大鼠睾丸痛模型中NMDA受体的表达研究
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摘要
目的:NMDA受体的NR1和NR2B亚基对于痛觉的产生和维持起着关键作用,本文旨在探究NMDA受体在大鼠睾丸痛模型中的表达及可能机制。方法:参照Yoshioka等的方法建立大鼠睾丸痛模型,分实验组和对照组,其中对照组大鼠睾丸注射0.2 ml生理盐水,实验组大鼠睾丸注射0.2 ml 2%醋酸溶液,后进行行为学检测。建模成功后4 h采用Western印迹、RT-qPCR、免疫荧光检测NMDA受体在背根神经节和脊髓背角中的表达。结果:建模后,实验组大鼠逃避反应潜伏期明显降低,于4 h达到最低值[(4.15±0.84) s],显著低于对照组[(12.32±1.05) s,P<0.05]。建模后4 h,RT-qPCR, Western印迹、免疫荧光结果显示:与对照组相比,实验组大鼠背根神经节中NR2B mRNA和蛋白表达明显升高(P<0.05),脊髓背角中NR2B的表达无明显变化;实验组和对照组大鼠的背根神经节和脊髓背角的NR1表达也无明显差异。结论:NMDA受体在大鼠睾丸痛发病过程中具有重要作用,疼痛前期便可通过上调NMDA受体NR2B亚基表达介导外周痛敏形成,介导睾丸疼痛的发生。
Objective: To explore the expression of the N-methyl-D-aspartate( NMDA) receptor in the rat model of orchialgia and its possible mechanisms. Methods: According to Yoshioka's method,the male rats in the control group were injected with 0. 2 ml saline,and those in the experimental group with 0. 2 ml 2% acetic acid solution. Then we tested the behavioral responses of the rats and determined the expressions of the subunits NR1 and NR2 B of the NMDA receptor in the dorsal root ganglion and spinal dorsal horn by Western blot,RT-qP CR and immunofluorescence staining. Results: The withdrawal latency was decreased in the model rats,reaching the lowest value at 4 hours after modeling,significantly lower than in the controls( [4. 15 ± 0. 84] vs [12. 32 ± 1. 05],P< 0. 05). Compared with the controls,the model rats showed remarkably increased mRNA and protein expressions of NR2 B in the dorsal root ganglion( P < 0. 05) but not in the spinal dorsal horn at 4 hours. However,no statistically significant difference was found in the expression of NR1 either in the dorsal root ganglion or in the spinal dorsal horn between the two groups( P > 0. 05). Conclusion: The NMDA receptor plays an important role in pathogenesis of orchialgia in rats. In the early stage of pain,upregulating the expression of the subunit NR2 B of the NMDA receptor can mediate peripheral hyperalgesia and consequently orchialgia. Natl J Androl,2019,25( 4) : 296-301
Objective: To explore the expression of the N-methyl-D-aspartate( NMDA) receptor in the rat model of orchialgia and its possible mechanisms. Methods: According to Yoshioka's method,the male rats in the control group were injected with 0. 2 ml saline,and those in the experimental group with 0. 2 ml 2% acetic acid solution. Then we tested the behavioral responses of the rats and determined the expressions of the subunits NR1 and NR2 B of the NMDA receptor in the dorsal root ganglion and spinal dorsal horn by Western blot,RT-qP CR and immunofluorescence staining. Results: The withdrawal latency was decreased in the model rats,reaching the lowest value at 4 hours after modeling,significantly lower than in the controls( [4. 15 ± 0. 84] vs [12. 32 ± 1. 05],P< 0. 05). Compared with the controls,the model rats showed remarkably increased mRNA and protein expressions of NR2 B in the dorsal root ganglion( P < 0. 05) but not in the spinal dorsal horn at 4 hours. However,no statistically significant difference was found in the expression of NR1 either in the dorsal root ganglion or in the spinal dorsal horn between the two groups( P > 0. 05). Conclusion: The NMDA receptor plays an important role in pathogenesis of orchialgia in rats. In the early stage of pain,upregulating the expression of the subunit NR2 B of the NMDA receptor can mediate peripheral hyperalgesia and consequently orchialgia. Natl J Androl,2019,25( 4) : 296-301
引文
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[7] 涂响安,余敬威.慢性睾丸痛的诊断与治疗.中华男科学杂志,2016,22(3):195-199.
[8] Sun WH,Chen CC.Roles of proton-sensing receptors in the transition from acute to chronic pain.J Dent Res,2016,95(2):135-142.
[9] 周利君,信文君,庞瑞萍,等.慢性疼痛的细胞因子微环境假说.中国疼痛医学杂志,2013,19(11):679-684.
[10] 宋学军.疼痛信号外周神经转导的分子生物学机制.中国疼痛医学杂志,2016,22(1):2-7.
[11] Woolf CJ,Salter MW.Neuronal plasticity:Increasing the gain in pain.Science,2000,288(5472):1765-1768.
[12] 余敬威,黄洁虹,吕坤龙,等.TRPV1和TRPA1在大鼠睾丸痛模型背神经节中的表达.中华男科学杂志,2017,23(4):296-301.
[13] Pagadala P,Park CK,Bang S,et al.Loss of NR1 subunit of NMDARs in primary sensory neurons leads to hyperexcitability and pain hypersensitivity:Involvement of Ca2+-activated small conductance potassium channels.J Neurosci,2013,33(33):13425-13430.
[14] Ivanusicl JJ,Beainil D,Hatchl RJ,et al.Peripheral N-methyl-d-aspartate receptors contribute to mechanical hypersensitivity in a rat model of inflammatory temporomandibular joint pain.Eur J Pain,2011,15(2):179-185.
[15] Zhuo M.Plasticity of NMDA receptor NR2B subunit in memory and chronic pain.Mol Brain,2009,2:4.
[16] Norcini M,Sideris A,Adler SM,et al.NR2B expression in rat DRG is differentially regulated following peripheral nerve injuries that lead to transient or sustained stimuli-evoked hypersensitivity.Front Mol Neurosci,2016,9:100.
[17] Zhao S,Liu F,Wu Y,et al.Upregulation of spinal NMDA receptors mediates hydrogen sulfide-induced hyperalgesia.J Neurol Sci,2016,363:176-181.
[18] Lee J,Ro JY.Differential regulation of glutamate receptors in trigeminal ganglia following masseter inflammation.Neurosci Lett,2007,421(2):91-95.
[19] Guo W,Zou S,Guan Y,et al.Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia.J Neurosci,2002,22(14):6208-6217.
[2] Kavoussi PK,Costabile RA.Orchialgia and the chronic pelvic pain syndrome.World J Urol,2013,31(4):773-778.
[3] Hardingham GE.Coupling of the NMDA receptor to neuroprotective and neurodestructive events.Biochem Soc Trans,2009,37(Pt 6):1147-1160.
[4] 郭张华,陈建平.NMDA受体在神经病理性疼痛中作用的研究进展.中国疼痛医学杂志,2013,19(3):173-176.
[5] Yoshioka K,Tanahashi M,Uchida W.Behavioral and urological evaluation of a testicular pain model.Urology,2010,75(4):943-948.
[6] Sigalos JT,Pastuszak AW.Chronic orchialgia:Epidemiology,diagnosis and evaluation.Transl Androl Urol,2017,6(Suppl 1):S37-S43.
[7] 涂响安,余敬威.慢性睾丸痛的诊断与治疗.中华男科学杂志,2016,22(3):195-199.
[8] Sun WH,Chen CC.Roles of proton-sensing receptors in the transition from acute to chronic pain.J Dent Res,2016,95(2):135-142.
[9] 周利君,信文君,庞瑞萍,等.慢性疼痛的细胞因子微环境假说.中国疼痛医学杂志,2013,19(11):679-684.
[10] 宋学军.疼痛信号外周神经转导的分子生物学机制.中国疼痛医学杂志,2016,22(1):2-7.
[11] Woolf CJ,Salter MW.Neuronal plasticity:Increasing the gain in pain.Science,2000,288(5472):1765-1768.
[12] 余敬威,黄洁虹,吕坤龙,等.TRPV1和TRPA1在大鼠睾丸痛模型背神经节中的表达.中华男科学杂志,2017,23(4):296-301.
[13] Pagadala P,Park CK,Bang S,et al.Loss of NR1 subunit of NMDARs in primary sensory neurons leads to hyperexcitability and pain hypersensitivity:Involvement of Ca2+-activated small conductance potassium channels.J Neurosci,2013,33(33):13425-13430.
[14] Ivanusicl JJ,Beainil D,Hatchl RJ,et al.Peripheral N-methyl-d-aspartate receptors contribute to mechanical hypersensitivity in a rat model of inflammatory temporomandibular joint pain.Eur J Pain,2011,15(2):179-185.
[15] Zhuo M.Plasticity of NMDA receptor NR2B subunit in memory and chronic pain.Mol Brain,2009,2:4.
[16] Norcini M,Sideris A,Adler SM,et al.NR2B expression in rat DRG is differentially regulated following peripheral nerve injuries that lead to transient or sustained stimuli-evoked hypersensitivity.Front Mol Neurosci,2016,9:100.
[17] Zhao S,Liu F,Wu Y,et al.Upregulation of spinal NMDA receptors mediates hydrogen sulfide-induced hyperalgesia.J Neurol Sci,2016,363:176-181.
[18] Lee J,Ro JY.Differential regulation of glutamate receptors in trigeminal ganglia following masseter inflammation.Neurosci Lett,2007,421(2):91-95.
[19] Guo W,Zou S,Guan Y,et al.Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia.J Neurosci,2002,22(14):6208-6217.