Unraveling morphine tolerance: CCL2 induces spinal cord apoptosis via inhibition of Nrf2 signaling pathway and PGC-1α-mediated mitochondrial biogenesis

BACKGROUND: Morphine effectively relieves severe pain but leads to analgesic tolerance with long-term use.The molecular mechanisms underlying morphine tolerance remain incompletely understood. Existing literature suggests that chemokine CCL2, present in the spinal cord, plays a role in central nervous system inflammation, including neuropathic pain. Nevertheless, the precise mechanism through which CCL2 mediates morphine tolerance has yet to be elucidated. Consequently, this study aims to investigate the molecular pathways by which CCL2 contributes to the development of morphine analgesic tolerance. METHODS: Rats were administered intrathecal morphine (10 μg/5 μl) twice a day for seven consecutive days to induce a model of morphine nociceptive tolerance. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression levels of CCL2 and its related mechanism molecules. Immunofluorescence was used to detect the localization of CCL2 in the spinal cord. Intrathecal injections of inhibitors or agonists to artificially regulate the expression of relevant molecules. The thermal tail-flick experiment was performed to evaluate morphine tolerance in rats. RESULTS: Morphine-induced CCL2 expression was significantly increased in spinal cord, while conversely, the expressions of Nrf2 and PGC-1a were downregulated. Immunofluorescence showed that the enhanced immune response of CCL2 mainly co-localized with neurons. In vivo, we confirmed that intrathecally injection of CCL2 inhibitor Bindarit could effectively alleviate the occurrence of apoptosis and alleviate morphine tolerance. Similarly, pretreatment with Nrf2 signaling pathway agonist Oltipraz and PGC-1α agonist ZLN005 also achieved similar results, respectively. ROS Fluorescence Assay Kit indicated that increasing the expression of PGC-1α could alleviate the occurrence of apoptosis by reducing the level of ROS. CONCLUSION: Our data emphasize that chemokine CCL2 inhibited the Nrf2 signaling pathway and PGC-1α-mediated mitochondrial biogenesis, alleviating the occurrence of apoptosis in spinal cord, thereby participating in morphine tolerance. This may provide new targets for the treatment of morphine tolerance.