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
AIM:To investigate the distribution and mechanism of M 1/M2 macrophages in inflammatory injury and repair process of renal tissues .METHODS: SD male rats ( n=45 ) were randomly divided into 2 parts: ischemia-reperfusion injury (IRI) and unilateral ureteral obstruction (UUO) renal injury.The rats with IRI were divided into sham operation group and operation groups (0, 6, 24, and 72 h after operation), and the rats with UUO were divided into sham operation group and operation groups (3, 7 and 14 d after operation).Automatic biochemical analyzer was used to detect serum levels of creatinine and urea nitrogen .The degree of renal injury in IRI group and UUO group were detected by HE staining.The expression of CD68 was examined by immunohistochemical staining .The levels of inducible nitric oxide syn-thase (iNOS), arginase-1 (Arg-1), transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α(TNF-α) were measured by ELISA.The polarizations of M1 ( CD68 +, F4/80 + and CD16/32 +) and M2 ( CD68 +, F4/80 +and CD206 +) macrophages were analyzed by flow cytometry .RESULTS:In IRI group, the infiltration of CD68 +macrophages and the degree of injury were increased with the prolongation of time in the renal tissues .At 24 h, the tissue injury and macrophage infiltration were the most serious , but then decreased .At 72 h, the tissue damage and CD68 +macrophage in-filtration were significantly reduced .In UUO group, obstructive injury was increased with the prolongation of time , and at 14 d, marked fibrous hyperplasia occurred .The infiltration of CD68 +macrophages at 7 d was the most serious , but then reduced at 14 d.Flow cytometry analysis showed that M 1 macrophages were the majority in the early stages of UUO and IRI, and the result of ELISA identified the higher level of iNOS .At the late stage of injury , the M1 macrophages were de-creased, while the M2 macrophages were increased with higher level of Arg-1.M1 macrophage-mediated early injury was due to the induction of TNF-αexpression, and M2 macrophage-mediated later recovery was due to enhancing TGF-β1 levels.CONCLUSION:The polarization of M1 and M2 macrophages is involved in the processes of UUO and IRI .M1 macrophages play a key role in early injury , and M2 macrophages contribute to the late stage of fibrotic repair .The polari-zation of macrophages during renal injury and repair provides a guiding significance for the clinical treatment .