摘要
目的探究食欲素A(orexin-A)、食欲素受体1(OX1R)及食欲素受体2(OX2R)是否参与慢性不可预测轻度应激(CUMS)抑郁大鼠铁死亡及脂质过氧化调控。方法将40只大鼠随机分为正常组(NC组),模型组(Mod组)、外源性Orexin-A组(Orexin-A组),OX1R/OX2R阻断剂组(TCS1102组),每组10只。造模后,采用旷场实验(OFT)、糖水偏好实验(SPT)及强迫游泳实验(FST)观察行为学变化,膜片钳技术检测动作电位(PA)和静息膜电位(Vm),采用蛋白免疫印迹法(WB)法检测眶额皮层(OFC)组织中Orexin-A/OX1R/OX2R蛋白表达,RT-PCR法检测谷胱甘肽过氧化物酶4(GPX4)、长链酰基辅酶A合成酶4(ACSL4)及半胱氨酸/谷氨酸转运体轻链(SLC7A11)的mRNA表达,免疫荧光标记大鼠胶质纤维酸性蛋白(GFAP)与脂质过氧化产物4-羟基壬烯醛(4-HNE)表达强度。结果与NC组比较,Mod组OFT、SPT和FST行为学均存在显著差异性(P<0.01),PA发放数量降低(P<0.001)、Vm升高(P<0.01),Orexin-A/OX1R/OX2R蛋白表达升高(P<0.01,P<0.001,P<0.001),GPX4/SLC7A11 mRNA表达降低(P<0.01)、ACSL4 m RNA表达升高(P<0.01),GFAP与4-HNE荧光强度表达均升高(P<0.001);与Mod组比较,Orexin-A组PA发放数量降低(P<0.05),Orexin-A/OX1R/OX2R蛋白表达升高(P<0.05,P<0.01),GPX4/SLC7A11 mRNA表达降低(P<0.05)、ACSL4 mRNA表达升高(P<0.05)、GFAP与4-HNE荧光强度表达升高(P<0.05,P<0.01);TCS1102组在行为学、Orexin-A/OX1R/OX2R蛋白表达、PA与Vm、GPX4/SLC7A11/ACSL4 mRNA及GFAP与4-HNE荧光强度表达方面均呈现逆转趋势。结论Orexin-A/OX1R/OX2R参与CUMS抑郁大鼠铁死亡及脂质过氧化调控,其机制可能是Orexin-A通过激活OX1R/OX2R信号通路增强OFC神经元兴奋性,上调铁死亡关键因子ACSL4/4-HNE表达、降低GPX4/SLC7A11表达,从而促进脂质过氧化和铁死亡的发生。
Objective To investigate whether orexin A(orexin-A),orexin receptor 1(OX1R)and orexin receptor 2(OX2R)are involved in iron death and lipid peroxidation regulation in chronically unpredictable mild stress(CUMS)depressed rats.Methods Forty rats were randomly divided into a normal group(NC group),a modeling group(Mod group),an exogenous Orexin-A group(Orexin-A group,),and an OX1R/OX2R blocker group(TCS1102 group),with 10 rats in each group.After modeling,behavioral changes were observed using the absent field test(OFT),sugar-water preference test(SPT)and forced swimming test(FST),action potential(PA)and resting membrane potential(Vm)were detected by diaphragm-clamp technique,Orexin-A/OX1R/OX2R protein expression in orbital frontal cortex(OFC)tissues was detected by protein immunoblotting(WB)method,RT-PCR The mRNA expression of glutathione peroxidase 4(GPX4),long chain acyl coenzyme A synthase 4(ACSL4)and cysteine/glutamate transporter light chain(SLC7A11)were detected by RT-PCR method,and the intensity of the expression of rat glial fibrillary acidic protein(GFAP)and lipid peroxidation product 4-hydroxynon-enal(4-HNE)was labeled by immunofluorescence.Results Compared with the NC group,there were significant differences in OFT,SPT and FST behavioral in the Mod group(P<0.01),with lower number of PA issuance(P<0.001),higher Vm(P<0.01),and higher expression of Orexin-A/OX1R/OX2R proteins(P<0.01,P<0.001,and P<0.001).GPX4/SLC7A11 mRNA expression was decreased(P<0.01),ACSL4 mRNA expression was elevated(P<0.01),and the fluorescence intensity expression of both GFAP and 4-HNE was elevated(P<0.001);the number of PA issuance was decreased in the Orexin-A group compared to the Mod group(P<0.05),and the Orexin-A/OX1R/OX2R protein expression was elevated(P<0.05,P<0.01),GPX4/SLC7A11 mRNA ex-pression was decreased(P<0.05),ACSL4 mRNA expression was elevated(P<0.05),and the fluorescence in-tensity of GFAP and 4-HNE expression was elevated(P<0.05,P<0.01);the TCS1102 group had higher expres-sion of GFAP and 4-HNE in the behavioral,Orexin-A/OX1R/OX2R protein expression,PA and Vm,GPX4/SLC7A11/ACSL4 mRNA,and GFAP and 4-HNE fluorescence intensity expression showed a reversed trend.Con⁃clusions Orexin-A/OX1R/OX2R is involved in the regulation of iron death and lipid peroxidation in CUMS de-pressed rats,and the mechanism may be that Orexin-A enhances the excitability of OFC neurons by activating the OX1R/OX2R signaling pathway,up-regulates the expression of the key factor of iron death,ACSL4/4-HNE,and decreases the expression of GPX4/SLC7A11,which promotes lipid peroxidation and iron death.
作者
张震
程茗
蒋召书
杨洁
罗振亮
曹峰
ZHANG Zhen;CHENG Ming;JIANG Zhaoshu;YANG Jie;LUO Zhenliang;CAO Feng(College of Traditional Chinese Medicine,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,Guizhou,China)
出处
《实用医学杂志》
北大核心
2025年第16期2507-2514,共8页
The Journal of Practical Medicine
基金
国家自然科学基金项目(编号:82160863,82160920,82360951)
贵州省自然科学基金项目(编号:黔科合基础-ZK[2022]一般511)
贵州省卫健委科学技术基金项目(编号:gzwkj2023-148)
贵州省中医药、民族医药科学技术研究课题(编号:QZYY-2024-011)
贵州中医药大学研究生教育创新计划项目(编号:YCXKYS2023027)。
关键词
食欲素
抑郁症
铁死亡
脂质过氧化
眶额皮层
orexin
depression
iron death
lipid peroxidation
orbitofrontal cortex
