[Effect and mechanism of NLRP3-mediated microglial pyroptosis on AT1R antagonists against neuropathic pain in rats].

Objective To investigate the role and mechanism of NLR family pyrin domain containing protein 3 (NLRP3) in the therapeutic effects of angiotensin type 1 receptor (AT1R) antagonists on neuropathic pain (NP) in rats. Methods The NP rat model was established through chronic compression injury of sciatic nerve. Successfully modeled rats were randomly assigned to the following groups: Model group, AT1R antagonist losartan potassium (LP) group, NLRP3 agonist nigericin (Nig) group and the combination group of LP and Nig. A Sham group was also included for comparison, with 8 rats in each group. The rats were administered intraperitoneal injections of either 100 mg/kg LP or 1 mg/kg Nig once daily for five consecutive days. Mechanical withdraw threshold (MWT) and thermal withdrawal latency (TWL) were assessed one day before surgery and on postoperative days 7 and 12. Histopathological alterations in the spinal cord tissue were evaluated using hematoxylin and eosin (HE) staining. The sciatic nerve conduction velocity (NCV) was measured using neuroelectrophysiological techniques. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the levels of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-18, in the spinal cord tissue of rats. The kit was used to detect the level of lactate dehydrogenase (LDH) in the spinal cord tissue of rats. Immunofluorescence staining was conducted to assess the co-localization of ionized calcium-binding adapter molecule 1 (Iba-1) and Gasdermin D (GSDMD) in the spinal cord tissue of rats. Western blot analysis was used to determine the expression levels of NLRP3, GSDMD, caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) proteins in the spinal cord tissues of rats. Results Compared with the Sham group, the MWT and TWL in the Model group rats were significantly reduced at both 7 and 12 days post-surgery. In addition, the NCV was decreased, while the levels of IL-1β, IL-18, TNF-α and LDH were significantly elevated. Simultaneously, the number of co-localized Iba-1 and GSDMD positive cells in the spinal cord tissue increased, along with upregulated expression levels of NLRP3, GSDMD, caspase-1 and ASC proteins. Compared with the Model group, the MWT and TWL in the LP-treated rats were significantly improved at 12 days postsurgery, accompanied by an increase in NCV and a significant reduction in the levels of IL-1β, IL-18, TNF-α and LDH. Concurrently, the number of Iba-1 and GSDMD co-localization cells decreased, and the expression levels of NLRP3, GSDMD, caspase-1 and ASC proteins were significantly downregulated. Notably, Nig intervention markedly exacerbated spinal cord inflammation in NP rats and attenuated the therapeutic effects of LP on neuropathic pain. Conclusion LP may alleviate spinal cord inflammatory injury and pain in NP rats by inhibiting NLRP3-mediated microglial pyroptosis.