111: TLR4-IRE1?/XBP1 Signaling and NLRP3 Inflammasome Activation in Morphine Withdrawal

Topic: Basic Science
Subtopic: Non-specific to any diagnosis (including studies in healthy animals), Neuroimmunology, Pharmacological interventions, Behavioral studies

Background and Aims: Morphine is one of the most commonly used analgesics in clinical practice, and abrupt cessation or substantial dose reduction after prolonged use can precipitate withdrawal syndrome. Previous studies have shown that chronic morphine exposure activates the unfolded protein response (UPR); however, whether IRE1α/XBP1 signaling is involved in morphine withdrawal and how it is associated with NLRP3 inflammasome activation remain unclear. This study aimed to investigate the role of TLR4-IRE1α/XBP1 signaling in morphine withdrawal and to explore the potential involvement of the NLRP3 inflammasome in this process.

Methods: Morphine dependence/withdrawal models were established in BV2 microglial cells, C57BL/6 mice, and SD rats. q-PCR and Western blotting were used to examine the expression of UPR-related molecules (GRP78, p-IRE1α/IRE1α, XBP1s, p-JNK/JNK, ATF4, and ATF6), neuroinflammation-related genes, and NLRP3 inflammasome-related molecules (NLRP3, pro-Caspase-1, and IL-1β) in BV2 cells and the spinal dorsal horn of mice. Pharmacological interventions were performed using the TLR4 inhibitor LPS-RS, the IRE1α inhibitors KIRA6, STF-083010, and 4μ8C, and the NLRP3 inhibitors β-HB and MCC950. Behavioral tests were conducted to evaluate their effects on morphine withdrawal responses.

Results: Morphine withdrawal significantly increased GRP78, p-IRE1α/IRE1α, XBP1s, p-JNK/JNK, ATF4, and ATF6 expression in BV2 cells and the mouse spinal dorsal horn, indicating activation of the unfolded protein response, particularly the IRE1α/XBP1 branch. Pharmacological inhibition of IRE1α suppressed these changes and partially alleviated withdrawal behaviors in mice. In BV2 cells, the TLR4 inhibitor LPS-RS reduced Grp78 and Xbp1s expression, suggesting that TLR4 may act upstream of IRE1α/XBP1 signaling. Morphine withdrawal also markedly upregulated multiple neuroinflammation-related genes in BV2 cells and the spinal dorsal horn, and these effects were partially reversed by IRE1α inhibitors or LPS-RS. Although mRNA levels of inflammasome-related factors were not significantly changed, protein levels of NLRP3, pro-Caspase-1, and IL-1β were significantly increased. Pharmacological inhibition or genetic deletion of NLRP3 attenuated morphine withdrawal behaviors.

Conclusions: Morphine withdrawal activates IRE1α/XBP1 signaling in the spinal dorsal horn. TLR4 may participate in its upstream regulation, and NLRP3 inflammasome activation may be involved in this process.

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Presenting Author: Sijia Liu
Submitting Author: Sijia Liu

Senior Author: Tong Liu

IASP 2026 Global Year theme of Neuropathic Pain: No