摘要
目的探讨蛋白激酶Cε(PKCε)在瑞芬太尼诱导的大鼠脊髓μ阿片受体(MOR)下调中的作用。方法成年雄性SD大鼠分为对照组(Naive组)、瑞芬太尼组(R组)、鞘内注射εV1-2组(εV1-2组)、瑞芬太尼+鞘内注射εV1-2组(R+εV1-2组)。静脉输注瑞芬太尼建立痛觉过敏模型,通过蛋白免疫印迹法检测瑞芬太尼输注后2 h至5 d大鼠脊髓PKCε和MOR总蛋白、膜浆蛋白以及磷酸化蛋白的表达变化。采用免疫荧光组织化学法观察PKCε和MOR在脊髓背角的分布。免疫共沉淀法观察瑞芬太尼输注后大鼠脊髓PKCε和MOR的相互作用情况。瑞芬太尼输注前30 min预先鞘内注射PKCε特异性抑制剂εV1-2,观察其对脊髓PKCε和MOR总蛋白、膜浆蛋白以及其磷酸化水平表达变化的影响。结果静脉输注瑞芬太尼后1 d,与Naive组相比,脊髓背角PKCε蛋白表达增加[(1.04±0.06)vs.(1.79±0.15),F=13.05,P<0.05],MOR表达下调[(1.07±0.08)vs.(0.57±0.04),F=17.85,P<0.05],差异均有统计学意义;同时,PKCε[(1.10±0.11)vs.(1.53±0.06),F=12.87,P<0.05]和MOR[(1.00±0.17)vs.(1.62±0.08),F=28.95,P<0.05]磷酸化水平显著升高,PKCε膜表达显著增加[(0.97±0.07)vs.(1.60±0.06),F=23.30,P<0.05],MOR膜表达显著减少[(0.97±0.07)vs.(0.38±0.04),F=28.50,P<0.05],差异均有统计学意义。免疫荧光双染结果显示,PKCε与MOR在脊髓背角存在共定位,且免疫共沉淀结果表明瑞芬太尼可增强大鼠脊髓背角PKCε和MOR的相互作用。而鞘内注射εV1-2后,与R1d组相比,(R1d+εV1-2)组大鼠脊髓磷酸化PKCε表达水平下降[(1.48±0.09)vs.(1.19±0.07),F=23.80,P<0.05],PKCε膜表达水平下降[(1.65±0.08)vs.(1.15±0.08),F=13.48,P<0.05],磷酸化MOR表达水平下降[(1.58±0.05)vs.(1.18±0.06),F=21.12,P<0.05],MOR蛋白表达水平升高[(0.53±0.09)vs.(1.11±0.05),F=18.18,P<0.05],差异均有统计学意义。结论瑞芬太尼可能通过诱导PKCε的激活,促进脊髓背角MOR磷酸化,进而在介导MOR内化和下调中起关键作用。
Objective To evaluate the role of protein kinase C epsilon(PKCε)in remifentanil-induced downregulation ofμopioid receptor(MOR)in the spinal cord of rats.MethodsThe adult male SD rats were randomly divided into control group(Naive group),remifentanil group(R group),intrathecalεV1-2 group(εV1-2 group)and remifentanil+intrathecal groupεV1-2(R+εV1-2 group).Hyperalgesia was induced by continuous infusion of remifentanil via vein tail in adult male SD rats.Western blot was used to detect the whole lysate and membrane/cytosolic protein fractions as well as phosphorylated protein levels changes of PKCεand MOR in spinal cord after remifentanil infusion from the 2nd hour to the 5th day.Immunohistochemistry was used to observe the distribution of PKCεand MOR in spinal dorsal horn.The interaction between spinal PKCεand MOR was investigated by co-immunoprecipitation.PKCεinhibitorεV1-2 was intrathecally administered 30min before remifentanil infusion.ResultsCompared with naive group,the expression of PKCεwas increased[(1.04±0.06)vs.(1.79±0.15),F=13.05,P<0.05]while the expression of MOR was decreased[(1.07±0.08)vs.(0.57±0.04),F=17.85,P<0.05]on the first day after remifentanil infusion.Membrane PKCε[(0.97±0.07)vs.(1.60±0.06),F=23.30,P<0.05]was significantly increased while membrane MOR[(0.97±0.07)vs.(0.38±0.04),F=28.50,P<0.05]was decreased in spinal cord induced by remifentanil with the phosphorylation levels of PKCε[(1.10±0.11)vs.(1.53±0.06),F=12.87,P<0.05]and MOR significantly increased[(1.00±0.17)vs.(1.62±0.08),F=28.95,P<0.05].In addition,PKCεco-located with MOR in the spinal dorsal horn.Continuous infusion of remifentanil enhanced the interaction between PKCεand MOR.Moreover,εV1-2 not only blocked remifentanil-induced increase in phosphorylation[(1.48±0.09)vs.(1.19±0.07),F=23.80,P<0.05]and the plasma membrane translocation of PKCε[(1.65±0.08)vs.(1.15±0.08),F=13.48,P<0.05],but also significantly attenuated the phosphorylation levels[(1.58±0.05)vs.(1.18±0.06),F=21.12,P<0.05]and up-regulation of MOR[(0.53±0.09)vs.(1.11±0.05),F=18.18,P<0.05].ConclusionPKCεcould mediate the remifentanil-induced spinal MOR internalization and downregulation by promoting the phosphorylation of MOR.
作者
陈彦梅
于萍
王晓娥
王文慧
李林芝
罗国娅
崔宇
陈元
CHEN Yan-mei;YU Ping;WANG Xiao-e;WANG Wen-hui;LI Lin-zhi;LUO Guo-ya;CUI Yu;CHEN Yuan(Neurobiology Research Center,School of Medicine,Sun Yat-sen University,Shenzhen,Guangdong 518107;Department of Anesthesiology,the First Affiliated Hospital,Sun Yat-sen University,Guangzhou,Guangdong 510080,China)
出处
《热带医学杂志》
CAS
2022年第5期606-611,596,共7页
Journal of Tropical Medicine
基金
广东省自然科学基金(2021A1515010588)
广东省科技攻关计划(2018B030335001)
