The role of ectopic P granules protein 5 homolog (EPG5) in DHPG‐induced pain sensitization in mice

• Published in: Journal of neurochemistry 2023-04, Vol.165 (2), p.196-210
• Main Authors: Mei, Xiangyang, Yin, Chengyu, Pan, Yushuang, Chen, Lei, Wu, Cheng, Li, Xiangyao, Feng, Zhiying
• Format: Article
• Language: English
• Subjects: Animals; Autophagy; Autophagy-Related Proteins; DHPG; EPG5; Genetic screening; Germ Cell Ribonucleoprotein Granules; Glutamic acid receptors (metabotropic); Granular materials; Homology; Hyperalgesia; Immunohistochemistry; Injection; metabolic glutamate receptor; Mice; mRNA; Pain; Pain - metabolism; Pain perception; pain sensitization; Proteins; Proteomics; Receptors, Metabotropic Glutamate - metabolism; Spinal cord; Vesicular Transport Proteins; Viruses; Western blotting
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/jnc.15779

Nociplastic pain is a severe health problem, while its mechanisms are still unclear. (R, S)‐3,5‐Dihydroxyphenylglycine (DHPG) is a group I metabotropic glutamate receptor (mGluR) agonist that can cause central sensitization, which plays a role in nociplastic pain. In this study, after intrathecal injection of 25 nmol DHPG for three consecutive days, whole proteins were extracted from the L4~6 lumbar spinal cord of mice 2 h after intrathecal administration on the third day for proteomics analysis. Based on the results, 15 down‐regulated and 20 up‐regulated proteins were identified in mice. Real‐time quantitative PCR (RT‐qPCR) and western blotting (WB) revealed that the expression of ectopic P granules protein 5 homolog (EPG5) mRNA and protein were significantly up‐regulated compared with the control group, which was consistent with the proteomics results. Originally identified in the genetic screening of Caenorhabditis elegans, EPG5 is mainly involved in regulating autophagy in the body, and in our study, it was mainly expressed in spinal neurons, as revealed by immunohistochemistry staining. After the intrathecal injection of 8 μL adeno‐associated virus (AAV)‐EPG5 short hairpin RNA (shRNA) to knock down spinal EPG5, the hyperalgesia caused by DHPG was relieved. Altogether, these results suggest that EPG5 plays an important role in DHPG‐induced pain sensitization in mice. Intrathecal injection of DHPG can induce mechanical and thermal hyperalgesia in mice, and compared with the control group, the intrathecal injection of DHPG not only caused hyperalgesia in mice, but also the results of proteomics also confirmed that 15 proteins were significantly down‐regulated, and 20 proteins were significantly up‐regulated, including EPG5; furthermore, RT‐qPCR was used to verify the DEPs at the gene level, and it was found that only Epg5 mRNA expression was significantly up‐regulated when compared with the control group. We further used western blotting to reconfirm the increased protein expression of EPG5. The immunohistochemistry results showed that EPG5 was mainly expressed in spinal cord neurons.