Jianguo Xu

NADPH oxidase inhibition improves neurological outcome in experimental traumatic brain injury

PURPOSEnTraumatic brain injury (TBI) is a worldwide health problem with oxidative stress recognized as a major pathogenetic factor. The present experimental study was designed to explore the neuroprotective effect of NADPH oxidase (NOX) inhibitor, apocynin, on mouse TBI.nnnMETHODSnModerately severe weight-drop impact head injury was induced in adult male mice, randomly divided into four groups: sham, TBI, TBI+vehicle and TBI+apocynin treatment. Apocynin (50 mg/kg) was injected intraperitoneally 30 min before TBI. The expression of NOX2 protein was investigated using immunoblotting techniques 1 and 24h after TBI. Neurological score was evaluated 24h after TBI. Blood-brain barrier disruption was detected by Evans blue extravasation and cortical apoptosis was analyzed by TUNEL assay. Additionally, we assessed tissue levels of malondialdehyde (MDA).nnnRESULTSnNOX2 expression increased rapidly following TBI in male mice, with an early peak at 1h, followed by a second peak at 24h. Pre-treatment with the NOX inhibitor, apocynin markedly inhibited NOX2 expression. Apocynin also attenuated MDA levels and TBI-induced blood-brain barrier dysfunction. In addition apocynin significantly attenuated TBI-induced neurological deficits and cortical apoptosis.nnnCONCLUSIONnPre-treatment with apocynin effectively attenuates markers of cerebral oxidative stress after TBI, thus supporting the hypothesis that apocynin is a potential neuroprotectant and adjunct therapy for TBI patients.

Melatonin protects the brain from apoptosis by enhancement of autophagy after traumatic brain injury in mice

Melatonin has been proven to possess neuroprotection property against various neurological diseases by decreasing cerebral oxidative stress and inhibiting inflammatory process. However, whether administration of melatonin influences the autophagy pathway, which has recently been reported playing a pivotal role in traumatic brain injury, is yet not fully understood. We supposed that treatment of melatonin enhances the autophagy pathway after traumatic brain injury (TBI) in mice and subsequently inhibited the mitochondrion apoptotic pathway. Firstly, we investigated the neurological severity score, brain water content and neuronal apoptosis in mice cortex to demonstrate the neuroprotection of melatonin. Then we determined the autophagy markers, namely Beclin1 and LC3-II, using western blot and immunofluorescence. Next, we evaluated the mitochondrial apoptotic pathway in the presence or absence of melatonin. More significantly, we employed 3-methyladenine (3-MA) to inhibit the autophagy pathway, to further confirm our hypothesis. The results showed that melatonin significantly ameliorated secondary brain injury induced by TBI. In addition, melatonin enhanced autophagy after TBI, which was accompanied by a decrease in both the translocation of Bax to mitochondria and the release of cytochrome C to cytoplasm. Furthermore, simultaneous treatment of 3-MA reversed the beneficial effects of melatonin on mitochondrial apoptotic pathway. Taken together, we conclude that melatonin enhances autophagy, which inhibits mitochondrial apoptotic pathway, thus protecting mice from secondary brain injury after TBI.

Puerarin ameliorates oxidative stress in a rodent model of traumatic brain injury

BACKGROUNDnA wealth of evidence has suggested that oxidative stress is involved in the secondary brain injury after traumatic brain injury (TBI). Recently, numerous in vivo and in vitro studies were reported that puerarin could inhibit oxidative stress through the activation of phosphatidylinositol 3-kinase (PI3K)-Akt pathway. It is unknown, however, whether puerarin can provide neuroprotection and reduce oxidative stress after TBI. The present study investigated the effects of puerarin on the TBI-induced neurodegeneration, oxidative stress, and the possible role of PI3K-Akt pathway in the neuroprotection of puerarin, in a rat model of TBI.nnnMATERIALS AND METHODSnRats were randomly distributed into various subgroups undergoing the sham surgery or TBI procedures. Puerarin (200 mg/kg) was given intraperitoneally at 10 min before injury and PI3K-Akt pathway inhibitor LY294002 was also administered intracerebroventricular in one subgroup. All rats were killed at 24 h after TBI for examination.nnnRESULTSnOur data indicated that puerarin could significantly reduce TBI-induced neuronal degeneration, accompanied by the partial restoration of the redox disturbance and enhanced expression of phospho-Akt in the pericontusional cortex after TBI. Moreover, PI3K-Akt pathway inhibitor LY294002 could partially abrogate the neuroprotection of puerarin in rats with TBI.nnnCONCLUSIONSnThese results indicate that puerarin can ameliorate oxidative neurodegeneration after TBI, at least in part, through the activation of PI3K-Akt pathway.

Posttraumatic administration of luteolin protects mice from traumatic brain injury: implication of autophagy and inflammation.

Secondary brain insult induced by traumatic brain injury (TBI), including excitotoxicity, oxidative stress, inflammatory response, and neuronal degeneration, is sensitive to therapeutic interventions; therefore, searching for neuroprotective agents represents a promising therapeutic strategy for TBI treatment. Luteolin, a member of the flavonoid family, has recently been proven to modulate autophagy. However, whether it activates autophagy after TBI thereby alleviating the secondary insult is not yet understood. Here, we aimed to evaluate the neuroprotection of luteolin against TBI and the potential role of autophagy where it is involved. For this purpose, mice were randomly divided into four groups and then subjected to TBI. The treatment mice received luteolin at a dose of 30mg/kg 30min post-TBI based on our previous study. We employed western blot, immunofluorescence and quantitative real-time PCR to determine autophagy process and inflammatory response among different groups. Autophagy was found to be enhanced after luteolin treatment according to the expressions of autophagic markers. Furthermore, luteolin decreased nuclear accumulation of p65 induced by TBI, indicating attenuation of inflammation. In line with these observations, luteolin decreased mRNA and protein expressions of pro-inflammatory factors IL-1b and TNF-a. At last, luteolin reduced neuronal degeneration, and alleviated brain edema and blood-brain barrier (BBB) disruption. In conclusion, these results implied that luteolin protected mice brain from traumatic brain injury by inhibiting inflammatory response, and luteolin-induced autophagy might play a pivotal role in its neuroprotection.

Rapamycin protects against apoptotic neuronal death and improves neurologic function after traumatic brain injury in mice via modulation of the mTOR-p53-Bax axis.

BACKGROUNDnRapamycin has proven to be a neuroprotective agent in traumatic brain injury (TBI). However, there is a lack of data regarding the effect of rapamycin on apoptotic neuronal death after TBI. Thus, the present study was designed to detect the modulatory role of rapamycin on apoptosis and explore the potential involvement of the mammalian target of rapamycin (mTOR)-p53-Bax axis after TBI.nnnMATERIAL AND METHODSnNeurologic severity score tests were performed to measure behavioral outcomes. The effect of rapamycin treatment on neuronal death was analyzed using immunofluorescence analysis of NeuN. Terminal deoxynucleotidyl transferase-mediated dUTP nick 3-end labeling was performed to detect apoptotic cells. The expression of Bax and phosphorylated protein of p53 was detected using Western blotting analyses and immunofluorescence staining. Phosphorylated protein of the mTOR in the ipsilateral cortex was detected using Western blotting analyses.nnnRESULTSnRapamycin administration after TBI was associated with an increased number of neurons, decreased apoptosis index, and improved neurobehavioral function, which was potentially mediated by inactivation of the mTOR-p53-Bax axis.nnnCONCLUSIONSnRapamycin can protect neurons from apoptotic neuronal death after TBI. This study presents a new insight into the antiapoptosis mechanisms, which are responsible for the neuroprotection of rapamycin, with the potential involvement of the mTOR-p53-Bax axis.

Mollugin induces tumor cell apoptosis and autophagy via the PI3K/AKT/mTOR/p70S6K and ERK signaling pathways.

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L., has shown preclinical anticancer efficacy in various cancer models. However the effects of mollugin in regulating cancer cell survival and death remains undefined. In the present study we found that mollugin exhibited cytotoxicity on various cancer models. The suppression of cell viability was due to the induction of mitochondria apoptosis. In addition, the presence of autophagic hallmarks was observed in mollugin-treated cells. Notably, blockade of autophagy by a chemical inhibitor or RNA interference enhanced the cytotoxicity of mollugin. Further experiments demonstrated that phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) and extracellular regulated protein kinases (ERK) signaling pathways participated in mollugin-induced autophagy and apoptosis. Together, these findings support further studies of mollugin as candidate for treatment of human cancer cells.

FTY720 induces autophagy-related apoptosis and necroptosis in human glioblastoma cells.

FTY720 is a potent immunosuppressant which has preclinical antitumor efficacy in various cancer models. However, its role in glioblastoma remains unclear. In the present study, we found that FTY720 induced extrinsic apoptosis, necroptosis and autophagy in human glioblastoma cells. Inhibition of autophagy by either RNA interference or chemical inhibitors attenuated FTY720-induced apoptosis and necrosis. Furthermore, autophagy, apoptosis and necrosis induction were dependent on reactive oxygen species-c-Jun N-terminal kinase-protein 53 (ROS-JNK-p53) loop mediated phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) pathway. In addition, receptor-interacting protein 1 and 3 (RIP1 and RIP3) served as an upstream of ROS-JNK-p53 loop. However, the phosphorylation form of FTY720 induced autophagy but not apoptosis and necroptosis. Finally, the in vitro results were validated in vivo in xenograft mouse of glioblastoma cells. In conclusion, the current study provided novel insights into understanding the mechanisms and functions of FTY720-induced apoptosis, necroptosis and autophagy in human glioblastoma cells.

Traumatic Brain Injury-Induced Neuronal Apoptosis is Reduced Through Modulation of PI3K and Autophagy Pathways in Mouse by FTY720

FTY720 is a synthetic compound produced by modification of metabolite from Isaria sinclairii. It is a novel type of immunosuppressive agent inhibiting lymphocyte egress from secondary lymphoid tissues, thereby causing peripheral lymphopenia. Growing evidences have suggested that apoptosis and autophagy were involved in the secondary brain injury after traumatic brain injury (TBI) although FTY720 exerted neuroprotective effects in a variety of neurological diseases except TBI. The present study was aimed to investigate the role of FTY720 in a mouse model of TBI. In experiment 1, ICR mice were divided into four groups: sham group, TBI group, TBIxa0+xa0vehicle group, and TBIxa0+xa0FTY720 group. And the injured cerebral cortex (including both contused and penumbra) was used for analysis. We found that FTY720 administration after TBI improved neurobehavioral function, alleviated brain edema, accompanied by modulation of apoptotic indicators such as Bcl-2, Bcl-xL, Bax, and cytochrome c. In experiment 2, ICR mice were also divided into four groups: sham group, TBIxa0+xa0vehicle group, TBIxa0+xa0FTY720 group, and TBIxa0+xa0FTY720xa0+xa0inhibitors group. And the injured cerebral cortex (including both contused and penumbra) was used for analysis. We found that FTY720 increased the expression of phospho-protein kinase B (AKT) and some autophagy markers such as LC3 and Beclin 1. In addition, the apoptosis inhibition effect of FTY720 was partly abrogated by the phosphatidylinositide 3-kinases (PI3K)/AKT pathway inhibitor LY294002 and autophagy inhibitor 3-methyladenine. Collectively, our data provide the first evidence that FTY720 exerted neuroprotective effects after TBI, at least in part, through the activation of PI3K/AKT pathway and autophagy.

Pretreatment with tert-butylhydroquinone attenuates cerebral oxidative stress in mice after traumatic brain injury

BACKGROUNDnTraumatic brain injury (TBI) is a worldwide health problem, identified as a major cause of death and disability. Increasing evidence has shown that oxidative stress plays an important role in TBI pathogenesis. The antioxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), is a known mediator in protection against TBI-induced brain damage. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), a novel Nrf2 activator, can protect against TBI-induced oxidative stress.nnnMETHODSnAdult male imprinting control region mice were randomly divided into three groups: (1) shamxa0+xa0vehicle group; (2) TBIxa0+xa0vehicle group; and (3) TBIxa0+xa0tBHQ group. Closed-head brain injury was applied using the Feeney weight-drop method. We accessed the neurologic outcome of mice at 24xa0h after TBI, and subsequently measured protein levels of Nrf2 and the NOX2 subunit of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), the concentration of malondialdehyde, superoxide dismutase activity, and brain edema.nnnRESULTnThe NOX2 protein level was increased fivefold in the TBIxa0+xa0vehicle group, whereas pretreatment with tBHQ markedly attenuated the NOX2 protein expression relative to that in the TBIxa0+xa0vehicle group. TBI increased Nrf2 formation by 5% compared with the sham group, whereas treatment with tBHQ further upregulated the Nrf2 protein level by 12% compared with the sham group. The level of the oxidative damage marker malondialdehyde was reduced by 29% in the TBIxa0+xa0tBHQ group compared with the TBIxa0+xa0vehicle group, Moreover, pretreatment with tBHQ significantly increased the antioxidant enzyme superoxide dismutase activity. Administration of tBHQ also significantly decreased TBI-induced brain edema and neurologic deficits.nnnCONCLUSIONSnPretreatment with tBHQ effectively attenuated markers of cerebral oxidative stress after TBI, thus supporting the testing of tBHQ as a potential neuroprotectant and adjunct therapy for TBI patients.

Baincalein alleviates early brain injury after experimental subarachnoid hemorrhage in rats: Possible involvement of TLR4/NF-κB-mediated inflammatory pathway

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) largely contributes to unfavorable outcomes. Hence, effective therapeutic strategies targeting on EBI have recently become a major goal in the treatment of SAH patients. Baicalein is a flavonoid that has been shown to offer neuroprotection in kinds of brain injury models. This study investigated the effects of baicalein on EBI in rats following SAH. SAH was inducted in male Sprauge-Dawley rats by injection of fresh non-heparinized arterial blood into the prechiasmatic cistern. Baicalein (30 or 100 mg/kg) or vehicle were administrated 30 min after injury. Neurological deficit, brain edema, blood-brain barrier (BBB) permeability and neural cell apoptosis were assessed. To explore the further mechanisms, the change of toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) signaling pathway and the levels of apoptosis associated proteins were also examined. Our study showed that treatment with baicalein (30 mg/kg) significantly improved neurological function at 24h after SAH and reduced brain edema at both 24h and 72 h after SAH. Baicalein also significantly reduced neural cell death, BBB permeability. These changes were associated with the remarkable reductions of TLR4 expression, IκB-α degradation, NF-κB translocation to nucleus, as well as the expressions of matrix metalloproteinase-9, tight junctions protein, interleukin-1β and tumor necrosis factor- ɑ. These findings suggest that baicalein may ameliorate EBI after SAH potentially via inhibition of inflammation-related pathway.