Targeting iNOS Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage via Promoting Ferroptosis of M1 Microglia and Reducing Neuroinflammation

Abstract

Numerous studies have demonstrated the role of neuroinflammation in mediating acute pathophysiological events of early brain injury after subarachnoid hemorrhage (SAH). However, it is not clear how to target this inflammatory cascade after SAH. M1 activation of microglia is an important pathological mechanism driving neuroinflammation in SAH, which is considered aggressive, leading to cytotoxicity and robust inflammation related to the release of proinflammatory cytokines and chemokines after SAH. Thus, reducing the number of M1 microglia represents a potential target for therapies to improve outcomes after SAH. Previous studies have found that inducible nitric oxide synthase (iNOS/NO•) plays an essential role in promoting the survival of M1 microglia by blocking ferroptosis. Ferroptosis is a new type of iron-dependent cellular procedural death associated with pathological cell death related to mammalian degenerative diseases, cerebral hemorrhage, and traumatic brain injury. Here, we investigated the effect of L-NIL, an inhibitor of iNOS, on M1 microglia, neuroinflammation, neuronal cell death, brain edema, and neurological function in an experimental SAH model in vivo and in vitro. We found that L-NIL reduced the number of M1 microglia and alleviated neuroinflammation following SAH. Notably, treatment with L-NIL relieves brain edema and neuronal injury and improves outcomes of neurological function after SAH in rats. Mechanistically, we found that L-NIL inhibited the expression of iNOS and promoted ferroptosis of M1 microglia by increasing the expression of ferroptosis-related proteins and lipid peroxidation in an in vitro model of SAH, which was reversed by a ferroptosis inhibitor, liproxstatin-1. In addition, inhibiting iNOS had no significant effect on ferroptosis of neurons after oxyhemoglobin stimulation in vitro. Thus, our research demonstrated that inhibition of iNOS might represent a potential therapeutic strategy to improve outcomes after SAH by promoting ferroptosis of M1 microglia and reducing neuroinflammation.

Keywords: Early brain injury; Ferroptosis; Neuroinflammation; Subarachnoid hemorrhage; iNOS.