Toll样受体4调节硫化氢引起腰椎间盘突出症模型鼠中枢痛敏的机制  

Toll-like receptor 4 regulates hydrogen sulfide and central pain sensitivity in rat lumbar disc herniation model

作　　者：朱伟卿 凌卓彦[1] 孙永明[1] 严军[1] Zhu Weiqing;Ling Zhuoyan;Sun Yongming;Yan Jun(Department of Orthopedics,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China)

机构地区：[1]苏州大学附属第二医院脊柱外科,215004

出　　处：《中华实验外科杂志》2020年第3期513-516,共4页Chinese Journal of Experimental Surgery

基　　金：江苏省自然科学研究基金(BK20191169);苏州市民生科技项目(SYSD2018205)。

摘　　要：目的探讨Toll样受体4(TLR4)和核因子-κB(NF-κB)调节硫化氢(H2S)和内源性胱硫醚B合成酶(CBS)蛋白表达的作用以及其参与腰椎间突出症(LDH)中枢痛敏的机制。方法利用大鼠(购自苏州大学实验动物中心)构建腰椎间盘突出症模型(LDH)组和假手术(Sham)组;使用膜片钳技术检测脊髓胶质区(SG)神经元兴奋性的产生和传导;蛋白质印迹法(Western blot)实验测定不同组别之间脊髓背角(SC)中TLR4、NF-κB、CBS蛋白表达发生的差异性变化;通过给予大鼠有害的热或机械等异常性疼痛增加的刺激,观察其行为的变化,评估大鼠痛觉过敏是否增强。分析电生理数据时,选取sEPSCs时长4 min,并保证至少包含300个events,并且使用Clampfit 10.3分析软件分析其频率和振幅的大小。结果造模后与Sham组(-30.86±3.55、4.34±0.64)比较,LDH组(-35.21±1.74、7.46±1.15)大鼠脊髓SG神经元的兴奋性和自发性兴奋性传递显著增强,差异有统计学意义(t=2.564,P<0.01;t=2.225,P<0.05)。在大鼠的脊髓背角中LDH组(0.41±0.02、0.40±0.03、4.61±0.33)比sham组(0.21±0.02、0.28±0.04、3.11±0.43)TLR4、NF-κB、CBS蛋白表达量显著上升,差异有统计学意义(t=7.071、2.400、2.767,P<0.05)。鞘内给予LDH组大鼠TLR4选择性抑制剂CL1095,与NC对照组(0.46±0.12、0.51±0.07)比较,CL1095组(0.21±0.11、0.18±0.11)NF-κB和CBS蛋白表达显著下调(t=10.07、2.531,P<0.05),并且与NC对照组(11.88±1.11、9.32±1.48)相比CL1095组(8.23±1.01、6.43±1.55)大鼠脊髓背角SG谷氨酸能神经元sEPSCs峰值振幅和频率缓解动物的病理性疼痛,差异均有统计学意义(t=4.334、2.437,P<0.05)。结论LDH诱导脊髓背角TLR4和NF-κB蛋白水平表达增高,进而激活下游CBS-H2S的表达,最终引发病理性疼痛。Objective To investigate the roles of toll-like receptor 4(TLR4)and nuclear factor-κB(NF-κB)in regulating the endogenous hydrogen sulfide-producing enzyme cystathionineβsynthetase(CBS)protein expression and the mechanism that was involved in increasing sciatic nerve hypersensitivity in lumbar disc herniation(LDH).Methods We generated a LDH rat model.We measured excitability with patch-clamp techniques.We recorded the synaptic neuron excitability and transmission in the substantia gelatinosa(SG)of spinal cord using patch clamping in vitro.Western blotting was used to detect the protein expression of TLR4,NF-κB,and CBS in dorsal horn of spinal cord(SC)from sham and LDH rats.The behavior study was used to measure the pain sensitive of animals.Results LDH enhanced the excitation(t=2.564,P<0.01)and spontaneous excitatory neurotransmission in SG neurons(t=2.225,P<0.05).We detected significant upregulation of TLR4,NF-κB,and CBS proteins in SC neurons in the LDH rat model(t=7.071,t=2.40,t=2.767,P<0.05).Intrathecal administration of the TLR4-selective inhibitor,CLI095,significantly suppressed the expression of NF-κB,and CBS proteins(t=10.070,P<0.01;t=2.531,P<0.05)and the frequency of glutamatergic synaptic activities of SG neurons(t=4.334,t=2.437,P<0.05)and attenuated sciatic hypersensitivity in LDH rats.Conclusion LDH increased the expression of TLR4 and NF-κB proteins,which in turn sensitized sciatic nerve hypersensitivity by upregulating CBS-H2S protein levels,finally leading to the pathological pain.

关 键 词：腰椎间盘突出症 脊髓 硫化氢 TOLL样受体4 核因子-ΚB 

分 类 号：R681.5[医药卫生—骨科学] R-332[医药卫生—外科学]