ABSTRACT
AIM:To investigate the effect of dopamine ( DA) on the glutamate ( Glu)-uptake ability of astro-cytes, and the role of mammalian target of rapamycin (mTOR)-excitatory amino acid transporter 2(EAAT2) pathway in this process .METHODS:Extracellular Glu levels in DA-treated primary cortical astrocytes ( PCAs) were measured by a fluorimetric method .The relative expression of EAAT 2 and mTOR at mRNA and protein levels was measured by RT-qPCR and Western blot .PCAs stimulated with or without DA in the presence or absence of mTOR antagonist rapamycin or mTOR agonist MHY1485 were used to determine the expression of mTOR and EAAT 2, and Glu content in the culture supernatant was also measured.RESULTS: The expression of mTOR in DA-treated PCAs was decreased, the expression of EAAT2 was also decreased .Extracellular Glu levels of DA-treated PCAs were elevated significantly .When the PCAs were stimula-ted with DA in the presence of rapamycin , the expression of EAAT2 was decreased , and the levels of extracellular Glu was significantly increased.In the presence of MHY1485, the expression of EAAT2 was elevated, and significant decrease in the levels of extracellular Glu was also observed .CONCLUSION:DA interacts with mTOR-EAAT2 pathway to reduce the Glu-uptake ability of the astrocytes , and causes extracellular Glu accumulation , ultimately destroys the function of astro-cytes.
ABSTRACT
AIM:To investigate the effect of dopamine ( DA) on the glutamate ( Glu)-uptake ability of astro-cytes, and the role of mammalian target of rapamycin (mTOR)-excitatory amino acid transporter 2(EAAT2) pathway in this process .METHODS:Extracellular Glu levels in DA-treated primary cortical astrocytes ( PCAs) were measured by a fluorimetric method .The relative expression of EAAT 2 and mTOR at mRNA and protein levels was measured by RT-qPCR and Western blot .PCAs stimulated with or without DA in the presence or absence of mTOR antagonist rapamycin or mTOR agonist MHY1485 were used to determine the expression of mTOR and EAAT 2, and Glu content in the culture supernatant was also measured.RESULTS: The expression of mTOR in DA-treated PCAs was decreased, the expression of EAAT2 was also decreased .Extracellular Glu levels of DA-treated PCAs were elevated significantly .When the PCAs were stimula-ted with DA in the presence of rapamycin , the expression of EAAT2 was decreased , and the levels of extracellular Glu was significantly increased.In the presence of MHY1485, the expression of EAAT2 was elevated, and significant decrease in the levels of extracellular Glu was also observed .CONCLUSION:DA interacts with mTOR-EAAT2 pathway to reduce the Glu-uptake ability of the astrocytes , and causes extracellular Glu accumulation , ultimately destroys the function of astro-cytes.
ABSTRACT
<p><b>OBJECTIVE</b>To establish the quality standards of the herbs of Peganum harmala.</p><p><b>METHOD</b>According to the Chinese Pharmacopoeia (2010 version, volume 1) and its appendix method, the water, total ash, acid insoluble ash, water-soluble extractives, and heavy metal were analyzed for herbs of P. harmala. TLC method was used to separate harmaline, harmine and vasicine from the herb samples by mixture of ethyl acetate-methanol-ammonia (10: 1.5: 0.5) as a developing solvent on high performance silica gel precoated plate (HSGF254) and to identify them inspected under UV 366 nm, visualized by spraying with both Dragendorff reagent, and by bioautographic assay. In the HPLC method, vasicine was separated on a C18 (4.6 mm x 250 mm, 5 microm) column with metnanol-0.1% trifluoroacetic acid (15:85) as the mobile phase and detected at at 280 nm.</p><p><b>RESULT</b>In the TLC procedures, 254 nm fluorescent and bioautographic assay for the detection of acetylcholinesterase inhibitor can be used for the qualitative identification of the active ingredients. For the HPLC quantitation method, the calibration curve of vasicine displayed ideal linearity over the range of 0.7923-792.3 mg x L(-1) with the regression equation of Y = 18,227X - 24.879 (r = 0.9999). The average recovery of vasicine was 101.6% with a RSD of 1.9%. The RSD values of intra-day and inter-day precision were less than 2%. The content of vasicine in 10 batches of herbs of P. harmala fluctuates between 0.23% and 1.47%.</p><p><b>CONCLUSION</b>The results indicated that the limit of vasicine was not lower than 0.6%, and the water, total ash, acid insoluble ash, and water-soluble extractives were not more than 10.0%, 20.0%, 1.7%, and 30.0%, respectively. The heavy metal of plumbum, cadmium, arsenic, mercury, and copper were not more than 5, 3, 2, 2, and 20 mg x kg(-1), respectively. The qualitative and quantitative method established was suitable for the quality evaluation and assessment of herbs of P. harmala.</p>