Diabetic Retinopathy (DR) is the most severe ocular complication of diabetes mellitus (DM) and the leading cause of blindness in working-age adults. By 2045, global DR cases are expected to rise from 103.1 million in 2020 to 160.5 million, with sight-threatening cases reaching 44.82 million [1]. Effective early intervention strategies to reduce the risk of blindness associated with DR are lacking [2]. The pathogenesis of DR is complex and has not yet been fully elucidated. Traditionally, DR has been considered a microvascular disease. Recent research has indicated that neurovascular unit (NVU) dysfunction, including glial activation and neurodegeneration, occurs before clinical signs and contributes to the progression of retinal microvascular damage [3]. Retinal microglia (RMG), a crucial component of the NVU, play a central role in mediating inflammation and neurovascular damage in DR [4]. In a high-glucose environment, RMG may undergo polarization towards a pro-inflammatory M1 phenotype, aggravating inflammation and tissue damage, whereas the M2 phenotype is associated with tissue repair and homeostasis [2,5]. Therefore, the regulation of RMG activation is an important area of current DR research.

Hyperglycemia induces the expression of Thioredoxin-interacting protein (TXNIP) in DR, which activates the NOD-like receptor protein 3 (NLRP3) inflammasome [[6], [7], [8]]. Our previous research suggested that hyperglycemia increases NLRP3 expression in the RMG, leading to Caspase-1 activation and increased secretion of pro-inflammatory factors [9]. Treatment with NLRP3 or Caspase-1 inhibitors can suppress NLRP3 inflammasome signaling in RMG. This may alleviate oxidative stress and retinal NVU damage. Concurrently, Nitric oxide (NO), a key regulator of neurovascular coupling, exhibits decreased levels in advanced diabetes, while initially increasing at earlier stages [10,11]. The role of NO in DR remains unclear. It acts as a vasodilator, regulating retinal blood flow and inhibiting NLRP3 inflammasome activation [12]. NO suppresses excessive inflammasome activation through S-nitrosylation of NLRP3 [13]. Recent studies have demonstrated that NO donor-loaded nanoparticles can suppress RMG-associated inflammation [14]. Nitric oxide synthase (NOS) inhibitors exacerbate diabetes-induced neural damage [15].

In this study, we investigated whether NO could regulate the TXNIP/NLRP3 inflammasome pathway in retinal cells under hyperglycemic conditions, and assessed the protective role of NO against NVU dysfunction in a rat model of early DR. Understanding this mechanism may offer new therapeutic targets for early intervention in DR.