Neuropathic pain,often featuring allodynia,imposes significant physical and psychological burdens on patients,with limited treatments due to unclear central mechanisms.Addressing this challenge remains a crucial unsol...Neuropathic pain,often featuring allodynia,imposes significant physical and psychological burdens on patients,with limited treatments due to unclear central mechanisms.Addressing this challenge remains a crucial unsolved issue in pain medicine.Our previous study,using protein kinase C gamma(PKCγ)-tdTomato mice,highlights the spinal feedforward inhibitory circuit involving PKCγ neurons in gating neuropathic allodynia.However,the regulatory mechanisms governing this circuit necessitate further elucidation.We used diverse transgenic mice and advanced techniques to uncover the regulatory role of the descending serotonin(5-HT)facilitation system on spinal PKCγ neurons.Our findings revealed that 5-HT neurons from the rostral ventromedial medulla hyperpolarize spinal inhibitory interneurons via 5-HT_(2C) receptors,disinhibiting the feedforward inhibitory circuit involving PKCγ neurons and exacerbating allodynia.Inhibiting spinal 5-HT_(2C) receptors restored the feedforward inhibitory circuit,effectively preventing neuropathic allodynia.These insights offer promising therapeutic targets for neuropathic allodynia management,emphasizing the potential of spinal 5-HT_(2C) receptors as a novel avenue for intervention.展开更多
目的:研究指出蛋白激酶Cγ(PKCγ)在脊髓水平上参与了伤害性信号的传递,文中拟构建大鼠PKCγ基因的小发卡RNA(shRNA)慢病毒载体,并在细胞水平鉴定其干扰效率。方法:根据PKCγ-mRNA序列,选择3条19nt的靶序列,设计并合成包含正、反义靶序...目的:研究指出蛋白激酶Cγ(PKCγ)在脊髓水平上参与了伤害性信号的传递,文中拟构建大鼠PKCγ基因的小发卡RNA(shRNA)慢病毒载体,并在细胞水平鉴定其干扰效率。方法:根据PKCγ-mRNA序列,选择3条19nt的靶序列,设计并合成包含正、反义靶序列的互补DNA链,退火后插入到pLVTHM载体的H1启动子后获得重组质粒,同时构建一个非特异性对照质粒。将所构建的质粒与pMDLg-pRRE、pR sv-REV、pMD2G共转染293T细胞,包装产生慢病毒后,分别感染C6细胞,经免疫印迹技术(W estern b lot)检测PKCγ基因的蛋白表达水平以评价慢病毒载体的抑制效率。结果:测序证实成功构建了3个大鼠PKCγ基因shRNA慢病毒载体,分别感染C6细胞后pLV-PKC2和pLV-PKC3可明显降低PKCγ蛋白的表达,其中以pLV-PKC2(靶向位点:+1913^+1931)的慢病毒抑制效率最高。结论:成功构建了大鼠PKCγ基因shRNA慢病毒载体,且慢病毒载体pLV-PKC2能特异、高效地抑制PKCγ基因的表达。展开更多
背景:内脏-内脏之间存在广泛的伤害性传入会聚和相互作用,在跨器官痛觉过敏的发生中起重要作用。目的:在内脏高敏感状态下建立跨器官敏化大鼠模型,探讨蛋白激酶Cγ(PKCγ)对跨器官内脏敏感性的调控作用。方法:Sprague-Dawley大鼠随机分...背景:内脏-内脏之间存在广泛的伤害性传入会聚和相互作用,在跨器官痛觉过敏的发生中起重要作用。目的:在内脏高敏感状态下建立跨器官敏化大鼠模型,探讨蛋白激酶Cγ(PKCγ)对跨器官内脏敏感性的调控作用。方法:Sprague-Dawley大鼠随机分为4组,其中3组在鸡卵清蛋白(OVA)基础致敏联合结直肠芥子油(MO)灌注建立结直肠-膀胱跨器官敏化模型的基础上,分别鞘内注射PKCγ特异性抑制剂GF109203X、0.9%NaCl溶液(NS)或不予鞘内注射,另一组为正常对照组。各组大鼠行梯度结直肠扩张/膀胱扩张(CRD/UBD),根据腹壁肌电活动曲线下面积(AUC)评估内脏敏感性的变化。结果:各组UBD梯度扩张刺激体积均与腹壁肌电活动AUC显著相关(P<0.05)。UBD扩张体积为1.0、1.5、2.0 ml以及CRD扩张压力为40、60、80 mm Hg时,OVA+MO组AUC显著高于正常对照组(P<0.05);UBD扩张体积为1.5、2.0 ml以及CRD扩张压力为60、80 mm Hg时,OVA+MO+GF109203X组AUC较OVA+MO+NS组显著降低(P<0.05)。结论:鞘内注射PKCγ特异性抑制剂能逆转跨器官交叉内脏高敏感性,提示PKCγ在跨器官敏化的产生和维持中起重要作用。展开更多
基金supported by the National Natural Science Foundation of China(81971058,82371226,82101295,82301398)the National Funded Postdoctoral Researcher Program(GZC20233585)The Boost Plan of Xijing Hospital(XJZT24QN25,XJZT25CX22).
文摘Neuropathic pain,often featuring allodynia,imposes significant physical and psychological burdens on patients,with limited treatments due to unclear central mechanisms.Addressing this challenge remains a crucial unsolved issue in pain medicine.Our previous study,using protein kinase C gamma(PKCγ)-tdTomato mice,highlights the spinal feedforward inhibitory circuit involving PKCγ neurons in gating neuropathic allodynia.However,the regulatory mechanisms governing this circuit necessitate further elucidation.We used diverse transgenic mice and advanced techniques to uncover the regulatory role of the descending serotonin(5-HT)facilitation system on spinal PKCγ neurons.Our findings revealed that 5-HT neurons from the rostral ventromedial medulla hyperpolarize spinal inhibitory interneurons via 5-HT_(2C) receptors,disinhibiting the feedforward inhibitory circuit involving PKCγ neurons and exacerbating allodynia.Inhibiting spinal 5-HT_(2C) receptors restored the feedforward inhibitory circuit,effectively preventing neuropathic allodynia.These insights offer promising therapeutic targets for neuropathic allodynia management,emphasizing the potential of spinal 5-HT_(2C) receptors as a novel avenue for intervention.
基金This work was supported by the Excellent Young Teachers Program of Ministry of Education of Chian Scientific Developing Program of Beijing Municipal Commission of Education (200KJ080+2 种基金 KM200310025100) Beijing Natural Science Foundation (7032005) Nati
文摘目的:研究指出蛋白激酶Cγ(PKCγ)在脊髓水平上参与了伤害性信号的传递,文中拟构建大鼠PKCγ基因的小发卡RNA(shRNA)慢病毒载体,并在细胞水平鉴定其干扰效率。方法:根据PKCγ-mRNA序列,选择3条19nt的靶序列,设计并合成包含正、反义靶序列的互补DNA链,退火后插入到pLVTHM载体的H1启动子后获得重组质粒,同时构建一个非特异性对照质粒。将所构建的质粒与pMDLg-pRRE、pR sv-REV、pMD2G共转染293T细胞,包装产生慢病毒后,分别感染C6细胞,经免疫印迹技术(W estern b lot)检测PKCγ基因的蛋白表达水平以评价慢病毒载体的抑制效率。结果:测序证实成功构建了3个大鼠PKCγ基因shRNA慢病毒载体,分别感染C6细胞后pLV-PKC2和pLV-PKC3可明显降低PKCγ蛋白的表达,其中以pLV-PKC2(靶向位点:+1913^+1931)的慢病毒抑制效率最高。结论:成功构建了大鼠PKCγ基因shRNA慢病毒载体,且慢病毒载体pLV-PKC2能特异、高效地抑制PKCγ基因的表达。
文摘背景:内脏-内脏之间存在广泛的伤害性传入会聚和相互作用,在跨器官痛觉过敏的发生中起重要作用。目的:在内脏高敏感状态下建立跨器官敏化大鼠模型,探讨蛋白激酶Cγ(PKCγ)对跨器官内脏敏感性的调控作用。方法:Sprague-Dawley大鼠随机分为4组,其中3组在鸡卵清蛋白(OVA)基础致敏联合结直肠芥子油(MO)灌注建立结直肠-膀胱跨器官敏化模型的基础上,分别鞘内注射PKCγ特异性抑制剂GF109203X、0.9%NaCl溶液(NS)或不予鞘内注射,另一组为正常对照组。各组大鼠行梯度结直肠扩张/膀胱扩张(CRD/UBD),根据腹壁肌电活动曲线下面积(AUC)评估内脏敏感性的变化。结果:各组UBD梯度扩张刺激体积均与腹壁肌电活动AUC显著相关(P<0.05)。UBD扩张体积为1.0、1.5、2.0 ml以及CRD扩张压力为40、60、80 mm Hg时,OVA+MO组AUC显著高于正常对照组(P<0.05);UBD扩张体积为1.5、2.0 ml以及CRD扩张压力为60、80 mm Hg时,OVA+MO+GF109203X组AUC较OVA+MO+NS组显著降低(P<0.05)。结论:鞘内注射PKCγ特异性抑制剂能逆转跨器官交叉内脏高敏感性,提示PKCγ在跨器官敏化的产生和维持中起重要作用。
