Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord

Background: The use of morphine in clinical medicine is severely constrained by tolerance. Therefore, it is essential to examine pharmacological therapies that suppress the development of morphine tolerance. Amiloride suppressed the expression of inflammatory cytokines by inhibiting microglial activation. Microglia play a crucial role in the establishment of morphine tolerance. Thus, we anticipated that amiloride might suppress the development of morphine tolerance. During this investigation, we assessed the impact of amiloride on mouse morphine tolerance.Methods: Mice received morphine (10 mg/kg, s.c.) twice daily with intrathecally injected amiloride (0.3 mu g/5 mu l, 1 mu g/5 mu l, and 3 mu g/5 mu l) for nine continuous days. To assess morphine tolerance, mice underwent the tail-flick and hot plate tests. BV-2 cells were used to investigate the mechanism of amiloride. By using Western blotting, real-time PCR, and immunofluorescence labeling methods, the levels of acid-sensing ion channels (ASICs), nu-clear factor kappa B (NF-kB) p65, p38 mitogen-activated protein kinase (MAPK) proteins, and neuroinflammation-related cytokines were determined.Results: The levels of ASIC3 in the spinal cord were considerably increased after long-term morphine administration. Amiloride was found to delay the development of tolerance to chronic morphine assessed via tail-flick and hot plate tests. Amiloride reduced microglial activation and downregulated the cytokines IL-113 and TNF-a by inhibiting ASIC3 in response to morphine. Furthermore, amiloride reduced p38 MAPK phosphorylation and inhibited NF-xB expression.Conclusions: Amiloride effectively reduces chronic morphine tolerance by suppressing microglial activation caused by morphine by inhibiting ASIC3.