Immunomodulatory effect of astragaloside IV on T cells of experimental autoimmune encephalomyelitis mice / 中国组织工程研究

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.

Regulatory effect of Ganoderma lucidum polysaccharides on H2O2-induced apoptosis and mitochondrial dysfunction in SH-SY5Y cells / 中国组织工程研究

BACKGROUND:Current studies have confirmed that Ganoderma lucidum polysaccharides can promote nerve regeneration in neurodegeneration-related diseases.The occurrence of neurodegenerative diseases is closely related to mitochondrial dysfunction,but the role of Ganoderma lucidum polysaccharides on the regulation of apoptosis and mitochondrial function in neurodegenerative diseases is not yet clarified. OBJECTIVE:To explore the regulatory effects and mechanisms of Ganoderma lucidum polysaccharides on apoptosis and mitochondrial dysfunction in H2O2-induced SH-SY5Y cells. METHODS:SH-SY5Y cells were divided into three groups:control group,H2O2 group,and Ganoderma lucidum polysaccharides group.Cells in the control group were normally cultured.Cells in the H2O2 group were treated with 300 μmol/L H2O2 for 24 hours.In the Ganoderma lucidum polysaccharides group,the intervention with 300 μg/L Ganoderma lucidum polysaccharides was conducted first for 1-2 hours,followed by the addition of 300 μmol/L H2O2 for 24 hours.The mitochondrial membrane potential was detected by JC-1 kit.Apoptosis was detected by TUNEL staining kit.The activities of malondialdehyde and superoxide dismutase were detected by malondialdehyde test kit and superoxide dismutase test kit,respectively.The apoptosis and expression of mitochondrial dynamics-related proteins were detected by immunofluorescence staining and western blot assay. RESULTS AND CONCLUSION:(1)Compared with the control group,the mitochondrial membrane potential and superoxide dismutase activity were significantly reduced,as well as apoptotic rate and malondialdehyde levels were significantly increased in the H2O2 group(P<0.05).After treatment with Ganoderma lucidum polysaccharides,the membrane potential and superoxide dismutase activities were significantly increased,and apoptotic rate and malondialdehyde levels were significantly reduced compared with the H2O2 group(P<0.05).(2)The expression levels of pro-apoptotic proteins Bax and Caspase-3 were significantly increased,but the expression of anti-apoptotic protein Bcl-2 was significantly decreased in the H2O2 group compared with the control group(P<0.05).Compared with the H2O2 group,the levels of Bax and Caspase-3 were significantly decreased,but the expression of anti-apoptotic protein Bcl-2 was significantly increased in the Ganoderma lucidum polysaccharides group(P<0.05).(3)Compared with the control group,the expression of mitochondrial splitting proteins Fis1 and p-Drp1 was significantly increased,but the expression of mitochondrial fusion proteins OPA1,Mfn1,and Mfn2 was decreased in the H2O2 group(P<0.05).Compared with the H2O2 group,Fis1 and p-Drp1 expression was significantly reduced,but the expression levels of OPA1,Mfn1,and Mfn2 were significantly increased in the Ganoderma lucidum polysaccharides group(P<0.05).(4)The above results confirm that Ganoderma lucidum polysaccharides can attenuate H2O2-induced oxidative stress damage and apoptosis in SH-SY5Y cells by ameliorating mitochondrial dysfunction.

Astragaloside inhibits astrocyte activation and inflammatory response induced by inflammation / 中国组织工程研究

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 μmol/L and the final concentration of DAPT was 50 μmol/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.

Knock-down of ROCK2 gene improves cognitive function and reduces neuronal apoptosis in AD mice by promoting mitochondrial fusion and inhibiting its division / 细胞与分子免疫学杂志

Objective To explore the effect of knocking down Rho-associated coiled-coil kinase (ROCK2) gene on the cognitive function of amyloid precursor protein/presenilin-1 (APP/PS1) double transgenic mice and its mechanism. Methods APP/PS1 double transgenic mice were randomly divided into AD model group (AD group), ROCK2 gene knock-down group (shROCK2 group), ROCK2 gene knock-down control group (shNCgroup), and wild-type C57BL/6 mice of the same age served as the wild-type control (WT group). Morris water maze and Y maze were employed to test the cognitive function of mice. Neuron morphology was detected by Nissl staining. Immunofluorescence histochemical staining was used to detect the expression of phosphorylated dynamin-related protein 1 (p-Drp1) and mitochondrial fusion 1 (Mfn1). Western blot analysis was used to detect the expression ROCK2, cleaved-caspase-3 (c-caspase-3), B-cell lymphoma 2 (Bcl2), Bcl2-related protein X (BAX), p-Drp1, mitochondrial fission 1 (Fis1), optic atrophy 1 (OPA1), Mfn1 and Mfn2. Results Compared with AD group mice, the expression of ROCK2 in shROCK2 group mice was significantly reduced; the cognitive function was significantly improved with the number of neurons in the hippocampal CA3 and DG areas increasing, and nissl bodies were deeply stained; the expression of c-caspase-3 and BAX was decreased, while the expression of Bcl2 was increased; the expression of mitochondrial division related proteins p-Drp1 and Fis1 were decreased, while the expression of mitochondrial fusion-related proteins OPA1, Mfn1 and Mfn2 were increased. Conclusion Knock-down of ROCK2 gene can significantly improve the cognitive function and inhibit the apoptosis of nerve cells of APP/PS1 mice. The mechanism may be related to promoting mitochondrial fusion and inhibiting its division.

Effects of astragaloside Ⅳ on inflammatory responses in astrocytes induced by lipopolysaccharide in vitro and its mechanism / 中华微生物学和免疫学杂志

Objective To investigate whether astragaloside ( AST) Ⅳ could inhibit lipopolysac-charide (LPS)-induced activation of astrocytes and the possible mechanism. Methods Effects of different concentrations of AST Ⅳ on astrocyte viability were determined by MTT to select the optimum concentration for the following experiments. Primary astrocytes were induced by LPS to construct the inflammatory model. Astrocytes were divided into three groups: PBS, LPS and LPS+ASTⅣ(LPS+ASTⅣ) groups. The release of nitric oxide (NO) was detected by Griess method. Expression of glial fibrillary acidic protein ( GFAP), an astrocyte marker, and TLR4 were analyzed by immunohistochemistry and Western blot. Cytokines of IL-6, TNF-α, IL-4 and IL-10 in the supernatants of cell culture for 24 h collected after stimulation were meas-ured by ELISA. Results ASTⅣsignificantly inhibited the LPS-induced activation of astrocytes and NO re-lease (P<0. 01), suppressed the expression of TLR4 (P<0. 05) and reduced the secretion of IL-6 (P<0. 01) and TNF-α (P<0. 01), but increased the secretion of IL-4 and IL-10 (P<0. 05 and P<0. 01). Con-clusion AST Ⅳ could significantly inhibit the LPS-induced activation of astrocytes and suppress inflamma-tory responses, and the possible mechanism might be related to reduced secretion of inflammatory factors af-ter blocking the expression of TLR4.