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.

BACKGROUND:A large number of studies have shown that neurodegenerative diseases are closely related to oxidative stress injury and the imbalance of mitochondrial dynamics.Lycium barbarum polysaccharides have a neuroprotective effect.However,it is not clear whether lycium barbarum polysaccharides can ameliorate apoptosis induced by oxidative stress injury by regulating abnormal mitochondrial dynamics.OBJECTIVE:To study the effect of lycium barbarum polysaccharides on apoptosis induced by H2O2 in SH-SY5Y human neuroblastoma cells.METHODS:SH-SY5Y cells were cultured in three groups.The control group was cultured for 24 hours.The hydrogen peroxide group was treated with H2O2 for 24 hours,and the lycium barbarum polysaccharide group was treated with lycium barbarum polysaccharide for 2 hours and then treated with H2O2 for 24 hours.After treatment,the levels of malondialdehyde,glutathione,and superoxide dismutase in the precipitation of the cells were detected by kit.Mitochondrial membrane potential was detected by JC-1 kit.Cell viability was detected by MTT assay.Apoptosis was detected by TUNEL.The expression levels of mitochondrial dynamics-related proteins (phosphorylated promoter protein 1,mitochondrial fission protein 1,mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1) and apoptotic proteins (Bax,Bcl-2,and Caspase-3) were detected by immunofluorescence staining and western blot assay.RESULTS AND CONCLUSION:(1) Compared with the control group,the levels of malondialdehyde were increased (P<0.05),and the levels of superoxide dismutase and glutathione were decreased (P<0.05) in the H2O2 group.Compared with the H2O2 group,the malondialdehyde level was decreased (P<0.05),and the superoxide dismutase and glutathione levels were increased (P<0.05) in the lycium barbarum polysaccharide group.(2) The mitochondrial membrane potential in the H2O2 group was lower than that in the control group (P<0.05),and that of lycium barbarum polysaccharide group was higher than that of the H2O2 group (P<0.05).(3) Compared with the control group,the apoptosis rate and the expression of Bax and Caspase-3 protein were increased (P<0.05),while the cell viability and the expression of Bcl-2 protein were decreased (P<0.05) in the H2O2 group.Compared with the H2O2 group,the apoptosis rate and the expression of Bax and Caspase-3 protein were decreased (P<0.05),while the cell viability and the expression of Bcl-2 protein were increased (P<0.05) in the lycium barbarum polysaccharide group.(4) Compared with the control group,the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were increased (P<0.05),and the protein expression levels of mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1 were decreased (P<0.05) in the H2O2 group.Compared with the H2O2 group,the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were decreased (P<0.05),and the protein expression levels of mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1 were increased (P<0.05) in the lycium barbarum polysaccharide group.(5) These results indicate that lycium barbarum polysaccharide can improve SH-SY5Y cell apoptosis caused by oxidative stress damage by regulating mitochondrial dynamics.

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.

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.

BACKGROUND:A large number of studies have shown that neurodegenerative diseases are closely related to oxidative stress injury and the imbalance of mitochondrial dynamics.Lycium barbarum polysaccharides have a neuroprotective effect.However,it is not clear whether lycium barbarum polysaccharides can ameliorate apoptosis induced by oxidative stress injury by regulating abnormal mitochondrial dynamics.OBJECTIVE:To study the effect of lycium barbarum polysaccharides on apoptosis induced by H2O2 in SH-SY5Y human neuroblastoma cells.METHODS:SH-SY5Y cells were cultured in three groups.The control group was cultured for 24 hours.The hydrogen peroxide group was treated with H2O2 for 24 hours,and the lycium barbarum polysaccharide group was treated with lycium barbarum polysaccharide for 2 hours and then treated with H2O2 for 24 hours.After treatment,the levels of malondialdehyde,glutathione,and superoxide dismutase in the precipitation of the cells were detected by kit.Mitochondrial membrane potential was detected by JC-1 kit.Cell viability was detected by MTT assay.Apoptosis was detected by TUNEL.The expression levels of mitochondrial dynamics-related proteins (phosphorylated promoter protein 1,mitochondrial fission protein 1,mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1) and apoptotic proteins (Bax,Bcl-2,and Caspase-3) were detected by immunofluorescence staining and western blot assay.RESULTS AND CONCLUSION:(1) Compared with the control group,the levels of malondialdehyde were increased (P<0.05),and the levels of superoxide dismutase and glutathione were decreased (P<0.05) in the H2O2 group.Compared with the H2O2 group,the malondialdehyde level was decreased (P<0.05),and the superoxide dismutase and glutathione levels were increased (P<0.05) in the lycium barbarum polysaccharide group.(2) The mitochondrial membrane potential in the H2O2 group was lower than that in the control group (P<0.05),and that of lycium barbarum polysaccharide group was higher than that of the H2O2 group (P<0.05).(3) Compared with the control group,the apoptosis rate and the expression of Bax and Caspase-3 protein were increased (P<0.05),while the cell viability and the expression of Bcl-2 protein were decreased (P<0.05) in the H2O2 group.Compared with the H2O2 group,the apoptosis rate and the expression of Bax and Caspase-3 protein were decreased (P<0.05),while the cell viability and the expression of Bcl-2 protein were increased (P<0.05) in the lycium barbarum polysaccharide group.(4) Compared with the control group,the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were increased (P<0.05),and the protein expression levels of mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1 were decreased (P<0.05) in the H2O2 group.Compared with the H2O2 group,the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were decreased (P<0.05),and the protein expression levels of mitochondrial fusion protein 1,mitochondrial fusion protein 2,and optic atrophy protein 1 were increased (P<0.05) in the lycium barbarum polysaccharide group.(5) These results indicate that lycium barbarum polysaccharide can improve SH-SY5Y cell apoptosis caused by oxidative stress damage by regulating mitochondrial dynamics.

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 study the efficacy evaluation of triptolide(TP) in rats with cerebral ischemia-reperfusion(I/R) injury and its mechanism. METHODS Rat brain I/R injury model was copied by middle cerebral artery wire embolism surgery, and TP (0.1, 0.2 mg·kg−1) was given to the treatment group, and set the sham surgery group. The Longa score method was used to measure the neural function of rats, and Niselferi staining was used to show the morphology of neurons in the ischemic side brain tissue of rats, immunofluorescence was used to detect the expression levels of MAP2 and Syn in ischemic lateral brain tissue. The expression levels of cAMP, PKA, BDNF, Syn and PSD-95 were detected by Western blotting. RESULTS Compared with the model group, the neurological scores of TP treatment group decreased significantly(P<0.01 or P<0.001), it had a protective effect on damaged neurons. Compared with the model group, cAMP, PKA, BDNF, Syn and PSD-95 in TP treatment group were significantly up-regulated. CONCLUSION TP treatment can significantly improve I/R injury, and the mechanism may be related to the activation of the cAMP/PKA/BDNF signaling pathway.

BACKGROUND:In the initial stage of multiple sclerosis,central immune cells activate and release a large number of inflammatory factors,causing white matter demyelination and even involving gray matter neurons.The equilibrium of differentiation between different subsets of CD4+ T cells plays an important role in the progression of experimental autoimmune encephalomyelitis.The previous results of the research group showed that the active ingredient astragalus glycoprotein in astragalus can regulate the immune response in experimental autoimmune encephalomyelitis mice,and whether it has a regulatory effect on the differentiation of T cell subsets has not been determined. OBJECTIVE:To explore the therapeutic effects and immune regulatory mechanisms of astragaloside IV on experimental autoimmune encephalomyelitis mice. METHODS:Female C57BL/6 mice were divided into the normal control group,experimental autoimmune encephalomyelitis disease model group,and astragaloside IV treatment group(n=8 per group).Myelin oligodendrocyte glycoprotein peptides 35-55 were used for experimental autoimmune encephalomyelitis model induction in the last two groups.On day 10 to 28 after immunization,the astragaloside IV treatment group was treated with 40 mg/kg per day astragaloside IV intragastrically.Body weight and clinical scores of mice in each group were recorded from the immunization day to the 28th day.On the 28th day after immunization,the mouse spinal cord was taken and made into frozen sections for hematoxylin-eosin staining and Lux fast blue staining to observe pathological changes in the spinal cord.Percentage of splenic T cell subsets was detected using flow cytometry.Western blot assay was used to determine the protein expression of interferon-γ,interleukin-17 and interleukin-6 in the spinal cord.Levels of interferon-γ,interleukin-17,interleukin-6 and interleukin-4 in supernatants of cultured splenocytes were determined by ELISA. RESULTS AND CONCLUSION:(1)Compared with the experimental autoimmune encephalomyelitis disease model group,astragaloside IV could reduce the degree of weight loss in experimental autoimmune encephalomyelitis mice(P<0.05),ameliorate clinical symptoms(P<0.05),inhibit the infiltration of inflammatory cells and alleviate myelin loss(P<0.01,P<0.05).(2)Compared with the experimental autoimmune encephalomyelitis disease model group,astragaloside IV could inhibit the proportion of CD4+T cell subsets expressing interferon-γ(P<0.001)and interleukin-17(P<0.001),but increase percentages of CD4+ interleukin-10+(P<0.001)and CD4+ transforming growth factor-β+(P<0.01)T cell subsets.(3)Astragaloside IV could inhibit the expression of interferon-γ(P<0.05,P<0.01),interleukin-17(P<0.05,P<0.05),and interleukin-6(P<0.05,P<0.05)in the spinal cord and spleen,and up-regulate the expression of interleukin-4(P<0.01)in spleen.(4)These findings confirm that astragaloside IV alleviates clinical symptoms in experimental autoimmune encephalomyelitis mice,which may be related to regulating the splenic T cell subsets,therefore,inhibiting the infiltration of inflammatory cells into the center and reducing the demyelination.

BACKGROUND:Astrocytes play an important role in the pathology of central nervous system diseases.The phenotypic and functional changes in astrocytes suggest that it may be an effective therapeutic target for central nervous system diseases.Our previous studies have confirmed that astragaloside can inhibit the lipopolysaccharide-induced astrocyte inflammatory response.Whether astragaloside can regulate the phenotype and function of astrocytes through Notch-1 and its downstream signaling pathway remains unclear. OBJECTIVE:To explore the effect of astragaloside on astrocyte activation and inflammatory response induced by inflammation and its possible mechanism. METHODS:Cerebral cortex astrocytes derived from neonatal C57BL/6 mouse were cultured in vitro.CCK-8 assay was used to determine the optimum concentration of astragaloside and Notch active inhibitor DAPT.The astrocytes were divided into five groups:PBS group,lipopolysaccharide group,lipopolysaccharide + astragaloside group,lipopolysaccharide + DAPT group and lipopolysaccharide + DAPT + astragaloside group.The secretion level of inflammatory factors was detected by ELISA,and the level of nitric oxide was detected by Griess method.The astrocytes and splenic mononuclear cells were co-cultured in Transwell chamber to observe the migration of CD4T cells.The expression of astrocyte activation marker GFAP,A1 marker C3 and A2 marker S100A10 as well as Notch 1 and Jag-1 was detected by immunofluorescence staining.The expressions of CFB,C3,S100A10,PTX3,Notch-1,Jag-1,and Hes were detected by western blot assay. RESULTS AND CONCLUSION:(1)According to the results of CCK8 assay,the final concentration of astragaloside was selected as 25 &mu;mol/L and the final concentration of DAPT was 50 &mu;mol/L for follow-up experiments.(2)Compared with PBS group,interleukin-6,interleukin-12 and nitric oxide secretion levels in the lipopolysaccharide group were significantly increased(P<0.05,P<0.05,P<0.01).Compared with the lipopolysaccharide group,interleukin-6(all P<0.05),interleukin-12(P>0.05,P<0.05,P<0.05)and nitric oxide(P<0.05,P<0.01,P<0.01)secretion significantly reduced in the lipopolysaccharide + astragaloside group,lipopolysaccharide +DAPT group,lipopolysaccharide + DAPT + astragaloside group.(3)Compared with the PBS group,the expression of GFAP that is the marker of activated astrocytes and the migration of CD4 T cells were significantly increased in the lipopolysaccharide group(P<0.01).Compared with the lipopolysaccharide group,astrocyte activation was significantly inhibited and CD4 T cell migration was significantly reduced in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group(P<0.05,P<0.05,P<0.01).(4)Compared with the PBS group,the expressions of A1 markers C3 and CFB in the lipopolysaccharide group were increased(P<0.01,P<0.05),and the expressions of A2 markers S100A10 and PTX3 were decreased(P<0.01,P<0.05).Compared with the lipopolysaccharide group,C3(all P<0.01)and CFB(both P<0.05)were significantly reduced and S100A10(all P<0.01)and PTX3(P<0.05,P<0.05 and P>0.05)were increased in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group.(5)Compared with the PBS group,the expressions of Jag-1,Notch-1 and Hes in the lipopolysaccharide group were significantly increased(all P<0.01).Compared with the lipopolysaccharide group,the expressions of Jag-1(all P<0.01),Notch-1(all P<0.01)and Hes(P<0.05,P<0.01 and P<0.01)were significantly reduced in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group.(6)The results indicate that astragaloside can promote the transformation of astrocytes from A1 to A2 by regulating Notch-1 signaling pathway,reduce the secretion of inflammatory factors and the migration of CD4 T cells,and thus inhibit astrocyte activation and inflammatory response.