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

https://doi.org/10.1016/j.expneurol.2021.113762Get rights and content

Highlights

  • Ferroptosis is involved in the pathological process of TBI.

  • Ruxo reduces ferroptosis induced by TBI.

  • Inhibition of ferroptosis exerts neuroprotection after TBI.

Abstract

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.

Introduction

Traumatic brain injury(TBI) is a major cause of death and disability worldwide, constituting a considerably portion of the global injury burden (Collaborators, 2019). TBI survivors suffer from chronic physical, cognitive, and psychological disorders, which will severely affect their quality of life (Stocchetti and Zanier, 2016). TBI often leads to the tissue displacement and destruction and further causes secondary brain damage such as aggravation of brain edema, accumulation of toxic by-products, excitatory intoxication, gliosis, neuroinflammation and oxidative stress (Maas et al., 2008; Prins et al., 2013). These pathophysiological changes ultimately result in the massive cell death, including necrosis and programmed cell death (PCD) such as apoptosis, autophagy, and ferroptosis (Zhang et al., 2005).

As a recently described manner of PCD, ferroptosis is mainly triggered by lipid peroxidation arising from an iron-dependent reactive oxygen species (ROS) accretion (Dixon et al., 2012). The biochemical characteristics of ferroptosis are abnormal iron metabolism, iron-dependent accumulation of active oxygen, inactivation or decreased expression of GPX4, and accumulation of lipid peroxides (Bersuker et al., 2019; Dixon et al., 2012). The abnormality of iron homeostasis is closely linked with TBI. The destruction of brain tissue after traumatic impact, damage of the blood-brain barrier integrity and increase of cerebrovascular permeability all lead to the infiltration of large amounts of iron from the blood into the brain parenchyma (Tang et al., 2020). Furthermore, neuronal cell membranes are rich in polyunsaturated fatty acids (PUFAs), which are easily oxidized by ROS (Shichiri, 2014). Lipid peroxidation is considered to be one of important mechanisms causing secondary injury after TBI (Hall et al., 2010). The abnormal iron metabolism after TBI leads to the increase of ions in labile iron pools of the cytoplasm that induces the iron-independent oxidative stress response and finally the demise of neurons (Ke and Qian, 2007).

Although there have been considerable researches upon TBI, there are still lacks of effective therapeutic treatments in the clinic. Ruxolitinib (Ruxo) is an inhibitor of the Janus kinase (JAK) 1 and 2 and a type of FDA approved drugs for treating myelofibrosis (Harrison et al., 2012). Previous studies have shown that TBI induced activation of the JAK-STAT pathway (Zhao et al., 2011), and inhibiting this signaling pathway contributed to the recovery of vestibular motor function (Raible et al., 2015). Additionally, our previous study also demonstrated that Ruxo played a neuroprotective effect via repressing pyroptotic cell death after TBI (Gao et al., 2020). However, little is known about the influence of Ruxo to ferroptosis after TBI.

To explore the problem, we firstly evaluated the expression levels of ferroptosis-related proteins at different time points after TBI. Subsequently, we used Fer-1 (a specific inhibitor for ferroptosis) as a positive control to evaluate the effect of Ruxo on the process of ferroptosis after TBI. We also verified the effect of Ruxo and Fer-1 on cerebral edema and neurodegeneration at the acute phase of TBI. Finally, we assessed the effect of these two interventions on the long-term outcomes of TBI using the corresponding behavioral tests including the wire-grip test, Morris water maze (MWM) and open field test and followed by the lesion volume and iron deposition.

Section snippets

Animal

Adult male C57BL/6 J mice (6–8 weeks, weighting 20–25 g) were used for all experiments. Mice were housed in pairs in a cage with access to food and water ad libitum. They were maintained in a room with controlled temperature (25 °C) and humidity (50%), and a 12-h light-dark cycle. All animal studies were approved by the Institutional Animal Use and Care Committee at Soochow University (reference number = SZUM2008031233) and were conducted in accordance with the guidelines of Animal Use and Care

The changes of time course of ferroptosis-related proteins post-TBI

To investigate the role of ferroptosis in the pathophysiological process of TBI, we firstly detected the expression levels of ferroptosis-related proteins in cerebral cortex at different time points by immunoblotting. The expression tendency of GPX4, which converts lipid hydrogen peroxide to non-toxic lipid alcohols (Bersuker et al., 2019; Yang et al., 2014), was decreased and then increased, and its level significantly decreased to valley at 12 h post-TBI. The overall tendency of TfR1 and COX2

Discussion

The major findings of this study were as follows: (1) TBI induces temporal changes of the molecules associated with ferroptosis; (2) Ruxo has the similar inhibition effect of ferroptosis with Fer-1; (3) Ruxo and Fer-1 both reduce the degree of neurodegeneration and alleviate brain edema at the acute phase of TBI; (4) Ruxo and Fer-1 respectively improve TBI-induced motor dysfunction, anxiety behavior and memory deficits; (5) Ruxo and Fer-1 finally ameliorate TBI-induced tissue loss and iron

Author contributions

X.C., C.G. and Y.Y. contributed equally to this paper. L.T., X.C. and T.W. comprehended the study, provided critical suggestions, contributed to manuscript preparation, oversaw the research program, and wrote the main manuscript. X.C., Z.C. and Y.Y. performed the Western blot and immunostaining experiments and analyzed the data. X.C., and Y.Y., C.G., G.C., T.R., C.L., M.Z., and Y.G. performed the Nissl staining, FJB staining, Perl's staining, the behavioral experiment, and the brain edema test

Declaration of Competing Interest

The authors declare no competing financial interest.

Acknowledgements

This study was supported by the National Nature Science Foundation of China (Nos. 81971800, 81871536, and 82072110), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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