Luteolin Exerts Neuroprotection Via Modulation Of The P62/Keap1/Nrf2 Pathway In Intracerebral Hemorrhage

Objective:Intracerebral hemorrhage(ICH)is a significant public health problem that has aroused worldwide concern due to its high mortality and morbidity rates.In addition to primary brain injury,ICH-induced secondary brain injury(SBI)often leads to severe neurological deficits or even death.As a member of the flavonoid family,Luteolin has been shown to exhibit multiple pharmacological effects,such as antioxidative and autophagic-regulatory effects in many disease models.However,the effects of Luteolin on ICH remain poorly understood.In previous studies,the sequestosome 1(p62)/kelch-like ECH-associated protein 1(Keapl)/Nuclear factor erythroid 2-related factor 2(Nrf2)pathway has been proved to be an important pathway for antioxidant stress and autophagy.Therefore,in this study,we investigated the neuroprotective effects of Luteolin in ICH-induced SBI,including potential underlying mechanisms related to regulation of antioxidative processes and autophagy.Methods:Adult male Sprague Dawley(SD)rats were divided into the following six groups randomly and equally:sham group,ICH group,ICH+vehicle group,and three ICH+Luteolin treatment groups(i.e.,5,10,and 20 mg/kg).By injecting 100 μl of non-heparinized autologous arterial blood into the right basal ganglia,a rat ICH model was induced.Neurobehavioral tests were examined to assess behavioral impairments.Brain water content was detected by the dry and wet method.Morris water maze was performed to assess cognitive function in rats.After induction of ICH for 24 h,brain samples from the right basal ganglia of each rat were collected and homogenized for Western blotting,and coimmunoprecipitation(CO-IP)analysis,to investigate the expression of the proteins related to the p62/keapl/Nrf2 pathway and the level of Nrf2 ubiquitination.The results were statistically analyzed.Meanwhile,primary rat cortical neurons were isolated from 1 7-day-old rat embryos and were cultured.An in vitro of ICH model was established by using OxyHb which was applied to emulate ICH pathophysiology in vitro.Primary neurons were treated with different concentrations of Luteolin(5,10,and 20 μM)and OxyHb(10 μM)for 24 h.To investigate the expressions of the proteins related to the p62/keapl/Nrf2 pathway,western blotting was used,and mitochondrial superoxide(MitoSOX)staining was applied to measure the level of mitochondrial superoxide.And the tetrachloro-tetraethylbenzimidazol carbocyanine iodide(JC-1)staining was applied to detect mitochondrial damage.Results:1.Luteolin treatment effectively alleviated brain edema and ameliorated neurobehavioral dysfunction and cognitive impairments in vivo.2.In vivo,we found that Luteolin promoted the activation of the p62/keapl/Nrf2 pathway by enhancing autophagy and increasing the translocation of Nrf2 to the nucleus.Meanwhile,Luteolin inhibited the ubiquitination of Nrf2 and increased the expression levels of downstream antioxidant proteins,such as heme oxygenase-1(HO-1)and NADPH:quinine oxidoreductase 1(NQO1).3.Luteolin inhibited the production of neuronal mitochondrial superoxides and alleviated neuronal mitochondrial injury in vitro.And the activation of p62/keapl/Nrf2 pathway was reversed by the autophagy inhibitor,chloroquine(CQ).Conclusions:Luteolin enhances autophagy and anti-oxidative processes in both in vivo and in vitro models of ICH,and that activation of the p62/keap1/Nrf2 pathway,is involved in such Luteolin-induced neuroprotection.Hence,Luteolin may represent a promising candidate for the treatment of ICH-induced SBI.