Involvement of endoplasmic reticulum stress in formalin-induced pain is attenuated by 4-phenylbutyric acid

Fan Zhou,1 Wei Zhang,1 Jianmei Zhou,1 Meirong Li,2 Feng Zhong,1 Yun Zhang,1 Yuezhu Liu,1 Yaping Wang1 1Department of Anesthesiology, 2Department of Pathology, Second Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China Background: 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.Methods: 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.Results: 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) (P<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 (P<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 (P<0.05). Compared with the formalin + vehicle group, 4-PBA inhibited the release of NO and PGE2 in the spinal cord (P<0.05).Conclusion: 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. Keywords: endoplasmic reticulum stress, formalin-induced pain, 4-phenylbutyric acid, central sensitizatio