO-1 expressions.CA increased the activities of CAT,SOD and the levels of GSH,suppressed the production of ROS,balancing cellular redox status.Furthermore,CA significantly inhibited the phosphorylation of MAPKs and AKT,restrained NFκB nucleus migration.The application of ROS scavenger N-acetyl-L-cysteine（NAC）revealed that ROS acted as an upstream signal involved in MAPKs,PI3K/Akt and NFκB signaling pathways in the anti-neuroinflammation activities of CA.（4）In order to study the effect of CA on the neuron damage induced by microglial inflammation,BV2 microglial conditioned medium and SH-SY5Y neuroblastoma cells co-culture system were conducted.Results showed that CA significantly reversed microglial cells conditioned medium-elicited cell viability decrease and morphological changes.Besides,CA notably decreased Bax/Bcl-2 ratio,inhibited cytochrome c release,caspase-3 activation,and PARP cleavage,suppressing inflammation-stimulated cell apoptosis.In addition,CA up-regulated autophagic extension related gene Atg5 and Atg12 mRNA levels,promoting the formation of autophagic vacuoles and overexpression of Beclin-1 and LC3 II.Results demonstrated that CA significantly restored defective mitochondrial membrane potential,promoted the protein expressions of mitochondrial respiratory chain complex I,IV and suppressed the production of ROS in SH-SY5Y cells,which improving mitochondrial function.And CA regulated redox homeostasis related signaling pathways such as MAPKs and PI3K/AKT.In addition,conditioned medium with CA notably increased the expressions of PGC1α,SIRT1 and enhanced the phosphorylation of AMPK,promoting mitochondria biogenesis.Furthermore,CA,NO scavenger,IL-1 receptor antagonist and TNF-αwere applied for further investigation.Results revealed that CA had directly neuroprotection effects on SH-SY5Y cells.On the other hand,the intervention of CA was partly depended on its inhibitory effects on released inflammatory cytokines by microglial cells.In conclusion,CA prevented neuroinflammation and the related cognitive disorder through improving mitochondria function and oxidative stress.This establishes a theoretical foundation for neuro-nutrition intervention studies of natural functional food components,and provides new clues for developing health foods containing CA.