To evaluate the antioxidant activities of different chemical constituents from Astragalus mongholicus Bunge and their protection against xanthine （XA）/xanthine oxidase （XO）-induced toxicity in PC12 cells. Methods ...To evaluate the antioxidant activities of different chemical constituents from Astragalus mongholicus Bunge and their protection against xanthine （XA）/xanthine oxidase （XO）-induced toxicity in PC12 cells. Methods The compounds of Astragalus mongholicus Bunge were isolated by chromatography and the structures were elucidated on the basis of spectral data interpretation. Their antioxidant activities were detected by 1, 1-diphenyl-2-picrylhydrazyl （DPPH） radical scavenging activities in a cell-free system. Meanwhile, the effects against XA/XO-induced toxicity were assessed using MTT assay in PC12 cells. Results Ten principal constituents were isolated and identified as formononetin （Ⅰ）, ononin （Ⅱ）, calycosin （Ⅲ）, calycosin-7-O-β-D-glucoside （Ⅳ）, 9,10-dimethoxypterocarpan-3-O-β-D-glucoside （Ⅴ）, adenosine （Ⅵ）, pinitol （Ⅶ）, daucosterol （Ⅷ）, β-sitoster （Ⅸ） and saccharose （Ⅹ） from Astragalus mongholicus Bunge. The compounds Ⅰ, Ⅲ, and Ⅳ scavenged DPPH free radicals in vitro. Formononetin and calycosin were found to inhibit XA/XO-induced cell injury significantly, with an estimated EC50 of 50 ng/mL. Conclusion Compound Ⅲ, Ⅵ, and Ⅶ are first reported in this plant. Calycosin exhibits the most potent antioxidant activity both in the cell-free system and in the cell system.展开更多
Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;ho...Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;however,it is unclear whether this neuroprotective effect involves autophagy.In this study,PC12 cells were treated with 1×10^-5–1×10^-9 M LIG for 0,3,12 or 24 hours,and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Treatment with 1×10^-6 M LIG for 3 hours had the greatest effect on cell proliferation,and was therefore used for subsequent experiments.PC12 cells were pre-treated with 1×10^-6 M LIG for 3 hours,cultured in 95%N2/5%CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours,and then cultured normally for 16 hours,to simulate oxygen-glucose deprivation/reoxygenation(OGD/R).Cell proliferation was assessed with the MTS assay.Apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2 and Bax,autophagy-related proteins,Beclin 1 and microtubule-associated protein l light chain 3B(LC3-II),and liver kinase B1(LKB1)-5′-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway-related proteins were assessed by western blot assay.Immunofluorescence staining was used to detect LC3-II expression.Autophagosome formation was observed by electron microscopy.LIG significantly decreased apoptosis,increased Bcl-2,Beclin 1 and LC3-II expression,decreased Bax expression,increased LC3-II immunoreactivity and the number of autophagosomes,and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R.The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG.Taken together,our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by展开更多
Objective The aim of this study is to investigate whether microwave exposure would affect the N-methyI-D-aspartate receptor （NMDAR） signaling pathway to establish whether this plays a role in synaptic plasticity imp...Objective The aim of this study is to investigate whether microwave exposure would affect the N-methyI-D-aspartate receptor （NMDAR） signaling pathway to establish whether this plays a role in synaptic plasticity impairment. Methods 48 male Wistar rats were exposed to 30 mW/cm^2 microwave for 10 min every other day for three times. Hippocampal structure was observed through H＆E staining and transmission electron microscope. PC12 cells were exposed to 30 mW/cm^2 microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated. Results Microwave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits （NR1, NR2A, and NR2B） and related signaling molecules （CaZ＋/calmodulin-dependent kinase II gamma and phosphorylated cAMP-response element binding protein） were examined. Conclusion 30 mW/cm^2 microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.展开更多
Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of B...Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.展开更多
Objective To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine （XA）/xanthine oxidase （XO）-induced injury to PC12 cells. Methods PC12 cells were damaged b...Objective To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine （XA）/xanthine oxidase （XO）-induced injury to PC12 cells. Methods PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isofavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry. Results Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-β-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/XO-induced injury to PC12 cells. Their ECs0values of formononetin and calycosin were 0.05 μg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px）, while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 μg/mL and 50 μg/mL. Formononetin and calycosin had no significant infuence on Bcl-2 or Bax protein contents. Conclusion Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.展开更多
Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson＇s disease. We hypothesized that necrostatin-1 c...Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson＇s disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before exposure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death process of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range（5–30 μM） elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Parkinson＇s disease.展开更多
An acidic polysaccharide, H2, was isolated from the alkali-extract CHC of seeds of Cuscuta chinensis Lam. with the molecular weight more than 1.0 x 10(6). Chemical and spectroscopic studies led to the structure determ...An acidic polysaccharide, H2, was isolated from the alkali-extract CHC of seeds of Cuscuta chinensis Lam. with the molecular weight more than 1.0 x 10(6). Chemical and spectroscopic studies led to the structure determination as follows: the backbone chain consists of 1, 6-linked-beta -D Galp, 1,4-linked-X -D Galp, 1,4-linked-beta -D GalA and 1,2- or 1,4-linked-beta -L Rhap having branching points at position O-3 of some 1,6-linked-beta -D Galp residues (one among eight) and O-4 or O-2 of 1,2- or 1,4-linked-beta -L Rhap residues to terminal beta -D-galactopyranose. The side chains composed of terminal Galp, 1,6-linked-beta -D Galp, 1,4-linked beta -D Galp and 1,3,6-linked-beta -D Galp also linked at position O-3 of 1,6-linked-beta -D Galp residues in the backbone chain. beta -L-arabinofuranosyl and terminal beta -L-rhamnopyranosyl residues existed in the periphery of this polysaccharide linked to O-3 of 1,6-linked-beta -D Galp residues in the backbone chain and the side chains. The polysaccharide H2 increased significantly the survival rate of PC12 cells indicating that it had protective effects against H2O2 insult.展开更多
Background Transient sublethal ischemia is known as ischemic preconditioning, which enables cells and tissues to survive subsequent prolonged lethal ischemic injury. Ischemic preconditioning exerts neuroprotection thr...Background Transient sublethal ischemia is known as ischemic preconditioning, which enables cells and tissues to survive subsequent prolonged lethal ischemic injury. Ischemic preconditioning exerts neuroprotection through phosphatidylinositol 3-kinase （PI3K）/Akt pathway. Cbl-b belongs to the Casitas B-lineage lymphoma （Cbl） family, and it can regulate the cell signal transduction.The roles of ubiquitin ligase Cbl-b and PI3K/Akt pathway and the relationship between them in oxygen-glucose deprivation preconditioninq （OGDPC） in PC12 cells were investiaated in the ore.~e.nt study展开更多
文摘To evaluate the antioxidant activities of different chemical constituents from Astragalus mongholicus Bunge and their protection against xanthine （XA）/xanthine oxidase （XO）-induced toxicity in PC12 cells. Methods The compounds of Astragalus mongholicus Bunge were isolated by chromatography and the structures were elucidated on the basis of spectral data interpretation. Their antioxidant activities were detected by 1, 1-diphenyl-2-picrylhydrazyl （DPPH） radical scavenging activities in a cell-free system. Meanwhile, the effects against XA/XO-induced toxicity were assessed using MTT assay in PC12 cells. Results Ten principal constituents were isolated and identified as formononetin （Ⅰ）, ononin （Ⅱ）, calycosin （Ⅲ）, calycosin-7-O-β-D-glucoside （Ⅳ）, 9,10-dimethoxypterocarpan-3-O-β-D-glucoside （Ⅴ）, adenosine （Ⅵ）, pinitol （Ⅶ）, daucosterol （Ⅷ）, β-sitoster （Ⅸ） and saccharose （Ⅹ） from Astragalus mongholicus Bunge. The compounds Ⅰ, Ⅲ, and Ⅳ scavenged DPPH free radicals in vitro. Formononetin and calycosin were found to inhibit XA/XO-induced cell injury significantly, with an estimated EC50 of 50 ng/mL. Conclusion Compound Ⅲ, Ⅵ, and Ⅶ are first reported in this plant. Calycosin exhibits the most potent antioxidant activity both in the cell-free system and in the cell system.
文摘Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;however,it is unclear whether this neuroprotective effect involves autophagy.In this study,PC12 cells were treated with 1×10^-5–1×10^-9 M LIG for 0,3,12 or 24 hours,and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Treatment with 1×10^-6 M LIG for 3 hours had the greatest effect on cell proliferation,and was therefore used for subsequent experiments.PC12 cells were pre-treated with 1×10^-6 M LIG for 3 hours,cultured in 95%N2/5%CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours,and then cultured normally for 16 hours,to simulate oxygen-glucose deprivation/reoxygenation(OGD/R).Cell proliferation was assessed with the MTS assay.Apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2 and Bax,autophagy-related proteins,Beclin 1 and microtubule-associated protein l light chain 3B(LC3-II),and liver kinase B1(LKB1)-5′-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway-related proteins were assessed by western blot assay.Immunofluorescence staining was used to detect LC3-II expression.Autophagosome formation was observed by electron microscopy.LIG significantly decreased apoptosis,increased Bcl-2,Beclin 1 and LC3-II expression,decreased Bax expression,increased LC3-II immunoreactivity and the number of autophagosomes,and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R.The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG.Taken together,our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by
基金supported by the National Natural Science Foundation of China(No.81172620)
文摘Objective The aim of this study is to investigate whether microwave exposure would affect the N-methyI-D-aspartate receptor （NMDAR） signaling pathway to establish whether this plays a role in synaptic plasticity impairment. Methods 48 male Wistar rats were exposed to 30 mW/cm^2 microwave for 10 min every other day for three times. Hippocampal structure was observed through H＆E staining and transmission electron microscope. PC12 cells were exposed to 30 mW/cm^2 microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated. Results Microwave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits （NR1, NR2A, and NR2B） and related signaling molecules （CaZ＋/calmodulin-dependent kinase II gamma and phosphorylated cAMP-response element binding protein） were examined. Conclusion 30 mW/cm^2 microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.
基金Project (No. 2005C30059) supported by the Science and TechnologyProgram of Zhejiang Province, China
文摘Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.
基金supported by the Natural Science Foundation of China(No.NSFC.30670415).
文摘Objective To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine （XA）/xanthine oxidase （XO）-induced injury to PC12 cells. Methods PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isofavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry. Results Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-β-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/XO-induced injury to PC12 cells. Their ECs0values of formononetin and calycosin were 0.05 μg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px）, while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 μg/mL and 50 μg/mL. Formononetin and calycosin had no significant infuence on Bcl-2 or Bax protein contents. Conclusion Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.
基金supported by grants from the Science and Technology Project of Xuzhou City in China,No.XM12B017the Priority Academic Program Development of Jiangsu Higher Education Institutions in China
文摘Necroptosis is characterized by programmed necrotic cell death and autophagic activation and might be involved in the death process of dopaminergic neurons in Parkinson＇s disease. We hypothesized that necrostatin-1 could block necroptosis and give protection to dopaminergic neurons. There is likely to be crosstalk between necroptosis and other cell death pathways, such as apoptosis and autophagy. PC12 cells were pretreated with necroststin-1 1 hour before exposure to 6-hydroxydopamine. We examined cell viability, mitochondrial membrane potential and expression patterns of apoptotic and necroptotic death signaling proteins. The results showed that the autophagy/lysosomal pathway is involved in the 6-hydroxydopamine-induced death process of PC12 cells. Mitochondrial disability induced overactive autophagy, increased cathepsin B expression, and diminished Bcl-2 expression. Necrostatin-1 within a certain concentration range（5–30 μM） elevated the viability of PC12 cells, stabilized mitochondrial membrane potential, inhibited excessive autophagy, reduced the expression of LC3-II and cathepsin B, and increased Bcl-2 expression. These findings suggest that necrostatin-1 exerted a protective effect against injury on dopaminergic neurons. Necrostatin-1 interacts with the apoptosis signaling pathway during this process. This pathway could be a new neuroprotective and therapeutic target in Parkinson＇s disease.
文摘An acidic polysaccharide, H2, was isolated from the alkali-extract CHC of seeds of Cuscuta chinensis Lam. with the molecular weight more than 1.0 x 10(6). Chemical and spectroscopic studies led to the structure determination as follows: the backbone chain consists of 1, 6-linked-beta -D Galp, 1,4-linked-X -D Galp, 1,4-linked-beta -D GalA and 1,2- or 1,4-linked-beta -L Rhap having branching points at position O-3 of some 1,6-linked-beta -D Galp residues (one among eight) and O-4 or O-2 of 1,2- or 1,4-linked-beta -L Rhap residues to terminal beta -D-galactopyranose. The side chains composed of terminal Galp, 1,6-linked-beta -D Galp, 1,4-linked beta -D Galp and 1,3,6-linked-beta -D Galp also linked at position O-3 of 1,6-linked-beta -D Galp residues in the backbone chain. beta -L-arabinofuranosyl and terminal beta -L-rhamnopyranosyl residues existed in the periphery of this polysaccharide linked to O-3 of 1,6-linked-beta -D Galp residues in the backbone chain and the side chains. The polysaccharide H2 increased significantly the survival rate of PC12 cells indicating that it had protective effects against H2O2 insult.
文摘Background Transient sublethal ischemia is known as ischemic preconditioning, which enables cells and tissues to survive subsequent prolonged lethal ischemic injury. Ischemic preconditioning exerts neuroprotection through phosphatidylinositol 3-kinase （PI3K）/Akt pathway. Cbl-b belongs to the Casitas B-lineage lymphoma （Cbl） family, and it can regulate the cell signal transduction.The roles of ubiquitin ligase Cbl-b and PI3K/Akt pathway and the relationship between them in oxygen-glucose deprivation preconditioninq （OGDPC） in PC12 cells were investiaated in the ore.~e.nt study
