Neuroprotective Effects Of Flavonoids Derived From Chinese Herbal Medicine And Their Underlying Mechanisms Exploration

ObjectiveAlzheimer's disease(AD)is one of intractably neurodegenerative diseases in the elderly.At present,the mainly clinical therapeutic drugs are acetylcholinesterase inhibitors(AchEIs)and N-methyl-D-aspartate receptors(NMDARs)antagonist,but their effects are limited.Flavonoids have been reported to some biological activities,including anti-inflammation,antioxidation,antitumor,estrogen-like effects,and the neuroprotective effects in neurodegeneration disease.In our present study,we prepared 15 flavonoids,which derived from Chinese herbal medicine,to choice better effects form two different models in vitro and research their underlying mechanisms.Our results suggest that 7MF and PIN might have beneficial effects for the treatment of neurodegenerative diseases such as AD and provide new clue for drug research.The flavonoids in our present study are comprised by seven flavanones including pinostrobin(PIN),(2S)-naringenin(2S-NAR),(±)-naringenin(NAR),7-methoxy-dihydroflavone(7MF),(2S)-alpinetin(ALP),ampelopsin(AMP)and liquiritigenin(LIQ),three flavones including myricetin(MYR),galangin(GAL)and wogonin(WOG),four isoflavones including genistein(GEN),daidzein(DAI),glycitein(GLY1)and glycitin(GLY2),and dihydrodaidzein(DDAI)as a dihydroisoflavone.Methods1.One of in vitro cell model was induced by glutamate in high-differentiated PC12 cells with neural phenotype to simulate the excitotoxic damage of neurons.PC12 cells were treated with different dosage flavonoids for 24 h and tested PC12 cell survival rate with CCK-8 kit.PC12 cells were pretreatment with or without flavonoids for 6 h then stimulated with glatamate for 24 h,PC12 cell survival rate were tested by CCK-8 kit.2.Another in vitro cell model was induced by lipopolysaccharide(LPS)in murine BV2 microglial cells to simulate neuroinflammation.BV2 cells were pretreatment with or without flavonoids for 30 min then stimulated with LPS for 24 h.The supernatants of the cell cultures were collected and measured by using ELISA kits for IL-6 and TNF-?.3.The mechanism of 7MF anti-neuroinflammatory effects in vivo and in vitro models.(1)LPS stimulated murine BV2 microglial cells and activated microglia.There are five groups including control group,LPS induced model group,and 7MF(10,20,40 ?M)pretreatment then induced by LPS group.BV2 cells were pretreatment with or without 7MF for 30 min then stimulated with LPS for 24 h.TNF-? and IL-6 mRNA levels were analyzed by qRT-PCR.The protein expression for iNOS,COX2,MCP-1,ICAM-1,TLR4,MyD88,p-p38,p38,p-ERK,ERK,p-JNK,JNK were analyzed by western blot assay.Intracellular reactive oxygen species(ROS)activity was detected with a fluorescent probe DCF-DA.LPS binding to the cell membrane receptor was observed by confocal microscope analysis.7MF treated BV2 cells with 5,10,20,40 ?M for 24 h,the protein expression for Nrf2,HO-1,NQO-1,p-AMPK,AMPK,p-LKB1 were analyzed by western blot assay.(2)Another microglia activated model was stimulated by the same dose of LPS in murine N9 cells.The following five groups including control group,LPS induced model group,and 7MF(10,20,40 ?M)pretreatment then induced by LPS group.N9 cells were pretreatment with or without 7MF for 30 min and then stimulated with LPS for 24 h.The key protein in MAPK signaling pathways including p-p38,p38,p-ERK,ERK,p-JNK,JNK were evaluated by western blot assay.(3)LPS was administered by intraperitoneal injection to induce neuroinflammation in C57BL/6J mice.Mice were divided randomly into control and LPS-treated groups with 7MF or vehicle,7MF was orally administered at a dose of 10,20 and 40 mg/kg for 3 days.With LPS treatment after 7MF treated 1 h on the third day,all mice were sacrificed 24 h later.IL-6 concentration in mouse serum was assayed for by ELISA.Activated microglia(markered with Iba-1 antibody)and astroglia(markered with GFAP antibody)were determined by immunohistochemical staining.The protein expression for p-AMPK,AMPK,p-LKB1,p-CaMK? were analyzed by western blot assay.4.The mechanism of PIN anti-neuroinflammatory effects in vivo and in vitro models.(1)LPS stimulated murine BV2 microglial cells and activated microglia.There are five groups including control group,LPS induced model group,and PIN(10,20,40 ?M)pretreatment then induced by LPS group.BV2 cells were pretreatment with or without PIN for 30 min then stimulated with LPS for 24 h.TNF-? and IL-6 mRNA levels were analyzed by qRT-PCR.The protein expression for COX2,p-p38,p38,p-ERK,ERK,p-JNK,JNK were analyzed by western blot assay.Intracellular ROS activity was detected with a fluorescent probe DCF-DA.LPS binding to the cell membrane receptor was observed by confocal microscope analysis.PIN treated BV2 cells with 5,10,20,40 ?M for 24 h,the protein expression for Nrf2,HO-1,NQO-1 were analyzed by western blot assay.(2)LPS was administered by intraperitoneal injection to induce neuroinflammation in C57BL/6J mice.Mice were divided randomly into control and LPS-treated groups with PIN or vehicle,PIN was orally administered at a dose of 10,20 and 40 mg/kg for 3 days.With LPS treatment after PIN treated 1 h on the third day,all mice were sacrificed 24 h later.IL-6 concentration in mouse serum was assayed for by ELISA.Activated microglia(markered with Iba-1 antibody)and astroglia(markered with GFAP antibody)were determined by immunohistochemical staining.Results1.Our results demonstrated that LIQ promoted PC12 cells growing and 2S-NAR,MYR,GAL,WOG and GEN inhibited growth within 15 flavonoids.PIN,2S-NAR,NAR,7MF,ALP,LIQ,GAL,WOG and DDAI protected PC12 cells from glutamate induced excitotoxic damage.AMP,MYR,GLY1 and GLY2 exacerbated glutamate neurotoxicity.But,2S-NAR,GAL and WOG protected PC12 cells from glutamate stimulating and the same dose inhibited normal growth.PIN and 7MF damaged PC12 cells at a dose of 80 ?M.2.Whole fifteen flavonoids inhibited the levels of TNF-? and IL-6 by LPS stimulated.PIN,2S-NAR,7MF,LIQ,GAL,GEN and DAI significantly inhibited pro-inflammatory cytokines TNF-? and IL-6 in a dose-dependent manner.NAR,MYR,WOG,GLY2 and DDAI significantly inhibited pro-inflammatory cytokines TNF-? and/or IL-6,but not in a concentration-dependent manner.ALP and AMP inhibited IL-6 production not in a dose-dependent manner,besides ALP,AMP and GLY1 have diminished inhibition for TNF-? at a higher dosage.3.Our results suggested that 7MF have anti-neuroinflammatory effects in vivo and in vitro.(1)LPS stimulated BV2 microglia cells leading to TLR4/MyD88/MAPK signaling pathway activation.The expression of iNOS,COX-2,MCP-1 and ICAM-1 were also detected.Our results showed that 7MF inhibited TNF-?and IL-6 expression at protein and transcription levels,7MF also inhibited iNOS,COX2,MCP-1 and ICAM-1 expression and decreased LPS-induced ROS production.7MF inhibited LPS binding to the cell membrane receptor and attenuated TLR4 and MyD88 expression in a dose-dependent manner.LPS induced BV2 cells increasing expression phosphorylates of p38 and JNK,but not ERK,in MAPK signaling pathway.7MF inhibited expressions of p-JNK,without effect for p-p38.Nrf2 is a redox-sensitive transcription factor and activate downstream anti-oxidative genes that include HO-1 and NQO1.AMPK stimulates antioxidant Nrf2 signaling.Our results showed that 7MF increased HO-1 and NQO1 protein levels through Nrf2 activation in BV2 microglia cells,which might be through increasing phosphorylation of AMPK and LKB1.(2)LPS induced N9 microglial cells increasing expression phosphorylates of p38,ERK and JNK in MAPK signaling pathway.Our results demonstrated that 7MF could inhibit expressions of p-ERK and p-JNK,without effect for p-p38 in N9 cells.(3)LPS successfully induced neuroinflammation in mice model by intraperitoneal injection,following microglia and astroglia were activated and phosphorylates of AMPK and CaMK? were decreased.Our results demonstrated that 7MF treatment could inhibit the microglia and astroglia activation and reduce serum IL-6 releasing.7MF promoted protein expression of p-AMPK and p-CaMK? in hippocampus,but without effect for p-LKB1.4.Our results suggested that PIN have anti-neuroinflammatory effects in vivo and in vitro.(1)LPS stimulated BV2 microglia cells leading to MAPK signaling pathway activation.The expression of COX-2 was also detected.Our results showed that PIN inhibited TNF-?and IL-6 expression at protein and transcription levels,PIN could also inhibit COX2 expression and decrease LPS-induced ROS production.PIN inhibited LPS binding to the cell membrane receptor.LPS induced BV2 cells increasing expression phosphorylates of p38 and JNK,but not ERK,in MAPK signaling pathway.PIN could inhibit expressions of p-JNK,without effect for p-p38.Nrf2 is a redox-sensitive transcription factor and activate downstream anti-oxidative genes that include HO-1 and NQO1.Our results showed that PIN increased HO-1 and NQO1 protein levels through Nrf2 activation in BV2 microglia cells.(2)LPS successfully induced neuroinflammation in mice model by intraperitoneal injection,following microglia and astroglia were activated.Our results demonstrated that PIN treatment could inhibit the microglia and astroglia activation and reduce serum IL-6 releasing.Conclusion1.PIN,7MF and LIQ have neuroprotective effects in PC12 cells damaged by glutamate and significantly inhibited pro-inflammatory cytokines TNF-? and IL-6 production in a dose-dependent manner stimulated by LPS in BV2 microglial cells.2.It is the first time to demonstrate 7MF attenuates the LPS-induced neuroinflammatory response in vitro and in vivo.7MF inhibited TNF-?and IL-6 expression at protein and transcription levels and also inhibited iNOS,COX2,MCP-1 and ICAM-1 expression and decreased LPS-induced ROS production.7MF exerts anti-neuroinflammatory effects induced by LPS via inhibiting TLR4/MyD88/MAPKs and activating AMPK/ Nrf2 signaling pathways.7MF also inhibited microglia and astroglia activation in mouse brain,these suggested that 7MF have anti-neuroinflammation effects in vivo.3.Our study demonstrated that PIN attenuates the LPS-induced neuroinflammatory response in vitro and in vivo.PIN inhibited TNF-?and IL-6 expression at protein and transcription levels and also inhibited COX2 expression and decreased LPS-induced ROS production.PIN inhibited LPS binding to cell membrane receptor and downregulated MAPKs and promoted Nrf2 signaling pathways activation.PIN also inhibited microglia and astroglia activation in mouse brain,these suggested that PIN have anti-neuroinflammation effects in vivo.Both in vivo and in vitro results suggested that 7MF have anti-neuroinflammatory effects and might be a potentially therapeutic for the prevention and therapy of neurodegenerative disorders.