Zhao Yang, Xin Yan, Chu Haichen
Department of Anesthesiology, The Affiliated Hospital of Qingdao University, Qingdao, China.
Department of Anesthesiology, Qingdao Municipal Hospital, Qingdao, China.
Front Neurosci. 2019 Sep 10;13:919. doi: 10.3389/fnins.2019.00919. eCollection 2019.
Neuropathic pain can develop after nerve injury, when deleterious changes occur in injured neurons and glia cells. Melanocortin 4 receptor (MC4R) is involved in the regulation of pain due to its high expressions in brain. Moreover, MC4R could mediate the c-Jun N-terminal kinase (JNK) signaling pathway, but whether the MC4R-regulated JNK signaling pathway participated in neuropathic pain after chronic constriction injury (CCI) is still unclear.
A total of 128 Sprague-Dawley rats were allocated into four experiment groups: the SHAM group, CCI + NaCl group, CCI + HS group, and CCI + SP + HS group. For the CCI + NaCl group, the sciatic nerves were ligated. For the SHAM group, an identical manner to the CCI without ligation was performed. For CCI + HS and CCI + SP + HS groups, rats were injected with MC4R inhibitor (HS014) and HS014 plus JNK inhibitor (SP600125), respectively, from days 3 to 14 after CCI. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were used to assess the nociceptive behavior. ELISA was used to detect the levels of inflammatory cytokines. qRT-PCR and Western blots (WB) were utilized to examine the mRNA and protein expressions of JNK signaling pathway-related genes. Meanwhile, the expression levels of MC4R and p-JNK were further evaluated by immunohistochemistry (IHC) and immunofluorescence (IF) experiments. Finally, in order to confirm the results, astrocytes were isolated and transfected with MC4R-overexpression plasmid. Furthermore, the protein expressions of JNK signaling pathway-related genes were tested by WB.
It was showed that the values of PWL and PWT were significantly increased in CCI + HS group and CCI + SP + HS group compared with CCI + NaCl group. The increased interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) secretion in CCI + NaCl group was lowered by HS and SP + HS. MC4R, p-JNK, ATF3, and c-Jun levels were up-regulated with CCI surgery, but down-regulated with HS and SP + HS treatments. Moreover, the IHC and IF results further revealed that MC4R and p-JNK expressions in CCI + NaCl group were remarkably higher than those in HS group and HS + SP group. data also indicated that HS, SP, and SP + HS could down-regulate the expressions of MC4R, p-JNK, ATF3, and c-Jun in M1830 astrocytes.
Our findings indicated that MC4R is involved in neuropathic pain by regulating JNK signaling pathway after CCI.
神经病理性疼痛可在神经损伤后发生，此时受损神经元和神经胶质细胞会出现有害变化。黑皮质素4受体（MC4R）因其在大脑中的高表达而参与疼痛调节。此外，MC4R可介导c-Jun氨基末端激酶（JNK）信号通路，但MC4R调节的JNK信号通路是否参与慢性缩窄性损伤（CCI）后的神经病理性疼痛仍不清楚。
将128只Sprague-Dawley大鼠分为四个实验组：假手术组、CCI+氯化钠组、CCI+HS组和CCI+SP+HS组。对于CCI+氯化钠组，结扎坐骨神经。对于假手术组，采用与CCI相同但不结扎的方式。对于CCI+HS组和CCI+SP+HS组，在CCI后第3天至第14天分别给大鼠注射MC4R抑制剂（HS014）和HS014加JNK抑制剂（SP600125）。采用爪部撤离潜伏期（PWL）和爪部撤离阈值（PWT）评估伤害性感受行为。采用酶联免疫吸附测定（ELISA）检测炎症细胞因子水平。采用定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法（WB）检测JNK信号通路相关基因的mRNA和蛋白表达。同时，通过免疫组织化学（IHC）和免疫荧光（IF）实验进一步评估MC4R和磷酸化JNK（p-JNK）的表达水平。最后，为了证实结果，分离星形胶质细胞并用MC4R过表达质粒转染。此外，通过WB检测JNK信号通路相关基因的蛋白表达。
结果显示，与CCI+氯化钠组相比，CCI+HS组和CCI+SP+HS组的PWL和PWT值显著升高。HS和SP+HS降低了CCI+氯化钠组中白细胞介素-6（IL-6）、白细胞介素-1β和肿瘤坏死因子-α（TNF-α）分泌的增加。MC4R、p-JNK、活化转录因子3（ATF3）和c-Jun水平在CCI手术时上调，但在HS和SP+HS处理时下调。此外，IHC和IF结果进一步显示，CCI+氯化钠组中MC4R和p-JNK的表达明显高于HS组和HS+SP组。数据还表明，HS、SP和SP+HS可下调M1830星形胶质细胞中MC4R、p-JNK、ATF3和c-Jun的表达。
我们的研究结果表明，MC4R通过在CCI后调节JNK信号通路参与神经病理性疼痛。
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