摘要
目的 探究依托咪酯(Eto)调节环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/cAMP反应元件结合蛋白(CREB)信号通路对糖氧剥夺/复氧(OGD/R)诱导的神经元损伤的影响.方法 将海马神经元细胞HT22随机分为对照组、模型组、Eto低、中、高剂量组、抑制剂组.MTT法计算细胞增殖抑制率,筛选Eto最佳干预浓度;EdU、流式细胞术分别检测细胞增殖、凋亡;ELISA法检测细胞中cAMP、白细胞介素-6(IL-6)、肿瘤坏死因子(TNF-α)水平;全自动生化分析仪检测细胞中乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)水平;Western blot方法检测细胞中增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、细胞抗凋亡因子B淋巴细胞瘤-2(Bcl-2)及cAMP/PKA/CREB信号通路蛋白表达.结果 与对照组比较,模型组 HT22 细胞 EdU 阳性细胞率、cAMP、GSH-Px、PCNA、Bcl-2、p-PKA/PKA、p-CREB/CREB 表达显著降低,凋亡率、IL-6、TNF-α、LDH、MDA、Bax表达显著升高(P＜0.05);与模型组比较,Eto低、中、高剂量HT22细胞 EdU 阳性细胞率、cAMP、GSH-Px、PCNA、Bcl-2、p-PKA/PKA、p-CREB/CREB 表达显著升高,凋亡率、IL-6、TNF-a、LDH、MDA、Bax表达显著降低(P＜0.05);cAMP/PKA/CREB信号通路抑制剂H-89可减弱Eto对神经细胞的保护作用(P＜0.05).结论 Eto可能通过激活cAMP/PKA/CREB信号通路,抑制氧化应激和炎症反应,促进细胞增殖,降低细胞凋亡,减弱OGD/R诱导的HT22细胞损伤.
Abstract
Objective To investigate the effect of etomidate(Eto)on neuronal damage induced by glucose ox-ygen deprivation/reoxygenation(OGD/R)by regulating the cyclic adenosine 3',5'-monophosphate(cAMP)/protein kinase A(PKA)/cAMP response element binding protein(CREB)signaling pathway.Methods Hip-pocampal neuronal cells HT22 were randomly separated into control group,model group,Eto low,medium,and high dose groups,and inhibitor group.MTT method was applied to calculate cell proliferation inhibition rate to screen for optimal intervention concentration of Eto.EdU and Flow cytometry were applied to detect cell proliferation and apoptosis,respectively.ELISA method was applied to detect the levels of cAMP,interleu-kin-6(IL-6),and tumor necrosis factor-α(TNF-α)in cells.Automatic Biochemistry Analyzer were applied to detect levels of lactate dehydrogenase(LDH),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)in cells.Western blot was applied to detect the expression of proliferating cell nuclear antigen(PCNA),Bcl-2 associated X protein(Bax),anti-apoptotic factor B cell lymphocyte tumor 2(Bcl-2),and cAMP/PKA/CREB signaling pathway proteins in cells.Results Compared with control group,EdU positive cell rate,cAMP,GSH-Px,PCNA,Bcl-2,p-PKA/PKA,p-CREB/CREB expression of HT22 cells in model group were greatly reduced,while apoptosis rate,IL-6,TNF-α,LDH,MDA,Bax expression were obviously increased(P＜0.05).Compared with model group,EdU positive cell rate,cAMP,GSH-Px,PCNA,Bcl-2,p-PKA/PKA,p-CREB/CREB expression of HT22 cells in Eto low,medium,and high dose groups were obviously increased,while apoptosis rate,IL-6,TNF-α,LDH,MDA,and Bax expression were greatly reduced(P＜0.05).The cAMP/PKA/CREB signaling pathway inhibitor H-89 attenuated protective effect of Eto on nerve cells(P＜0.05).Conclusion Eto may activate cAMP/PKA/CREB signaling pathway,inhibit oxidative stress and in-flammatory response,promote cell proliferation,reduce cell apoptosis,and weaken OGD/R induced HT22 cell damage.
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