Studies On The Neuroprotective Components Of Five Medicinal Mushrooms And Molecular Mechanism

Alzheimer’s disease（AD）is a neurodegenerative disease related to aging,with a prevalence rate of 5%to 10%in the elderly over 60 years old,which has become the main threat to the elderly health disease,caused by heavy social and economic burden.So far,the AD pathogenesis is unclear,inflammation,oxidative stress mitochondrial damage plays a key role in the pathogenesis of AD.Existing drug efficacy is limited,can only ease the symptoms.It has been reported that most crude extracts of medicinal mushrooms have anti-AD effects,but the effective components and the molecular mechanism are not clear.Natural products are important resources for discovering anti-AD drugs with novel structure and significant activity,and have become a hot spot in the field of natural product chemistry.In this study,the metabolites from mushrooms such as I.obliquus,G.resinaceum,G.lucidum,I.hispidus and C.africanus,using a variety of chromatographic methods,combined with the modern spectrum technology,isolated and identified 98compounds,including 27 new compounds.High active compounds are studied via cell experiments on the effect of nerve inflammation,oxidative stress and mitochondrial dysfunction,reveals the underlying molecular mechanism of neuroprotective effect.The main conclusions are as follows:（1）17 compounds were isolated and identified from the fruiting bodies of I.obliquus,including eight new compounds,involving lanostane triterpenoids and steroids.Using LPS-induced BV-2 microglial cells inflammation model,the nitric oxide（NO）inhibitory activities of all compounds were screened,and inonotusol K（5）showed significant NO inhibition with IC₅₀ value of 2.32μM.Western blot and molecular docking showed that inonotusol I（2）and 5 could bind to i NOS to inhibit the expression of i NOS protein,and then inhibit the production of NO.In conclusion,compound 2 and 5 could improve neuroinflammation by inhibiting the expression of i NOS protein.In addition,the neuroprotective effect of 2α-hydroxy-inotodiol（2α-HI,9）was studied.The oxidative stress injury model of SH-SY5Y cells was induced by H₂O₂.The results showed that pre-treatment with 2α-HI for 12 h significantly reduced the levels of MDA and ROS,increased the activity of SOD,and promoted the nuclear translocation of Nrf2 and the expression of downstream related antioxidant genes.Immunofluorescence staining,JC-1 staining and Western blot results,2α-HI could improve mitochondrial dysfunction and significantly inhibit cell apoptosis.Through inhibitors and si RNA experiments,it was first demonstrated that the neuroprotective effect of 2α-HI may depend on the BDNF/Trk B/ERK/CREB and Nrf2 signaling pathways.Therefore,2α-HI had a potential protective effect on oxidative stress injury,improved mitochondrial function and oxidative stress injury,and inhibited cell apoptosis.The BDNF/Trk B/ERK/CREB and Nrf2 signaling pathways was found to mediate the neuroprotective effect of the 2α-HI.（2）32 compounds were isolated and identified from the fruiting bodies of G.resinaceum,including five new compounds,the structure types involve lanostane triterpenoids,meroterpenoids and steroids.Using LPS-induced BV-2 microglial cells inflammation model,the intervention of all compounds on neuroinflammation was investigated.The results showed that the new compound ganoresinoid A（1）had the best inhibitory effect on NO,with IC₅₀ value of 5.41μM.Immunofluorescence staining,q RT-PCR and Western blot analysis showed that ganoresinoid A significantly inhibited the activation of NF-κB and MAPK,decreased the expression of inflammatory related proteins i NOS and COX-2,and decreased the expression of TLR-4 receptor.Further ROS and JC-1staining analysis showed that the compound significantly inhibited ROS generation and effectively improved mitochondrial membrane potential.In addition,the ganoresinoid A intervention also played a neuroprotective role by activating the antioxidant related Akt/GSK-3β/Nrf2 signaling pathway.By inhibitor intervention,the effect of the compound on neuroinflammation depended on the TLR-4/NF-κB/MAPK signaling pathway.In this study,we found for the first time that ganoresinoid A played a neuroprotective role through TLR-4/NF-κB/MAPK and Akt/GSK-3β/Nrf2 pathways,which may be closely related to its effective improvement of mitochondrial dysfunction and inhibition of apoptosis.（3）13 compounds were isolated and identified from the fruiting bodied of G.lucidum,including one new compound.The structures involved mainly included lanostane triterpenoids and steroids.LPS-induced BV-2 microglial cells inflammatory model showed that the new compound ganoderterpene A（1）significantly inhibited intracellular NO production with IC₅₀ value of 7.15μM.By immunofluorescence staining,q RT-PCR and Western blot analysis showed that ganoderterpene A inhibited the activation of TLR-4/NF-κB/MAPK signaling pathway and down-regulated the expression of inflammatory cytokines IL-6,IL-1β,TNF-α,i NOS and COX-2.Further studies showed that the compound could improve MMP,inhibit ROS production,increase Bcl-2/Bax ratio,reduce cytochrome c release,inhibit caspase pathway,and then inhibit apoptosis.In addition,the inhibitor experiments showed that TLR-4/NF-κB/MAPK signaling pathway played an important mediating role in the regulation of neuroinflammation by this compound.Therefore,ganoderterpene A played a neuroprotective role by improving mitochondrial dysfunction,inhibiting apoptosis and inhibiting TLR-4/NF-κB/MAPK signaling pathway.（4）15 compounds were isolated and identified from the fruiting bodies of I.hispidus,including four new compounds.The structures involved mainly included phenolic compounds,lanostane triterpenoids,sesquiterpenoids and steroids.From the perspective of neuroinflammation,catechol congeners pre-treatment significantly inhibited the activation of NF-κB and the expression of downstream inflammatory proteins i NOS and COX-2,and decreased the protein level of TLR-4 receptor.Further studies also showed that these compounds could significantly promote the neurite growth-promoting in PC-12 cells,and had significant scavenging ability on DPPH free radical.（5）26 compounds,including nine new compounds were isolated and identified from the metabolites of C.africanus 5.1117 rice medium.The structures involved mainly included cyathane-type diterpenoids and sesquiterpenoids.Among them,neocyathin L（1）and neocyathin K（8）were novel compounds with open ring skeleton,and the drimane-type sesquiterpenes were isolated from C.africanus for the first time.In the presence of nerve growth factor,compounds 1 and 8 showed the most significant neurotrophic activity,while the drimane-type sesquiterpenoids 3β,6α-dihydroxycinnamolide（6-19）、2-keto-3β,6β-dihydroxycinnamolide（6-20）and 3β,6β-dihydroxycinnamolide（6-21）showed moderate neurotrophic activity.In conclusion,from the perspectives of neuroinflammation,oxidative stress and mitochondrial dysfunction in a variety of vitro cells models,the highly active compounds2α-hydroxy-inotodiol、ganoresinoid A、ganoderterpene A were revealed the neuroprotective effect for the first time via BDNF/Trk B/ERK/CREB、Nrf2 and TLR-4/NF-κB/MAPK signaling pathway,providing a new theoretical basis for the development and recycle of these five medicinal mushrooms and neuroprotective active substances.