Autophagy inhibition facilitates wound closure partially dependent on the YAP/IL-33 signaling in a mouse model of skin wound healing.

Autophagy is a self-phagocytic and highly evolutionarily conserved intracellular lysosomal catabolic system, which plays a vital role in a variety of trauma models, including skin wound healing (SWH). However, the roles and potential mechanisms of autophagy in SWH are still controversial. We firstly investigated the role of autophagy in SWH-induced wound closure rate, inflammatory response, and histopathology, utilizing an inhibitor of autophagy 3-methyladenine (3-MA) and its agonist rapamycin (RAP). As expected, we found 3-MA treatment remarkably increased the wound closure rate, combated inflammation response, and mitigated histopathological changes, while RAP delivery aggravated SWH-induced pathological damage. To further exploit the underlying mechanism of autophagy regulating inflammation, the specific inhibitors of yes-associated protein (YAP), Verteporfin, and Anti-IL-33 were applied. Herein, treating with 3-MA markedly suppressed the expression of tumor necrosis factor-α (TNF-α), IL-1ß, and IL-6, promoted that of IL-10, IL-33, and ST2, while RAP administration reverted SWH-induced the up-regulation of these inflammatory cytokines mentioned above. Importantly, Verteporfin administration not only down-regulated the expression levels of YAP, TNF-α, and IL-6 but also up-regulated that of IL-33 and IL-10. Unexpectedly, 3-MA or RAP retreatment did not have any impact on the changes in IL-33 among these inflammatory indicators. Furthermore, elevated expression of IL-33 promoted wound closure and alleviated the pathological damage, whereas, its antagonist Anti-IL-33 treatment overtly reversed the above-mentioned effects of IL-33. Moreover, 3-MA in combination with anti-IL-33 treatment reversed the role of 3-MA alone in mitigated pathological changes, but they failed to revert the effect of anti-IL-33 alone on worsening pathological damage. In sum, emerging data support the novel contribution of the YAP/IL-33 pathway in autophagy inhibition against SWH-induced pathological damage, and highlight that the autophagy/YAP/IL-33 signal axis is expected to become a new therapeutic target for SWH.

Deletion of ferritin H in neurons counteracts the protective effect of melatonin against traumatic brain injury-induced ferroptosis.

Accumulating evidence demonstrates that ferroptosis may be important in the pathophysiological process of traumatic brain injury (TBI). As a major hormone of the pineal gland, melatonin exerts many beneficial effects on TBI, but there is no information regarding the effects of melatonin on ferroptosis after TBI. As expected, TBI resulted in the time-course changes of ferroptosis-related molecules expression and iron accumulation in the ipsilateral cortex. Importantly, we found that treating with melatonin potently rescued TBI induced the changes mentioned above and improved functional deficits versus vehicle. Similar results were obtained with a ferroptosis inhibitor, liproxstatin-1. Moreover, the protective effect of melatonin is likely dependent on melatonin receptor 1B (MT2). Although ferritin plays a vital role in iron metabolism by storing excess cellular iron, its precise function in the brain, and whether it involves melatonin's neuroprotection remain unexplored. Considering ferritin H (Fth) is expressed predominantly in the neurons and global loss of Fth in mice induces early embryonic lethality, we then generated neuron-specific Fth conditional knockout (Fth-KO) mice, which are viable and fertile but have altered iron metabolism. In addition, Fth-KO mice were more susceptible to ferroptosis after TBI, and the neuroprotection by melatonin was largely abolished in Fth-KO mice. In vitro siFth experiments further confirmed the results mentioned above. Taken together, these data indicate that melatonin produces cerebroprotection, at least partly by inhibiting neuronal Fth-mediated ferroptosis following TBI, supporting the notion that melatonin is an excellent ferroptosis inhibitor and its anti-ferroptosis provides a potential therapeutic target for treating TBI.

Mdivi-1 alleviates brain damage and synaptic dysfunction after intracerebral hemorrhage in mice.

As a selective inhibitor of mitochondrial fission protein dynamin-related protein-1 (Drp1), mitochondrial division inhibitor 1 (mdivi-1) can cross the blood-brain barrier (BBB) and exert neuroprotection. However, it remains unclear whether mdivi-1 can attenuate intracerebral hemorrhage (ICH)-induced secondary brain injury. This study was undertaken to characterize the roles of mdivi-1 in short-term and long-term behavioral outcomes, along with synaptic plasticity changes in mice after ICH. The results indicated mdivi-1 reversed Drp1 translocation and the morphologic changes of mitochondria, as well as ameliorated short-term neurobehavioral deficits, the BBB disruption and brain edema remarkably. In addition, mdivi-1 could rescue ICH-induced motor and memory dysfunctions. Mdivi-1 could also prevent ICH-induced reductions in synaptic proteins (synapsin I, PSD95) and phosphorylated cAMP-response element binding (p-CREB). In vitro, mdivi-1 inhibited hemin-induced hippocampal neuron death and improved neurite outgrowth. In conclusion, we found that mdivi-1 can alleviate short-term and long-term neurological deficits, synaptic dysfunction. These findings demonstrate that mdivi-1 may be beneficial in the treatment of secondary brain injury, synaptic dysfunction and neurological outcomes caused by ICH.

TrkB agonist N-acetyl serotonin promotes functional recovery after traumatic brain injury by suppressing ferroptosis via the PI3K/Akt/Nrf2/Ferritin H pathway.

Ferroptosis is a form of regulated cell death that is mainly triggered by iron-dependent lipid peroxidation. A growing body of evidence suggests that ferroptosis is involved in the pathophysiology of traumatic brain injury (TBI), and tropomyosin-related kinase B (TrkB) deficiency would mediate TBI pathologies. As an agonist of TrkB and an immediate precursor of melatonin, N-acetyl serotonin (NAS) exerts several beneficial effects on TBI, but there is no information regarding the role of NAS in ferroptosis after TBI. Here, we examined the effect of NAS treatment on TBI-induced functional outcomes and ferroptosis. Remarkably, the administration of NAS alleviated TBI-induced neurobehavioral deficits, lesion volume, and neurodegeneration. NAS also rescued TBI-induced mitochondrial shrinkage, the changes in ferroptosis-related molecule expression, and iron accumulation in the ipsilateral cortex. Similar results were obtained with a well-established ferroptosis inhibitor, liproxstatin-1. Furthermore, NAS activated the TrkB/PI3K/Akt/Nrf2 pathway in the mouse model of TBI, while inhibition of PI3K and Nrf2 weakened the protection of NAS against ferroptosis both in vitro and in vivo, suggesting that a possible pathway linking NAS to the action of anti-ferroptosis was TrkB/PI3K/Akt/Nrf2. Given that ferritin H (Fth) is a known transcription target of Nrf2, we then investigated the effects of NAS on neuron-specific Fth knockout (Fth-KO) mice. Strikingly, Fth deletion almost abolished the protective effects of NAS against TBI-induced ferroptosis and synaptic damage, although Fth deletion-induced susceptibility toward ferroptosis after TBI was reversed by an iron chelator, deferoxamine. Taken together, these data indicate that the TrkB agonist NAS treatment appears to improve brain function after TBI by suppressing ferroptosis, at least in part, through activation of the PI3K/Akt/Nrf2/Fth pathway, providing evidence that NAS is likely to be a promising anti-ferroptosis agent for further treatment for TBI.

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

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.

Ferristatin II, an Iron Uptake Inhibitor, Exerts Neuroprotection against Traumatic Brain Injury via Suppressing Ferroptosis.

As a specific ferroptosis marker, transferrin receptor 1 (TfR1) expression is increased following traumatic brain injury (TBI), but the precise role of TfR1 in TBI-induced ferroptosis and neurodegeneration remains to be determined. To further identify more potent ferroptosis inhibitors and effective targets for treating TBI, our study aims at investigating the effects of TfR1 on ferroptosis in a mouse TBI model using ferristatin II (an iron uptake and TfR1 inhibitor). The effect of ferristatin II was first verified in the HT-22 cell line in vitro and showed antiferroptotic action when exposed to ferric citrate (FAC), which is in parallel with the results obtained from the positive controls, including deferoxamine (DFO) and liproxstatin-1 (Lip-1). In vivo, ferristatin II administration reduced the expression of TfR1 at 12 h after TBI, and immunofluorescence experiments further confirmed that this decreased TfR1-positive cells were neurons. Importantly, ferristatin II suppressed TBI-induced iron homeostatic imbalance by decreasing the content of Fe (III) and iron-positive deposits and reversed the expression of iron homeostasis-related proteins. Moreover, ferristatin II attenuated TBI-induced lipid peroxidation by reversing the expression of lipid peroxidative genes and proteins, as well as the increase in malondialdehyde (MDA) level following TBI. Finally, ferristatin II alleviated TBI-induced neuronal injury and neurodegeneration, as detected by staining with Nissl and Fluoro-Jade B, thereby exerting a neuroprotective effect. In summary, these data indicated that ferristatin II might be a potential strategy to restrain ferroptosis and develop novel therapeutic agents against TBI.

Targeting NRF2 to suppress ferroptosis in brain injury.

Brain injury is accompanied by serious iron metabolism disorder and oxidative stress. As a novel form of regulated cell death (RCD) depending on lipid peroxidation caused by iron overload, ferroptosis (FPT) further aggravates brain injury, which is different from apoptosis, autophagy and other traditional cell death in terms of biochemistry, morphology and genetics. Noteworthy, transcriptional regulator NRF2 plays a key role in the cell antioxidant system, and many genes related to FPT are under the control of NRF2, including genes for iron regulation, thiol-dependent antioxidant system, enzymatic detoxification of RCS and carbonyls, NADPH regeneration and ROS sources from mitochondria or extra-mitochondria, which place NRF2 in the key position of regulating the ferroptotic death. Importantly, NRF2 can reduce iron load and resist FPT. In the future, it is expected to open up a new way to treat brain injury by targeting NRF2 to alleviate FPT in brain.

Ruxolitinib exerts neuroprotection via repressing ferroptosis in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Various forms of cells death are involved in the pathological process of TBI, without exception to ferroptosis, which is mainly triggered by iron-dependent lipid peroxidation. Although there have been studies on ferroptosis and TBI, the effect of ruxolitinib (Ruxo), one type of FDA approved drugs for treating myelofibrosis, on the process of ferroptosis post-TBI is remained non-elucidated. Therefore, using a controlled cortical impact device to establish the mouse TBI model, we examined the effect of Ruxo on TBI-induced ferroptosis, in which the inhibitor of ferroptosis, Ferrostatin-1 (Fer-1) was used as a positive control. Moreover, we also respectively explored the effects of these two interventions on neurological deficits caused by TBI. We firstly examined the expression patterns of ferroptosis-related markers at protein level at different time points after TBI. And based on the expression changes of these markers, we chose 12 h post-TBI to prove the effect of Ruxo on ferroptosis. Importantly, we found the intensely inhibitory effect of Ruxo on ferroptosis, which is in parallel with the results obtained after Fer-1-treatment. In addition, these two treatments both alleviated the content of brain water and degree of neurodegeneration in the acute phase of TBI. Finally, we further confirmed the neuroprotective effect of Ruxo or Fer-1 via the wire-grip test, Morris water maze and open field test, respectively. Thereafter, the lesion volume and iron deposition were also measured to certificate their effects on the long-term outcomes of TBI. Our results ultimately demonstrate that inhibiting ferroptosis exerts neuroprotection, and this is another neuroprotective mechanism of Ruxo on TBI.

Ferroptosis-relevant mechanisms and biomarkers for therapeutic interventions in traumatic brain injury.

Traumatic brain injury (TBI) is one of the most significant health care problems worldwide, causing disability and death especially among young individuals. Although a large range of agents and therapies have been proved beneficial to lesions post-TBI to some extent, effective treatments have not been translated to the clinic. As a newly discovered form of iron-dependent regulated cell death, ferroptosis has been implicated in TBI. In this review, we update the current state of knowledge related to second injuries post-TBI, including ferroptosis, oxidative stress, mitochondrial dysfunction, neuroinflammation and so on, which often lead to chronic symptoms and long-term disability. This review systematically summarizes the latest progress in the pathophysiological mechanisms of TBI, with a focus on providing references for proposing new multi-molecular targets for comprehensive therapeutic strategies based on ferroptosis-relevant mechanisms. In addition, biomarkers are essential diagnostic and prognostic tools in TBI. Several biomarkers associated with the outcome of TBI have been listed in this article, such as Pde10a, MDA, UCH-L1, S100A9, S100B, ALDOC, ACSL4, MBP and F2-Isoprostane. Therefore, the understating of ferroptosis-relevant mechanisms and biomarkers may contribute to development of promising therapies for TBI clinical trials.

A TrkB receptor agonist N-acetyl serotonin provides cerebral protection after traumatic brain injury by mitigating apoptotic activation and autophagic dysfunction.

Tropomyosin-related kinase B (TrkB) has emerged as a key mediator in the pathophysiology of traumatic brain injury (TBI). However, it is not known whether TrkB's agonist N-acetyl serotonin (NAS) involves in neuronal damage and brain dysfunction caused by TBI that is known as one of the most important causes of disability and death worldwide. Here, we investigated the effects of NAS on brain edema, blood-brain barrier (BBB), apoptosis activation and autophagy dysfunction after experimental TBI. A mouse TBI model was applied, NAS and ANA-12 (an antagonist of TrkB) were administered. Here, we first found that NAS administration ameliorated TBI-induced brain edema, blood-brain barrier (BBB) disruption, increase of matrix metalloproteinase-9 (MMP-9) expression and decrease of claudin-5 expression. NAS treatment decreased TBI-induced cell death and apoptosis activation (detected by propidium iodide labeling, TUNEL staining, blots for Bcl-2, Bax and caspase-3). In addition, NAS treatment decreased the expression of Beclin-1 and LC3, along with ratio of Beclin-1/Bcl-2, but increased p62 expression following TBI. NAS also enhanced the activation of the TrkB/Akt pathway following TBI. Whereas, the above protective effect of NAS following TBI was blocked by ANA-12 addition. Thus, we conclude that NAS-initiating the TrkB/Akt signaling cascade provides neuroprotection after experimental TBI in mice, which at least in part through inhibiting apoptosis activation and autophagic dysfunction.