孕酮对氧糖剥夺损伤的PC12细胞的保护作用

目的: 观察孕酮(PROG)对氧糖剥夺(OGD)PC12细胞活力及葡萄糖转运蛋白3(GLUT3)表达的影响,在培养细胞证明孕酮抗缺氧缺血损伤的神经保护作用。方法: 神经生长因子诱导分化良好的PC12细胞,随机分为3组:(1)正常对照组:常规培养,不进行OGD处理;(2)OGD组:无糖培养基、低氧环境(95%N₂和5%CO₂持续通气)进行OGD 30 min处理,然后复氧复糖培养24 h;(3)PROG +OGD组:培养液含终浓度为10 nmol/L 孕酮,余同OGD组。WST-8法检测各组PC12细胞的活力;检测培养液中乳酸脱氢酶(LDH)的活性,判断细胞损伤的程度;反转录-聚合酶链反应(RT-PCR)检测PC12细胞GLUT1 mRNA和GLUT3 mRNA表达;Western blotting法检测PC12细胞GLUT3蛋白表达。结果: 孕酮可以减轻OGD引起的PC12细胞肿胀,降低LDH漏出,减轻细胞损伤,显著增加OGD组PC12细胞的活力(P<0.01)。RT-PCR显示PROG +OGD组GLUT3 mRNA的表达明显高于OGD组(P<0.05),Western blotting显示PROG +OGD组GLUT3蛋白的表达显著高于OGD组(P<0.01)。结论: 孕酮对体外培养OGD损伤的PC12细胞具有保护作用,其机制可能与上调GLUT3蛋白的表达有关。

Protective effect of progesterone on PC12 cells injured by oxygen-glucose deprivation

AIM: To investigate the effects of progesterone on the cell viability and expression of glucose transporter type 3(GLUT3) in PC12 cells injured by oxygen-glucose deprivation(OGD) in attempt to prove the neuroprotection of progesterone(PROG) against the hypoxic-ischemic injury in cultured cells in vitro.METHODS: Well-differentiated PC12 cells induced by nerve growth factor were randomly divided into 3 groups.In normal group,the cells were cultured without OGD treatment.In OGD group,the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N2 and 5% CO2 for 30 min.After that,the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h.In PROG+OGD group,the cells were given the same treatments as those in OGD group except that the medium contained progesterone at concentration of 10 nmol/L.Cellular morphological changes were observed after OGD for 30 min.The cell viability was assessed by WST-8 assay.The degree of the cell damage was evaluated by determining lactate dehydrogenase(LDH) leakage.The expression of GLUT3 at mRNA and protein levels was examined by RT-PCR and Western blotting,respectively.RESULTS: Progesterone attenuated the cellular swelling,decreased the leakage of LDH and improved the viability of PC12 cells injured by OGD(P<0.01).The expression of GLUT3 at mRNA and protein levels in PC12 cells in PROG+OGD group was significantly higher than that in OGD group(P<0.05).CONCLUSION: Progesterone has protective effect on in vitro cultured PC12 cells injured by OGD.The mechanism may be related to the up-regulation of GLUT3 protein.