Involvement of spinal NADPH oxidase 4 and endoplasmic reticulum stress in morphine-tolerant rats

Morphine tolerance (MT) is currently a challenging issue related to intractable pain treatment. Studies have shown that reactive oxygen species (ROSs) derived from NADPH oxidase (NOX) and produced in response to endoplasmic reticulum (ER) stress participate in MT development. However, which NOX subtype initiates ER stress during MT development is unclear. NOX4 is mainly expressed on intracellular membranes, such as the ER and mitochondrial membranes, and its sole function is to produce ROS. Whether NOX4 is activated during MT development and the mechanisms underlying the association between NOX4 and ER stress during this process still need to be confirmed. In our study, we used the classic morphine-tolerant rat model and evaluated the analgesic effect of intrathecally injected morphine through a hot water tail-flick assay. Our research demonstrated for the first time that chronic morphine administration upregulates NOX4 expression in the spinal cord by activating three ER stress sensors, protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), subsequently leading to the activation of microtubule-associated protein 1 light chain 3 b (LC3B) and P62 (a well-known autophagy marker) in GABAergic neurons. Our results may suggest that regulating NOX4 and the key mechanism underlying ER stress or autophagy may be a promising strategy to treat and prevent MT development.