Gene therapy by lentivirus‐mediated RNA interference targeting extracellular‐regulated kinase alleviates neuropathic pain in vivo

Abstract

Backgrounds Neuropathic pain is an abnormal pain, which is related to the activation of extracellular‐regulated kinase (ERK) signaling. This study was to investigate the effects of ERK knockdown via lentivirus‐mediated RNA interference on allodynia in rats with chronic compression of the dorsal root ganglia (DRG) and to uncover the potential mechanisms. Methods The model of chronic compression of the dorsal root ganglia (CCD) was established in rats by surgery. Gene silence was induced by injecting rats with lentivirus expressing ERK short hairpin RNA (shRNA). Behavioral test was performed by calculating paw withdrawal mechanical threshold (PWMT) and thermal paw withdrawal latency (TPWL). Results We firstly generated lentivirus expressing ERK shRNA to downregulate ERK gene expression both in vitro and in vivo by using Western blot analysis and quantitative reverse transcription polymerase chain reaction. In CCD, ERK mRNA, and protein levels in DRG neurons were dramatically increased, accompanied with decreased PWMT and TPWL. Lentivirus‐mediated RNA interference decreased ERK gene expression in DRG neurons and normalized the PWMT and TPWL in CCD rats, but not in rats infected with lentivirus expressing negative control shRNA. Further, calcium responses of DRG neurons to the hypotonic solution and 4α‐phorbol 12,13‐didecanoate were enhanced in CCD rats, which were suppressed by lentivirus‐mediated ERK gene silence. Finally, the levels of transient receptor potential vanilloid 4 gene expressions in DRG neurons and L4 to L5 spinal cord isolated from CCD rats were dramatically upregulated, which were reversed by lentivirus‐mediated ERK gene knockdown. Conclusion Lentivirus‐mediated RNA interference (RNAi) silencing targeting ERK might reverse CCD‐induced neuropathic pain in rats through transient receptor potential vanilloid 4.

Obesity increases neuropathic pain via the AMPK-ERK-NOX4 pathway in rats

“…There are not enough studies on obesity-induced neuropathic pain, and therefore the precise mechanism is still not fully elucidated, and the effective clinical treatments are lacking. Our previous studies have confirmed that MAPK/ERK contributes to CCD-induced neuropathic pain, and inhibition of ERK phosphorylation with U0126 and interference of ERK expression with shRNA can alleviate pain [ 8 , 9 ]. Combined with previous studies showing that in smooth muscle cells and adipocytes, treatment with lipids can lead to ERK activation [ 10 , 11 ], we hypothesized that the elevated neuropathic pain in obese rats might be related to the abnormal activation of ERK in nervous tissue.…”

“…The test method is based on our previous reports [ 8 ]. The rats were fully accustomed to the test environment before the test.…”

“…More importantly, the MAPK/ERK pathway has a pivotal role in many neurodegenerative diseases [ 6 ], and targeting the ERK signaling pathway is an innovative therapeutic strategy for treating neurodegeneration [ 7 ]. Our previous study has reported that the ERK pathway is an important mechanism in neuropathic pain in rats with a chronic compression of dorsal root ganglia (CCD) model, and targeting ERK with U0126 or lentivirus expressing ERK shRNA significantly alleviates chronic compression of dorsal root ganglia (DRG)-induced neuropathic pain [ 8 , 9 ].…”

Identification of Differentially Expressed Genes and Key Pathways in the Dorsal Root Ganglion After Chronic Compression

“…CCD was induced as described earlier (Jia et al, 2018). In summary, after the rats were anesthetized, the paraspinal muscles were separated to unilaterally expose the transverse processes and intervertebral foramina of L4 and L5.…”

“…Thermal paw withdrawal latency (TPWL) was measured using the BME-410C thermal analgesia tester (CAMS) as described previously (Jia et al, 2018). The light source was a 12 V/10 W halogen lamp and the thermal intensity was set at 60 • C. Radiant light was focused on the center of the hind paw at about 1 cm, and TPWL was recorded when the rat lifted or licked the hind paw.…”