目的:基于电压门控钠离子通道NAV1.7探究运气方苁蓉牛膝汤对膝骨关节炎(knee osteoarthritis,KOA)慢性疼痛的影响及作用机制。方法:将18只8周龄C57BL/6J雄性小鼠随机分为3组,每组6只。模型组和苁蓉牛膝汤组采用内侧半月板失稳术于小鼠...目的:基于电压门控钠离子通道NAV1.7探究运气方苁蓉牛膝汤对膝骨关节炎(knee osteoarthritis,KOA)慢性疼痛的影响及作用机制。方法:将18只8周龄C57BL/6J雄性小鼠随机分为3组,每组6只。模型组和苁蓉牛膝汤组采用内侧半月板失稳术于小鼠右后肢构建KOA模型,假手术组暴露关节腔后缝合皮肤。造模手术后第2天起,苁蓉牛膝汤组按0.02 mL·g^(-1)以苁蓉牛膝汤浓缩液(生药浓度1 g·mL^(-1))灌胃,模型组和假手术组以等量生理盐水灌胃,每天1次,连续灌胃12周。造模后6周、8周、10周、12周分别采用Von Frey测痛纤维丝和智能热板仪测定小鼠机械性疼痛阈值和热敏性疼痛阈值。造模后12周药物干预结束后,取小鼠右侧膝关节和L_(4)~L_(6)右侧背根神经节,采用Micro-CT进行膝关节骨微结构检测;采用阿尔辛蓝-苏木素-橙黄G染色进行膝关节软骨组织病理学观察,并采用国际骨关节炎研究学会(Osteoarthritis Research Society International,OARSI)评分系统对膝关节软骨病理变化进行评价;采用免疫组织化学染色检测膝关节软骨中Ⅱ型胶原、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、白细胞介素(interleukin,IL)-1β、IL^(-1)8表达情况;采用免疫荧光技术检测L_(4)~L_(6)背根神经节中NAV1.7和c-fos表达情况及膝关节软骨中NAV1.7表达情况。结果:①机械性疼痛阈值测定结果。模型组机械性疼痛阈值随时间变化不明显(F=1.764,P=0.186),假手术组和苁蓉牛膝汤组机械性疼痛阈值随时间变化均呈先升高后降低的趋势(F=6.764,P=0.002;F=4.188,P=0.019)。造模后6周、8周、10周、12周,模型组的机械性疼痛阈值均低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.042;P=0.002,P=0.009;P=0.000,P=0.000;P=0.000,P=0.001)。②热敏性疼痛阈值测定结果。热敏性疼痛阈值随时间变化总体呈先升高后降低的趋势(F=14.383,P=0.000)。模型组的热敏性疼痛阈值低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.000)。③膝关节骨微结构检测结果。Micro-CT检查结果显示,假手术组膝关节结构基本正常,模型组可见明显骨赘和软骨下骨钙化,苁蓉牛膝汤组可见轻微骨赘和软骨下骨钙化。模型组的骨体积分数、骨小梁厚度均高于假手术组和苁蓉牛膝汤组(P=0.000,P=0.012;P=0.000,P=0.000),骨小梁分离度低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.027)。④膝关节软骨组织病理学观察结果。模型组的膝关节软骨OARSI评分高于假手术组和苁蓉牛膝汤组(P=0.000,P=0.004)。⑤膝关节软骨中Ⅱ型胶原、MMP-9、IL-1β、IL-18表达情况检测结果。模型组的Ⅱ型胶原阳性表达面积小于假手术组和苁蓉牛膝汤组(P=0.000,P=0.000),MMP-9、IL-1β、IL-18阳性表达面积均大于假手术组和苁蓉牛膝汤组(P=0.000,P=0.001;P=0.000,P=0.002;P=0.000,P=0.000)。⑥L_(4)~L_(6)背根神经节中NAV1.7和c-fos表达情况检测结果。模型组L_(4)~L_(6)背根神经节中NAV1.7和c-fos阳性表达面积均大于假手术组(NAV1.7:P=0.001,P=0.000,P=0.000;c-fos:P=0.001,P=0.000,P=0.000)和苁蓉牛膝汤组(NAV1.7:P=0.006,P=0.013,P=0.003;c-fos:P=0.019,P=0.018,P=0.006)。⑦膝关节软骨中NAV1.7表达情况检测结果。模型组膝关节软骨中NAV1.7阳性表达面积大于假手术组和苁蓉牛膝汤组(P=0.005,P=0.006)。结论:运气方苁蓉牛膝汤可改善KOA模型小鼠的慢性疼痛症状,其机制可能与下调NAV1.7表达水平,以及减轻炎症反应和延缓软骨退变有关。展开更多
Objective:To evaluate the effect of down-regulation of Nav1.7 on the activation of astrocytes and microglia in DRG of rats with cancer pain,and explore the transmission of the nociceptive information.Methods:Lentivira...Objective:To evaluate the effect of down-regulation of Nav1.7 on the activation of astrocytes and microglia in DRG of rats with cancer pain,and explore the transmission of the nociceptive information.Methods:Lentiviral vector harboring RNAi sequence targeting the Navl.7 gene was constructed,and Walker 256 breast cancer cell and morphine was injected to build the bone cancer pain model and morphine tolerance model in rats.Lentiviral vector was injected.Rats in each model were divided into 4 groups:model group,PBS group,vehicle group and LV-Nav1.7 group.The expression levels of GFAP and OX42 in dorsal root ganglia(DRG) were measured.Results:After the animal model was built,the level of Navl.7,GFAP and OX42 was improved obviously with the time prolonged,which was statistically significant(P<0.05).The expression level of GFAP and OX42 in the DRG in the LV-Navl.7 group declined obviously compared to the model group,PBS group and vehicle group(P<0.05).Conclusions:Intrathecal injection of Navl.7 shRNA lentiviral vector can reduce the expression of Nav1.7and inhibit the activation of astrocytes and microglia in DRG.The effort is also effective in morphine tolerance bone cancer pain model rats.展开更多
Various peptide toxins in animal venom inhibit voltage-gated sodium ion channel Nav1.7, including Nav-targeting spider toxin(NaSpTx) Family I. Toxins in NaSpTx Family I share a similar structure, i.e., Nterminal, loop...Various peptide toxins in animal venom inhibit voltage-gated sodium ion channel Nav1.7, including Nav-targeting spider toxin(NaSpTx) Family I. Toxins in NaSpTx Family I share a similar structure, i.e., Nterminal, loops 1–4, and C-terminal. Here, we used Mu-theraphotoxin-Ca2a(Ca2a), a peptide isolated from Cyriopagopus albostriatus, as a template to investigate the general properties of toxins in NaSpTx Family I. The toxins interacted with the cell membrane prior to binding to Nav1.7 via similar hydrophobic residues. Residues in loop 1, loop 4,and the C-terminal primarily interacted with the S3–S4 linker of domain II, especially basic amino acids binding to E818. We also identified the critical role of loop 2 in Ca2a regarding its affinity to Nav1.7.Our results provide further evidence that NaSpTx Family I toxins share similar structures and mechanisms of binding to Nav1.7.展开更多
Objective The aim of this study was to examine the effects of photodynamic therapy(PDT)on the expression of Nav1.7 in spinal dorsal root ganglion(DRG)neurons.Methods The primary DRG neurons from newborn SD rats were c...Objective The aim of this study was to examine the effects of photodynamic therapy(PDT)on the expression of Nav1.7 in spinal dorsal root ganglion(DRG)neurons.Methods The primary DRG neurons from newborn SD rats were cultured.The cells were identified by neuron-specific enolase immunofluorescence staining.DRG neurons were divided into four groups:control group,photosensitizer group,laser group,and PDT group.The cell viability was detected by a cell counting kit-8(CCK8)assay.qRT-PCR and Western blotting were used to determine the mRNA and protein expression levels of Nav1.7 in DRG neurons.Results The purity of the cultured primary DRG neurons was greater than 90%.Compared with the control group,no significant change was found in the cell viability of the photosensitizer group,while the viability in the laser group and the PDT group was significantly reduced.The mRNA and protein expression levels of Nav1.7 were significantly greater in the laser group and the PDT group than in the control group.At the same time,the mRNA and protein expression levels of Nav1.7 were greater in the laser group than in the PDT group.Conclusion Both laser and PDT could upregulate the expression of Nav1.7 in DRG neurons,and the promoting effect might be related to the pain induced by clinical treatment.This study provides a research basis for the use of laser and PDT to treat pain.A better understanding of the relationship between Nav1.7 and PDT can help clinicians better manage PDT-related pain.展开更多
文摘目的:基于电压门控钠离子通道NAV1.7探究运气方苁蓉牛膝汤对膝骨关节炎(knee osteoarthritis,KOA)慢性疼痛的影响及作用机制。方法:将18只8周龄C57BL/6J雄性小鼠随机分为3组,每组6只。模型组和苁蓉牛膝汤组采用内侧半月板失稳术于小鼠右后肢构建KOA模型,假手术组暴露关节腔后缝合皮肤。造模手术后第2天起,苁蓉牛膝汤组按0.02 mL·g^(-1)以苁蓉牛膝汤浓缩液(生药浓度1 g·mL^(-1))灌胃,模型组和假手术组以等量生理盐水灌胃,每天1次,连续灌胃12周。造模后6周、8周、10周、12周分别采用Von Frey测痛纤维丝和智能热板仪测定小鼠机械性疼痛阈值和热敏性疼痛阈值。造模后12周药物干预结束后,取小鼠右侧膝关节和L_(4)~L_(6)右侧背根神经节,采用Micro-CT进行膝关节骨微结构检测;采用阿尔辛蓝-苏木素-橙黄G染色进行膝关节软骨组织病理学观察,并采用国际骨关节炎研究学会(Osteoarthritis Research Society International,OARSI)评分系统对膝关节软骨病理变化进行评价;采用免疫组织化学染色检测膝关节软骨中Ⅱ型胶原、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、白细胞介素(interleukin,IL)-1β、IL^(-1)8表达情况;采用免疫荧光技术检测L_(4)~L_(6)背根神经节中NAV1.7和c-fos表达情况及膝关节软骨中NAV1.7表达情况。结果:①机械性疼痛阈值测定结果。模型组机械性疼痛阈值随时间变化不明显(F=1.764,P=0.186),假手术组和苁蓉牛膝汤组机械性疼痛阈值随时间变化均呈先升高后降低的趋势(F=6.764,P=0.002;F=4.188,P=0.019)。造模后6周、8周、10周、12周,模型组的机械性疼痛阈值均低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.042;P=0.002,P=0.009;P=0.000,P=0.000;P=0.000,P=0.001)。②热敏性疼痛阈值测定结果。热敏性疼痛阈值随时间变化总体呈先升高后降低的趋势(F=14.383,P=0.000)。模型组的热敏性疼痛阈值低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.000)。③膝关节骨微结构检测结果。Micro-CT检查结果显示,假手术组膝关节结构基本正常,模型组可见明显骨赘和软骨下骨钙化,苁蓉牛膝汤组可见轻微骨赘和软骨下骨钙化。模型组的骨体积分数、骨小梁厚度均高于假手术组和苁蓉牛膝汤组(P=0.000,P=0.012;P=0.000,P=0.000),骨小梁分离度低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.027)。④膝关节软骨组织病理学观察结果。模型组的膝关节软骨OARSI评分高于假手术组和苁蓉牛膝汤组(P=0.000,P=0.004)。⑤膝关节软骨中Ⅱ型胶原、MMP-9、IL-1β、IL-18表达情况检测结果。模型组的Ⅱ型胶原阳性表达面积小于假手术组和苁蓉牛膝汤组(P=0.000,P=0.000),MMP-9、IL-1β、IL-18阳性表达面积均大于假手术组和苁蓉牛膝汤组(P=0.000,P=0.001;P=0.000,P=0.002;P=0.000,P=0.000)。⑥L_(4)~L_(6)背根神经节中NAV1.7和c-fos表达情况检测结果。模型组L_(4)~L_(6)背根神经节中NAV1.7和c-fos阳性表达面积均大于假手术组(NAV1.7:P=0.001,P=0.000,P=0.000;c-fos:P=0.001,P=0.000,P=0.000)和苁蓉牛膝汤组(NAV1.7:P=0.006,P=0.013,P=0.003;c-fos:P=0.019,P=0.018,P=0.006)。⑦膝关节软骨中NAV1.7表达情况检测结果。模型组膝关节软骨中NAV1.7阳性表达面积大于假手术组和苁蓉牛膝汤组(P=0.005,P=0.006)。结论:运气方苁蓉牛膝汤可改善KOA模型小鼠的慢性疼痛症状,其机制可能与下调NAV1.7表达水平,以及减轻炎症反应和延缓软骨退变有关。
基金supported by National Natural Science Foundation of China(NO.81201395)
文摘Objective:To evaluate the effect of down-regulation of Nav1.7 on the activation of astrocytes and microglia in DRG of rats with cancer pain,and explore the transmission of the nociceptive information.Methods:Lentiviral vector harboring RNAi sequence targeting the Navl.7 gene was constructed,and Walker 256 breast cancer cell and morphine was injected to build the bone cancer pain model and morphine tolerance model in rats.Lentiviral vector was injected.Rats in each model were divided into 4 groups:model group,PBS group,vehicle group and LV-Nav1.7 group.The expression levels of GFAP and OX42 in dorsal root ganglia(DRG) were measured.Results:After the animal model was built,the level of Navl.7,GFAP and OX42 was improved obviously with the time prolonged,which was statistically significant(P<0.05).The expression level of GFAP and OX42 in the DRG in the LV-Navl.7 group declined obviously compared to the model group,PBS group and vehicle group(P<0.05).Conclusions:Intrathecal injection of Navl.7 shRNA lentiviral vector can reduce the expression of Nav1.7and inhibit the activation of astrocytes and microglia in DRG.The effort is also effective in morphine tolerance bone cancer pain model rats.
基金supported by the National Natural Science Foundation of China (31971190)Science Fund for Distinguished Young Scholars of Hunan Province (2021JJ10035)Education Department of Hunan Province (19A321)。
文摘Various peptide toxins in animal venom inhibit voltage-gated sodium ion channel Nav1.7, including Nav-targeting spider toxin(NaSpTx) Family I. Toxins in NaSpTx Family I share a similar structure, i.e., Nterminal, loops 1–4, and C-terminal. Here, we used Mu-theraphotoxin-Ca2a(Ca2a), a peptide isolated from Cyriopagopus albostriatus, as a template to investigate the general properties of toxins in NaSpTx Family I. The toxins interacted with the cell membrane prior to binding to Nav1.7 via similar hydrophobic residues. Residues in loop 1, loop 4,and the C-terminal primarily interacted with the S3–S4 linker of domain II, especially basic amino acids binding to E818. We also identified the critical role of loop 2 in Ca2a regarding its affinity to Nav1.7.Our results provide further evidence that NaSpTx Family I toxins share similar structures and mechanisms of binding to Nav1.7.
基金supported by Natural Science Foundation of Beijing Municipality(No.7202200)Central Military Commission Health Care Project(No.20BJZ13)Air Force Equipment Scientific Research Project(No.KJ20201A050226).
文摘Objective The aim of this study was to examine the effects of photodynamic therapy(PDT)on the expression of Nav1.7 in spinal dorsal root ganglion(DRG)neurons.Methods The primary DRG neurons from newborn SD rats were cultured.The cells were identified by neuron-specific enolase immunofluorescence staining.DRG neurons were divided into four groups:control group,photosensitizer group,laser group,and PDT group.The cell viability was detected by a cell counting kit-8(CCK8)assay.qRT-PCR and Western blotting were used to determine the mRNA and protein expression levels of Nav1.7 in DRG neurons.Results The purity of the cultured primary DRG neurons was greater than 90%.Compared with the control group,no significant change was found in the cell viability of the photosensitizer group,while the viability in the laser group and the PDT group was significantly reduced.The mRNA and protein expression levels of Nav1.7 were significantly greater in the laser group and the PDT group than in the control group.At the same time,the mRNA and protein expression levels of Nav1.7 were greater in the laser group than in the PDT group.Conclusion Both laser and PDT could upregulate the expression of Nav1.7 in DRG neurons,and the promoting effect might be related to the pain induced by clinical treatment.This study provides a research basis for the use of laser and PDT to treat pain.A better understanding of the relationship between Nav1.7 and PDT can help clinicians better manage PDT-related pain.
