OBJECTIVE: To observe the effects of electroacupuncture (EA) on improving motor function and regulating microglial activation based on Notch receptor 1 (Notch1)/Hes family bHLH transcription factor 1 (Hes1) pathway in mice with Parkinson's disease (PD). METHODS: Thirty-six male C57BL/6 mice were randomly divided into a control group, a model group and an EA group, 12 mice in each group. PD model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 consecutive days in the model group and the EA group. From the 1st day of modeling, EA was applied at "Baihui" (GV20) and bilateral "Shenshu" (BL23) in the EA group, with continuous wave, in frequency of 2 Hz and current of 2 mA, 15 min a time, once a day for 14 days continuously. The behavioral performance was evaluated by gait test, pole climbing test and hanging test, the number of positive cells of tyrosine hydroxylase (TH) and the co-expression positive cells of Notch1/ionized calcium binding adaptor molecule 1 (Iba-1) in the substantia nigra of midbrain was assessed by immunofluorescence, the protein expression of TH, α-synuclein (α-syn), Notch1, Hes1, Iba-1, inducible nitric oxide synthase (iNOS), Arginase-1 (ARG1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-10 was detected by Western blot, the mRNA expression of Notch1 and Hes1 was detected by real-time PCR. RESULTS: Compared with the control group, in the model group, the stride frequency was accelerated (P<0.001) and the stride length was shortened (P<0.001) for the four limbs, the pole climbing test time was prolonged (P<0.01) and the grip level was reduced (P<0.01); in the substantia nigra of midbrain, the number of positive cells of TH was decreased (P<0.001), the number of co-expression positive cells of Notch1/Iba-1 was increased (P<0.001), the protein expression of α-syn, Notch1, Hes1, Iba-1, iNOS, TNF-α, IL-1βand IL-6 was increased (P<0.01, P<0.05, P<0.001), the protein expression of TH, ARG1 and IL-10 was decreased (P<0.01, P<0.001), the mRNA expression of Notch1 and Hes1 was increased (P<0.01). Compared with the model group, in the EA group, the stride frequency was decelerated (P<0.001) and the stride length was increased (P<0.05, P<0.01, P<0.001) for the four limbs, the pole climbing test time was shortened (P<0.05) and the grip level was increased (P<0.05); in the substantia nigra of midbrain, the number of positive cells of TH was increased (P<0.01), the number of co-expression positive cells of Notch1/Iba-1 was decreased (P<0.001), the protein expression of α-syn, Notch1, Hes1, Iba-1, iNOS, TNF-α, IL-6 and IL-1β was decreased (P<0.05, P<0.01), the protein expression of TH, ARG1 and IL-10 was increased (P<0.05, P<0.001, P<0.01), the mRNA expression of Notch1 and Hes1 was decreased (P<0.05). CONCLUSION EA can improve the behavioral performance and protect the dopaminergic neurons in PD mice, its mechanism may relate to the inhibition of Notch1/Hes1-mediated neuroinflammation, thus inhibiting the microglial activation.
Animals ; Electroacupuncture ; Microglia/metabolism* ; Male ; Receptor, Notch1/metabolism* ; Parkinson Disease/physiopathology* ; Transcription Factor HES-1/metabolism* ; Mice ; Mice, Inbred C57BL ; Humans ; Signal Transduction

Objective:To investigate effect and mechanism of hydroxysafflor yellow A(HSYA)activating neuronal autophagy on cerebral ischemia-reperfusion injury through a combination of in vitro and in vivo experiments.Methods:SD rat MCAO/R model was established by improved suture method.Rats were randomly divided into sham surgery(Sham)group,MCAO/R group and MCAO/R+HSYA group,following indicators were detected to determine extent of cerebral ischemia-reperfusion nerve damage:Z-Longa neu-rological function score was detected,TTC staining to measure cerebral infarction area,and TUNEL staining to measure cell apopto-sis;Western blot was used to detect protein expressions of autophagy related markers LC3,Beclin1,P62 and SIRT1 in rat brain tis-sue;immunofluorescence staining was used to observe expression of LC3 co-localization with neurons.OGD/R injury model of SH-SY5Y cells was established and randomly divided into Normal group,OGD/R group,OGD/R+HSYA group,OGD/R+SIRT1 inhibitor(EX-527)group and OGD/R+EX-527+HSYA group.Western blot was used to detect protein expressions of LC3,Beclin1,P62 and SIRT1.Results:Compared with Sham group,model group rats showed impaired neurological function,significantly increased neu-robehavioral scores,widespread cerebral infarction,significantly increased neuronal cell apoptosis,significantly increased autophagy related protein Beclin1 expression and LC3-Ⅱ/LC3-Ⅰ,significantly decreased P62 expression,significantly increased LC3/NeuN co-stained cells,and decreased SIRT1 expression;compared with model group,HSYA intervention group showed a significant decrease in neurological functional scores,a significant reduction in cerebral infarction area,a significant decrease in neuronal cell apoptosis,a further increase in Beclin1 expression and LC3-Ⅱ/LC3-Ⅰ,a further decrease in P62 expression,number of LC3/NeuN and P62/NeuN co-stained cells also increased,and SIRT1 expression significantly increased.Expression trends of Beclin1,LC3-Ⅱ/LC3-Ⅰ,P62 and SIRT1 of cells between normal group,model group and HSYA intervention group were same as animal experiment;compared with model group,expressions of SIRT1,Beclin1 and LC3-Ⅱ/LC3-Ⅰ in OGD/R+EX-527 group were significantly reduced,while expression of P62 was significantly increased;compared with OGD/R+EX-527 group,there was no significant change in SIRT1 expression in OGD/R+EX-527+HSYA group,LC3-Ⅱ/LC3-Ⅰ and Beclin1 expression were significantly increased,and P62 expres-sion was significantly decreased.Conclusion:HSYA can significantly improve neurological deficits in rats after cerebral ischemia-reperfusion,reduce cerebral infarction area,and decrease neuronal cell apoptosis rate,whose neuroprotective effect may be related to its activation of SIRT1,which significantly enhances neuronal autophagy.

BACKGROUND:In the occurrence and development of demyelinating diseases of the central nervous system,neuroinflammation caused by microglia is the main pathological feature,so inhibiting the inflammatory response is very important to alleviate demyelination.Hydroxysafflor yellow A can protect the blood-brain barrier,inhibit neuronal apoptosis,and improve neurological function.OBJECTIVE:To explore the mechanism of hydroxysafflor yellow A inhibiting bicyclohexanone oxalyl dihydrazone-induced demyelination in mice.METHODS:(1)In vivo:Thirty healthy male C57BL/6 mice were randomly divided into three groups:normal group,cuprizone group,and hydroxysafflor yellow A group.The mice in the cuprizone group and the hydroxysafflor yellow A group were fed with 0.2％cuprizone diet for 6 weeks to establish mouse models of demyelination.The mice in the normal group were fed with normal diet.At the end of the 4th week,the mice in the hydroxysafflor yellow A group were intraperitoneally injected with hydroxysafflor yellow A 20 mg/kg per day.The mice in the normal and cuprizone groups were intraperitoneally injected with normal saline for 2 weeks.The behavioral changes of mice were evaluated by open field test and elevated plus maze test.The loss of myelin sheath in corpus callosum was detected by black gold staining,myelin basic protein and degraded myelin basic protein immunofluorescence staining.The activation of microglia and the expression of inflammatory factors were detected by I ba-1 immunofluorescence staining and ELISA,respectively.The protein expression levels of Toll-like receptor 4,myeloid differentiation factor 88,and nuclear factor κB p65 in the brain of mice in each group were detected by western blot assay.(2)In vitro experiment:The inflammation model of BV2 microglia was established by lipopolysaccharide induction.BV2 cells were divided into normal group,lipopolysaccharide group(1 μg/mL),and lipopolysaccharide(1 μg/mL)+hydroxysafflor yellow A(25 μmol/L)group.The expression levels of tumor necrosis factor α and interleukin 6 in the supernatant were detected by ELISA.RESULTS AND CONCLUSION:(1)Compared with the normal group,the mice in the cuprizone group had severe anxiety,abnormal autonomic movement ability,and a large amount of myelin sheath loss in the corpus callosum.The average fluorescence intensity of myelin basic protein was significantly reduced,and the average fluorescence intensity of degraded myelin basic protein was significantly increased.The number of lba1+microglia increased,the contents of interleukin 1β,tumor necrosis factor α,and interleukin 6 in the brain increased,and the protein expression levels of Toll-like receptor 4,myeloid differentiation factor 88,and nuclear factor κB p65 increased significantly.The above symptoms and indexes of mice were reversed after hydroxysafflor yellow A treatment.(2)Hydroxysafflor yellow A significantly inhibited the expression of inflammatory factors such as tumor necrosis factor α,and interleukin 6 induced by lipopolysaccharide in BV2 microglia.(3)The above results demonstrate that hydroxysafflor yellow A can significantly improve cuprizone-induced demyelination in mice.The mechanism of action is related to the inhibition of microglial activation-mediated inflammatory response through the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor κB p65 signaling pathway.

BACKGROUND:Neurodegenerative diseases are closely related to the imbalance of mitochondrial autophagy regulation.Previous studies by the research group have shown that lycium barbarum polysaccharide has neuroprotective effects,but whether it can improve the damage of SH-SY5Y cells induced byβ-amyloid protein 1-42 by regulating mitochondrial autophagy is still unclear.OBJECTIVE:To explore the protective effect and mechanism of Lycium barbarum polysaccharide on SH-SY5Y cells induced by β-amyloid protein 1-42.METHODS:An Alzheimer's disease cell model was established by inducing SH-SY5Y cells with β-amyloid protein 1-42,and then intervening with Lycium barbarum polysaccharide.SH-SY5Y cells were divided into three groups:control group,β-amyloid protein 1-42 group(20 μmol/L β-amyloid protein 1-42 for 24 hours),and Lycium barbarum polysaccharide group(1 g/L Lycium barbarum polysaccharide was added 1 hour in advance to form a protective effect,and then 20 μmol/L β-amyloid protein 1-42 was added to intervene with Lycium barbarum polysaccharide for 24 hours).CCK8 assay was used to detect cell viability.Mitochondrial membrane potential was detected by JC-1.TUNEL staining was used to detect cell apoptosis.Immunofluorescence and western blot assay were used to detect the expression of synaptic,apoptosis,and mitophagy-related indicators.RESULTS AND CONCLUSION:(1)Compared with the control group,the cell viability of the β-amyloid protein 1-42 group decreased(P＜0.05);cell apoptosis rate increased(P＜0.05);mitochondrial membrane potential decreased(P＜0.05);the expressions of pro-apoptotic proteins Bax and Caspase3 increased(P＜0.05);the expression of anti-apoptotic protein Bcl-2 decreased(P＜0.05);the expression levels of synaptic-related proteins Syn and PSD-95 decreased(P＜0.05);the expression levels of mitochondrial autophagy-related proteins Pink1,LC3A/B,Parkin,and Beclin-1 decreased(P＜0.05);and the expression of P62 increased(P＜0.05).(2)Compared with the β-amyloid protein 1-42 group,the cell viability in the Lycium barbarum polysaccharide group was increased(P＜0.05);the apoptosis rate was decreased(P＜0.05);the mitochondrial membrane potential was increased(P＜0.05);the expression levels of Bax and Caspase3 were decreased(P＜0.05);the expression of Bcl-2 was increased(P＜0.05);the expressions of Syn and PSD-95 were increased(P＜0.05);the expression levels of Pink1,LC3A/B,Parkin,and Beclin-1 were increased(P＜0.05),and the expression of P62 was decreased(P＜0.05).These findings indicate that Lycium barbarum polysaccharide may inhibit β-amyloid protein 1-42-induced damage to SH-SY5Y cells by regulating mitophagy,reduce cell apoptosis,and increase neuronal synaptic plasticity.

Objective:To investigate the effect and mechanism of lycium barbarum polysaccharide(LBP)on lipopolysaccharide(LPS)-induced inflammatory response of NLRP3 inflammasome in BV2 microglial cells.Methods:BV2 microglial cells were routinely cultured.CCK-8 assay was used to detect the effect of different concentrations(0.5,1,1.5,2 g/L)LBP on cell activity.Cells were di-vided into three groups:control group,LPS group and LBP+LPS group.Effect of LBP on LPS-induced cell activity was detected by CCK-8 method;RT-qPCR and immunofluorescence assay were used to detect NLRP3,ASC,Caspase-1,IL-18 and IL-1β expressions.Western blot was used to detect expressions of NLRP3,ASC,Caspase-1,TLR4,MyD88,NF-κB p65,IL-18,IL-1β and TNF-α pro-teins.Results:CCK-8 assay showed that 1 g/L LBP was the most applicable.Compared with control group,cell viability in LPS group was decreased;RT-qPCR,immunofluorescence and Western blot results showed that fluorescence intensity,mRNA and protein expres-sions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β were increased in LPS group.Western blot results showed that TLR4,MyD88,NF-κB p65 and TNF-α protein expressions were increased in LPS group.After LBP treatment,cell viability was increased;expres-sions of NLRP3,ASC,Caspase-1,NF-κB p65,TLR4,MyD88,IL-18,IL-1β and TNF-α were decreased.Conclusion:LBP may in-hibit LPS-induced NLRP3 inflammatory vesicles in BV2 cells via TLR4/MyD88/NF-κB signaling pathway.

Objective:To investigate the effect of ginkgolide B(GB)on astrocyte(AST)phagocytosis of myelin debris,and to investigate the mechanism of this functional therapy to demyelination by targeting AST.Methods:In vitro culture of AST,and divided AST into three groups:Control group,Myelin debris(Debris)group and Debris+GB group,incubed them in a constant temperature CO2 cell culture incubator for 24 hours,and then detected relevant indicators to observe the effect of GB on AST on phagocytic myelin debris.Results:Compared to the phagocytosis of myelin debris by primary AST,GB could effectively promote the phagocytosis by AST and show enhanced ABCA-1 expression(both P<0.05).Phagocytosis of myelin debris had no effect on the secretion of inflammatory cytokines by in vitro cultured AST.Debris+GB increased the expressions of neurotrophic CNTF and B-FGF compared to Debris(both P<0.05).Furthermore,Debris+GB decreased Bax and Caspase-3,while increased Bcl-2 expression(all P<0.05).Conclusion:GB can promote the phagocytosis of myelin debris by AST,which may be related to the upregulation of ABCA-1.Meanwhile,phagocytosis of myelin debris by AST increases the expression of the neurotrophic factors and the inhibits the apoptosis of AST themselves.

Neurodegenerative diseases are a group of chronic progressive diseases characterized by inflammation,degenera-tion and apoptosis.Chronic neuroinflammation is gradually becoming a potential pathogenic and predisposing factor.As a widely expressed non-histone nucleoprotein,HMGB1 participates in inflammatory process of human body through receptors of advanced glycation end products and Toll-like receptors while maintaining chromosome homeostasis.As a key factor of neuroinflammation,HMGB1 is widely involved in development of neurodegenerative diseases and may become a biomarker and a potential therapeutic target of neurodegenerative diseases.This article reviews the role of HMGB1 in neurodegenerative diseases and tries to provide ground-work for basic research and clinical application for targeting HMGB1 in the treatment of neurodegenerative diseases.

Objective:To explore effects and mechanism of Hydroxylsafflower Yellow A(HSYA)on expression of NLRP3 in glial cells after cerebral ischemic injury.Methods:A middle cerebral artery occlusion/reperfusion(MCAO/R)model was established in male SD rats.After successfully modeling for 24 h,Longa scoring and corner test were used to evaluate degree of neurological dys-function.Western blot and immunofluorescence were used to detect expressions of JAK2/STAT3 molecules and NLRP3,ELISA was used to measure IL-1β,IL-6 and TNF-α levels.A glucose-oxygen deprivation/reperfusion(OGD/R)model was established in microg-lia,and JAK2 and STAT3 inhibitor AG490 was used to further verify action of HSYA on NLRP3.Results:Compared with sham group,neurological dysfunction aggravated in MCAO/R group(P<0.01),HSYA treatment improved neurological function(P<0.01).Expres-sions of p-JAK2,p-STAT3 and NLRP3 in MCAO/R group were higher than those in the sham group(P<0.01);and HSYA treatment reduced expressions of p-JAK2,p-STAT3 and NLRP3(P<0.01).Levels of inflammatory factors IL-1β,IL-6 and TNF-α were higher in MCAO/R group than sham group(P<0.01),and HSYA inhibited expressions of IL-1β,IL-6 and TNF-α(P<0.01 or P<0.05).In vi-tro experiments showed expressions of p-JAK2,p-STAT3 and NLRP3 in OGD/R group were significantly higher than normal control group(P<0.01),after adding AG490,phosphorylation of JAK2 and STAT3 decreased,NLRP3 expression was inhibited(P<0.01).Inflammatory cytokines IL-1β,IL-6 and TNF-α levels were higher in OGD/R group than normal control group(P<0.01),and HSYA inhibited expressions of IL-1β,IL-6 and TNF-α(P<0.01 or P<0.05).Conclusion:HSYA alleviates brain damage,probably by regu-lating JAK2/STAT3 signaling pathway and inhibiting NLRP3 expression in microglia after cerebral ischemia and hypoxia.

BACKGROUND:In the occurrence and development of demyelinating diseases of the central nervous system,neuroinflammation caused by microglia is the main pathological feature,so inhibiting the inflammatory response is very important to alleviate demyelination.Hydroxysafflor yellow A can protect the blood-brain barrier,inhibit neuronal apoptosis,and improve neurological function.OBJECTIVE:To explore the mechanism of hydroxysafflor yellow A inhibiting bicyclohexanone oxalyl dihydrazone-induced demyelination in mice.METHODS:(1)In vivo:Thirty healthy male C57BL/6 mice were randomly divided into three groups:normal group,cuprizone group,and hydroxysafflor yellow A group.The mice in the cuprizone group and the hydroxysafflor yellow A group were fed with 0.2％cuprizone diet for 6 weeks to establish mouse models of demyelination.The mice in the normal group were fed with normal diet.At the end of the 4th week,the mice in the hydroxysafflor yellow A group were intraperitoneally injected with hydroxysafflor yellow A 20 mg/kg per day.The mice in the normal and cuprizone groups were intraperitoneally injected with normal saline for 2 weeks.The behavioral changes of mice were evaluated by open field test and elevated plus maze test.The loss of myelin sheath in corpus callosum was detected by black gold staining,myelin basic protein and degraded myelin basic protein immunofluorescence staining.The activation of microglia and the expression of inflammatory factors were detected by I ba-1 immunofluorescence staining and ELISA,respectively.The protein expression levels of Toll-like receptor 4,myeloid differentiation factor 88,and nuclear factor κB p65 in the brain of mice in each group were detected by western blot assay.(2)In vitro experiment:The inflammation model of BV2 microglia was established by lipopolysaccharide induction.BV2 cells were divided into normal group,lipopolysaccharide group(1 μg/mL),and lipopolysaccharide(1 μg/mL)+hydroxysafflor yellow A(25 μmol/L)group.The expression levels of tumor necrosis factor α and interleukin 6 in the supernatant were detected by ELISA.RESULTS AND CONCLUSION:(1)Compared with the normal group,the mice in the cuprizone group had severe anxiety,abnormal autonomic movement ability,and a large amount of myelin sheath loss in the corpus callosum.The average fluorescence intensity of myelin basic protein was significantly reduced,and the average fluorescence intensity of degraded myelin basic protein was significantly increased.The number of lba1+microglia increased,the contents of interleukin 1β,tumor necrosis factor α,and interleukin 6 in the brain increased,and the protein expression levels of Toll-like receptor 4,myeloid differentiation factor 88,and nuclear factor κB p65 increased significantly.The above symptoms and indexes of mice were reversed after hydroxysafflor yellow A treatment.(2)Hydroxysafflor yellow A significantly inhibited the expression of inflammatory factors such as tumor necrosis factor α,and interleukin 6 induced by lipopolysaccharide in BV2 microglia.(3)The above results demonstrate that hydroxysafflor yellow A can significantly improve cuprizone-induced demyelination in mice.The mechanism of action is related to the inhibition of microglial activation-mediated inflammatory response through the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor κB p65 signaling pathway.

BACKGROUND:Neurodegenerative diseases are closely related to the imbalance of mitochondrial autophagy regulation.Previous studies by the research group have shown that lycium barbarum polysaccharide has neuroprotective effects,but whether it can improve the damage of SH-SY5Y cells induced byβ-amyloid protein 1-42 by regulating mitochondrial autophagy is still unclear.OBJECTIVE:To explore the protective effect and mechanism of Lycium barbarum polysaccharide on SH-SY5Y cells induced by β-amyloid protein 1-42.METHODS:An Alzheimer's disease cell model was established by inducing SH-SY5Y cells with β-amyloid protein 1-42,and then intervening with Lycium barbarum polysaccharide.SH-SY5Y cells were divided into three groups:control group,β-amyloid protein 1-42 group(20 μmol/L β-amyloid protein 1-42 for 24 hours),and Lycium barbarum polysaccharide group(1 g/L Lycium barbarum polysaccharide was added 1 hour in advance to form a protective effect,and then 20 μmol/L β-amyloid protein 1-42 was added to intervene with Lycium barbarum polysaccharide for 24 hours).CCK8 assay was used to detect cell viability.Mitochondrial membrane potential was detected by JC-1.TUNEL staining was used to detect cell apoptosis.Immunofluorescence and western blot assay were used to detect the expression of synaptic,apoptosis,and mitophagy-related indicators.RESULTS AND CONCLUSION:(1)Compared with the control group,the cell viability of the β-amyloid protein 1-42 group decreased(P＜0.05);cell apoptosis rate increased(P＜0.05);mitochondrial membrane potential decreased(P＜0.05);the expressions of pro-apoptotic proteins Bax and Caspase3 increased(P＜0.05);the expression of anti-apoptotic protein Bcl-2 decreased(P＜0.05);the expression levels of synaptic-related proteins Syn and PSD-95 decreased(P＜0.05);the expression levels of mitochondrial autophagy-related proteins Pink1,LC3A/B,Parkin,and Beclin-1 decreased(P＜0.05);and the expression of P62 increased(P＜0.05).(2)Compared with the β-amyloid protein 1-42 group,the cell viability in the Lycium barbarum polysaccharide group was increased(P＜0.05);the apoptosis rate was decreased(P＜0.05);the mitochondrial membrane potential was increased(P＜0.05);the expression levels of Bax and Caspase3 were decreased(P＜0.05);the expression of Bcl-2 was increased(P＜0.05);the expressions of Syn and PSD-95 were increased(P＜0.05);the expression levels of Pink1,LC3A/B,Parkin,and Beclin-1 were increased(P＜0.05),and the expression of P62 was decreased(P＜0.05).These findings indicate that Lycium barbarum polysaccharide may inhibit β-amyloid protein 1-42-induced damage to SH-SY5Y cells by regulating mitophagy,reduce cell apoptosis,and increase neuronal synaptic plasticity.