Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene...Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene, was amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector. The recombinant plasmid pGL3-THprom was transfected into a dopammergic cell line MES23.5 or a α-Syn over-expressed MES23.5 （named MES23.5/hα-Syn^＋）. The promoter activity was detected by the Dual Luciferase Assay System. Results The luciferase activities in the MES23.5 cells transfected with pGl.,3-Basic, pGL3-THprom, and pGL3-Control vectors were 5.60±0.67, 26.80±4.11, and 32.90±4.75, respectively. On the other hand, the luciferase activity of pGL3-THprom in the MES23.5 （26.80±4.11） was significantly higher than that in the MES23.5/hα-Syn^＋（14.40±0.61） （P〈0.01）. Conclusion These results indicate that the -495 to ＋25 region in the TH gene possesses promoter activity for controlling the gene expression, and that α-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.展开更多
To study the neuro protective effect of flavonoids extracts from immature bitter orange(Citrus aurantium L.),the PC12 cells treated with 6-hydroxydopamine(6-OHDA)were used as the Parkinson’s disease(PD)model.To deter...To study the neuro protective effect of flavonoids extracts from immature bitter orange(Citrus aurantium L.),the PC12 cells treated with 6-hydroxydopamine(6-OHDA)were used as the Parkinson’s disease(PD)model.To determine the optimal dose of 6-OHDA for constructing a PD model,PC12 cells were incubated with different concentrations of 6-OHDA for 24 h.After 24 h incubation,PC12 cells of drug groups were added 6-OHDA and different concentrations of flavonoids extracts were measured cell viability by CCK8 for selecting effective concentration of flavonoids extracts;the ROS level was determined using flow cytometry;the levels of MDA,CAT,SOD and GSH-Px were assayed by Colorimetric kit for oxidative stress investigation.Compared with the model group,PC12 cell viability was significantly enhanced(P<0.05),the levels of ROS and MDA were reduced significantly(P<0.05),and the activities of SOD,CAT and GSH-Px were significantly enhanced(P<0.05)in drug groups.In conclusion,immature bitter orange flavonoids extracts could protect PC12 cells against 6-OHDA-induced oxidative stress.展开更多
Hybrid antioxidants cinnamoyldopamine（2a）, p-coumaroyldopamine（2b）, caffeoyldopamine（2c）, feruloyldopamine（2d） and sinapoyldopamine（2e） were synthesized by conjugation of dopamine（DA） and hydroxycinnamic a...Hybrid antioxidants cinnamoyldopamine（2a）, p-coumaroyldopamine（2b）, caffeoyldopamine（2c）, feruloyldopamine（2d） and sinapoyldopamine（2e） were synthesized by conjugation of dopamine（DA） and hydroxycinnamic acids（HCAs）. The stabilities were studied in buffers at p H 1.3, p H 5.0, and p H 7.4 including the human plasma. All the compounds were found highly stable at acidic p H, but underwent hydrolysis at neutral p H. Furthermore, the hydrolysis proceeded much faster in plasma in the following order as indicated by half-life values（t1/2）, 2c（1.21 h）〈2e（1.52 h）〈2d（1.85 h）〈2b（3.38 h）〈2a（3.88 h）, correlating with the number of electron-donating groups. It has been proven by UV spectrum that 2c, 2d, and 2e displayed red shift of more than 50 nm as compared to 2a and 2b, because of the presence of OH and OCH3 groups. In addition, the compounds（2b–e） showed no cytotoxicity on normal HUVEC cells as DA, although 2a displayed a 16% inhibition of proliferation at 40 μM following 48 h incubation. Their free radical-scavenging activities were evaluated using ABTS^＊＋ and superoxide anion assays and the mechanisms were proposed. It was found that they all exhibited higher activities than trolox, a recognized antioxidant. Amazingly, in the case of the hybrids（2a–e）, their activity was higher than that of HCAs while lower or comparable to that of DA, suggesting that there may be a ＂saturation effect＂ with the hybrid molecules in the antioxidant activities.展开更多
基金This work was supported by the Key Project of National Natural Science Foundation of China （No. 30430280）the National Natural Science Foundation of China （No. 30271437, No.30270482）the Natural Science Foundation of Beijing Municipality （No. 7022011 ）.
文摘Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene, was amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector. The recombinant plasmid pGL3-THprom was transfected into a dopammergic cell line MES23.5 or a α-Syn over-expressed MES23.5 （named MES23.5/hα-Syn^＋）. The promoter activity was detected by the Dual Luciferase Assay System. Results The luciferase activities in the MES23.5 cells transfected with pGl.,3-Basic, pGL3-THprom, and pGL3-Control vectors were 5.60±0.67, 26.80±4.11, and 32.90±4.75, respectively. On the other hand, the luciferase activity of pGL3-THprom in the MES23.5 （26.80±4.11） was significantly higher than that in the MES23.5/hα-Syn^＋（14.40±0.61） （P〈0.01）. Conclusion These results indicate that the -495 to ＋25 region in the TH gene possesses promoter activity for controlling the gene expression, and that α-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.
文摘To study the neuro protective effect of flavonoids extracts from immature bitter orange(Citrus aurantium L.),the PC12 cells treated with 6-hydroxydopamine(6-OHDA)were used as the Parkinson’s disease(PD)model.To determine the optimal dose of 6-OHDA for constructing a PD model,PC12 cells were incubated with different concentrations of 6-OHDA for 24 h.After 24 h incubation,PC12 cells of drug groups were added 6-OHDA and different concentrations of flavonoids extracts were measured cell viability by CCK8 for selecting effective concentration of flavonoids extracts;the ROS level was determined using flow cytometry;the levels of MDA,CAT,SOD and GSH-Px were assayed by Colorimetric kit for oxidative stress investigation.Compared with the model group,PC12 cell viability was significantly enhanced(P<0.05),the levels of ROS and MDA were reduced significantly(P<0.05),and the activities of SOD,CAT and GSH-Px were significantly enhanced(P<0.05)in drug groups.In conclusion,immature bitter orange flavonoids extracts could protect PC12 cells against 6-OHDA-induced oxidative stress.
基金The National Natural Science Foundation of China(Grant No.21302079)the Fundamental Research Funds for the Central Universities(Grant No.lzujbky-2014-151)
文摘Hybrid antioxidants cinnamoyldopamine（2a）, p-coumaroyldopamine（2b）, caffeoyldopamine（2c）, feruloyldopamine（2d） and sinapoyldopamine（2e） were synthesized by conjugation of dopamine（DA） and hydroxycinnamic acids（HCAs）. The stabilities were studied in buffers at p H 1.3, p H 5.0, and p H 7.4 including the human plasma. All the compounds were found highly stable at acidic p H, but underwent hydrolysis at neutral p H. Furthermore, the hydrolysis proceeded much faster in plasma in the following order as indicated by half-life values（t1/2）, 2c（1.21 h）〈2e（1.52 h）〈2d（1.85 h）〈2b（3.38 h）〈2a（3.88 h）, correlating with the number of electron-donating groups. It has been proven by UV spectrum that 2c, 2d, and 2e displayed red shift of more than 50 nm as compared to 2a and 2b, because of the presence of OH and OCH3 groups. In addition, the compounds（2b–e） showed no cytotoxicity on normal HUVEC cells as DA, although 2a displayed a 16% inhibition of proliferation at 40 μM following 48 h incubation. Their free radical-scavenging activities were evaluated using ABTS^＊＋ and superoxide anion assays and the mechanisms were proposed. It was found that they all exhibited higher activities than trolox, a recognized antioxidant. Amazingly, in the case of the hybrids（2a–e）, their activity was higher than that of HCAs while lower or comparable to that of DA, suggesting that there may be a ＂saturation effect＂ with the hybrid molecules in the antioxidant activities.
