Zhou Fan, Zhang Wei, Zhou Jianmei, Li Meirong, Zhong Feng, Zhang Yun, Liu Yuezhu, Wang Yaping
Department of Anesthesiology.
Department of Pathology, Second Xiangya Hospital of Central South University, Changsha, Hunan, People's Republic of China.
J Pain Res. 2017 Mar 20;10:653-662. doi: 10.2147/JPR.S125805. eCollection 2017.
Endoplasmic reticulum (ER) stress is involved in many neurological and inflammatory responses. Peripheral inflammatory responses can induce central sensitization and trigger inflammatory pain. However, there is little research on the relationship between ER stress and inflammatory pain. In this study, we examined whether the ER stress response is involved in peripheral inflammatory pain using a formalin-induced rat pain model.
Rats were divided into the following five groups: control, formalin, formalin + vehicle, formalin + 4-phenylbutyric acid (4-PBA) (40 mg/kg) and formalin + 4-PBA (100 mg/kg). Formalin-induced pain was assessed behaviorally by recording licking activity. The expression levels of immunoglobulin-binding protein (BIP), activating transcription factor-6 (ATF6), phosphorylated inositol-requiring enzyme-1 (p-IRE1), phosphorylated protein kinase RNA-like ER kinase (p-PERK) and c-fos were quantitatively assessed by Western blot, and the distribution of BIP, ATF6 and c-fos in the lumbar enlargement of spinal cord were identified by immunohistochemistry in spinal dorsal horn slices. In addition, the concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) in the spinal cord were tested by biochemical measurement and enzyme-linked immunosorbent assay (ELISA), respectively.
Intraperitoneal injection of 4-PBA at the dose of 100 mg/kg before formalin injection significantly decreased nociceptive behavior in the second phase compared with control, formalin, formalin + vehicle and formalin + 4-PBA (40 mg/kg) (<0.05). Western blot showed that formalin injection significantly upregulated the expression of BIP, ATF6, p-PERK and c-fos in the spinal cord. This upregulation was reduced by peritoneal injection of 4-PBA (<0.05), while expression of p-IRE1 was not altered by formalin treatment. Immunohistochemistry revealed markedly increased staining density for BIP, ATF6 and c-fos in the superficial spinal dorsal horn after formalin injection. This was significantly decreased by administration of 4-PBA (<0.05). Compared with the formalin + vehicle group, 4-PBA inhibited the release of NO and PGE2 in the spinal cord (<0.05).
These results suggest that ER stress is involved in formalin-induced inflammatory pain and that inhibition of ER stress may attenuate central sensitization induced by peripheral inflammatory stimulation.
内质网（ER）应激参与多种神经和炎症反应。外周炎症反应可诱导中枢敏化并引发炎性疼痛。然而，关于内质网应激与炎性疼痛之间的关系研究较少。在本研究中，我们使用福尔马林诱导的大鼠疼痛模型来研究内质网应激反应是否参与外周炎性疼痛。
将大鼠分为以下五组：对照组、福尔马林组、福尔马林+溶剂组、福尔马林+4-苯基丁酸（4-PBA）（40mg/kg）组和福尔马林+4-PBA（100mg/kg）组。通过记录舔舐活动行为学评估福尔马林诱导的疼痛。采用蛋白质免疫印迹法定量评估免疫球蛋白结合蛋白（BIP）、活化转录因子6（ATF6）、磷酸化肌醇需求酶1（p-IRE1）、磷酸化蛋白激酶RNA样内质网激酶（p-PERK）和c-fos的表达水平，并通过免疫组织化学法在脊髓背角切片中鉴定脊髓腰膨大处BIP、ATF6和c-fos的分布。此外，分别通过生化检测和酶联免疫吸附测定（ELISA）检测脊髓中一氧化氮（NO）和前列腺素E2（PGE2）的浓度。
在注射福尔马林前腹腔注射100mg/kg剂量的4-PBA，与对照组、福尔马林组、福尔马林+溶剂组和福尔马林+4-PBA（40mg/kg）组相比，第二阶段的伤害性行为明显降低（<0.05）。蛋白质免疫印迹法显示，注射福尔马林显著上调脊髓中BIP、ATF6、p-PERK和c-fos的表达。腹腔注射4-PBA可降低这种上调（<0.05），而福尔马林处理未改变p-IRE1的表达。免疫组织化学显示，注射福尔马林后脊髓背角浅层BIP、ATF6和c-fos的染色密度显著增加。给予4-PBA后显著降低（<0.05）。与福尔马林+溶剂组相比，4-PBA抑制脊髓中NO和PGE2的释放（<0.05）。
这些结果表明内质网应激参与福尔马林诱导的炎性疼痛，抑制内质网应激可能减轻外周炎症刺激诱导的中枢敏化。
