Knockdown of PAR2 alleviates cancer-induced bone pain by inhibiting the activation of astrocytes and the ERK pathway

Abstract

Objective Cancer-induced bone pain (CIBP) is a kind of pain with complex pathophysiology. Proteinase-activated receptor 2 (PAR-2) is involved in CIBP. This study explored the effects of PAR-2 on CIBP rats. Methods CIBP rat model was established by injecting Walker 256 rat breast cancer cells into the left tibia of female Sprague-Dawley rats and verified by tibial morphology observation, HE staining, and mechanical hyperalgesia assay. CIBP rats were injected with PAR-2 inhibitor, ERK activator, and CREB inhibitor through the spinal cord sheath on the 13th day after operation. CIBP behaviors were measured by mechanical hyperalgesia assay. On the 14th day after operation, L4-5 spinal cord tissues were obtained. PAR-2 expression, co-expression of PAR-2 and astrocyte marker GFAP, GFAP mRNA and protein levels and the ERK pathway-related protein levels were detected by Western blot, immunofluorescence double staining, RT-qPCR, and Western blot. Results CIBP rats had obvious mechanical hyperalgesia and thermal hyperalgesia from the 7th day after modeling; mechanical hyperalgesia threshold and thermal threshold were decreased; PAR-2 was increased in spinal cord tissues and was co-expressed with GFAP. PAR-2 silencing alleviated rat CIBP by inhibiting astrocyte activation. p-ERK/t-ERK and p-CREB/t-CREB levels in CIBP spinal cord were elevated, the ERK/CREB pathway was activated, while the ERK/CREB pathway was inhibited by PAR-2 silencing. The alleviating effect of PAR-2 inhibitor on hyperalgesia behaviors in CIBP rats were weakened by ERK activator, while were partially restored by CREB inhibitor. Conclusions PAR-2 knockdown inhibited the ERK/CREB pathway activation and astrocyte activation, thus alleviating CIBP in rats.

Mechanism of ERK/CREB pathway in pain and analgesia

“…Consistent with this, the silencing of PAR-2 can reduce the pain and inhibit the activation of astrocytes and activated ERK/CREB pathway in rats with cancerous bone pain, revealing that the silencing of PAR-2 inhibits the activation of ERK/CREB pathway and astrocytes, thus reduces the pain of rats (Tang et al, 2022). This further demonstrates that blocking the ERK/CREB pathway can have analgesic effects.…”

STING Contributes to Cancer-Induced Bone Pain by Promoting M1 Polarization of Microglia in the Medial Prefrontal Cortex

“…On day 14 after the model was established, the ipsilateral tibia of CIBP rats was fixed in 4% paraformaldehyde for 48 h, followed by decalcification in 10% EDTA for 3–4 weeks, and 6-μm sections were prepared on a rotating microtome. Paraffin-embedded sections were deparaffinized in xylene, rehydrated, and stained with hematoxylin-eosin (H&E; Sigma, St. Louis, MO, USA) according to the manufacturer’s protocol [ 40 ].…”