Sestrin2 protects against lethal sepsis by suppressing the pyroptosis of dendritic cells.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sestrin2 (SESN2), a highly evolutionarily conserved protein, is critically involved in the cellular response to various stresses and has been confirmed to maintain the homeostasis of the internal environment. However, the potential effects of SESN2 in regulating dendritic cells (DCs) pyroptosis in the context of sepsis and the related mechanisms are poorly characterized. In this study, we found that SESN2 was capable of decreasing gasdermin D (GSDMD)-dependent pyroptosis of splenic DCs by inhibiting endoplasmic reticulum (ER) stress (ERS)-related nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-mediated ASC pyroptosome formation and caspase-1 (CASP-1) activation. Furthermore, SESN2 deficiency induced NLRP3/ASC/CASP-1-dependent pyroptosis and the production of proinflammatory cytokines by exacerbating the PERK-ATF4-CHOP signaling pathway, resulting in an increase in the mortality of septic mice, which was reversed by inhibiting ERS. These findings suggest that SESN2 appears to be essential for inhibiting NLRP3 inflammasome hyperactivation, reducing CASP-1-dependent pyroptosis, and improving sepsis outcomes through stabilization of the ER. The present study might have important implications for exploration of novel potential therapeutic targets for the treatment of sepsis complications.

RSL3 induced autophagic death in glioma cells via causing glycolysis dysfunction.

RSL3 is a type of small molecular compound which can inactivate glutathione peroxidase 4 (GPX4) and induce ferroptosis, but its role in glioma cell death remains unclear. In this study, we found RSL3 inhibited the viabilities of glioma cells and induced glioma cell death in a dose-dependent manner. In vitro studies revealed that RSL3-induced cell death was accompanied with the changes of autophagy-associated protein levels and was alleviated by pretreatment of 3-Methyladenine, bafilomycin A1 and knockdown of ATG5 with siRNA. The ATP and pyruvate content as well as the protein levels of HKII, PFKP, PKM2 were decreased in cells treated by RSL3, indicating that RSL3 induced glycolysis dysfunction in glioma cells. Moreover, supplement of exterior sodium pyruvate, which was a final product of glycolysis, not only inhibited the changes of autophagy-associated protein levels caused by RSL3, but also prevented RSL3-induced cell death. In vivo data suggested that the inhibitory effect of RSL3 on the growth of glioma cells was associated with glycolysis dysfunction and autophagy activation. Taken together, RSL3 induced autophagic cell death in glioma cells via causing glycolysis dysfunction.

Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1/RIP3 necrosome formation.

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 (RIP1) and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species (ROS), but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin (2-10 µmol/L) dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose-dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 (100 µmol/L) or the specific RIP3 inhibitor GSK-872 (5 µmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 µmol/L), which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.

Traumatic hemorrhage within a cerebellar dermoid cyst.

Intracranial dermoid cysts with hemorrhage are fairly rare. Herein, we reported a 28-year-old female patient with a cerebellar dermoid cyst, which was found accidently on neuro-imaging after head trauma. MR scanning revealed that the lesion was located within the cerebellar vermis and was measured 3.5cm×3.9cm×3.0cm, with hyper-intensity on T1WI and hypo-intensity on T2WI. However, on CT imaging, it showed hyper-dense signals. It was removed completely via midline sub-occipital approach under surgical microscope. Histological examination proved it was a dermoid cyst with internal hemorrhage. In combination with literature review, we discussed the factors that might be responsible for the hemorrhage within dermoid cysts.

Gross total resection of glioma with the intraoperative fluorescence-guidance of fluorescein sodium.

OBJECTIVE: High dose fluorescein sodium has been utilized for fluorescence-guided tumor resection with conflicting reports on the efficacy of this procedure. The aim of this study was to reevaluate the utility and clinical limitations of using fluorescein sodium for the treatment and resection of glioma brain tumors. METHODS: Patients diagnosed with glioma were divided into two groups with a total of 22 patients enrolled in the study: 1) the study group (n=10), patients that received intravenous injection of fluorescein sodium and 2) the control group (n=12), patients that did not receive injections during surgical resection. Quality of life was evaluated according to Karnofsky Performance Scale (KPS) score and neurological status. Fluorescein sodium was intravenously injected at a dose of 15-20mg/kg of body weight. Glioma resection was evaluated preoperative and postoperatively with enhanced Magnetic Resonance Imaging (MRI). RESULTS: Significant differences in the gross total resection (GTR) rates were observed between the two patient groups (Fisher's Exact Test p=0.047). Progressive free survival was significantly longer in the study group (Student's T-Test p=0.033) as well as in the GTR group (Student's T-Test p=0.0001) compared to the control and non-GTR groups, respectively. Three patients in the study group and four patients in the control group had transient neurological deterioration. One patient in the control group had permanent hemiplegia. CONCLUSION: The intraoperative utility of using fluorescein sodium can significantly increase the GTR rate without obvious deterioration. In addition, we find that it is better to apply the fluorescein sodium in the cases with BBB (blood-brain barrier) disruption, which had been enhanced in preoperative MRI.

Ischemic postconditioning alleviates neuronal injury caused by relief of carotid stenosis in a rat model of cerebral hypoperfusion.

The effects of early relief of heavy bilateral carotid stenosis and ischemic postconditioning on hippocampus CA1 neurons are still unclear. In this study, we used a rat model to imitate severe bilateral carotid stenosis in humans. The rats were divided into sham group, carotid stenosis group, stenosis relief group and ischemic postconditioning group. Ischemic postconditioning consisted of three cycles of 30 s ischemia and 30 s reperfusion. The cerebral blood flow was measured with a laser Doppler flowmeter. Neuronal death in the CA1 region was observed by hematoxylin-eosin staining, and the number of live neurons was assessed by cell counting under a light microscope. The levels of oxidative products MDA and 8-iso-PGF2α, inflammatory factors IL-1ß and TNF-α, and the activities of anti-oxidative enzymes SOD and CAT were assayed by specific enzyme-linked immunosorbent assay (ELISA) kits, respectively. We found that relief of carotid stenosis and ischemic postconditioning could increase cerebral blood flow. When stenosis was relieved, the percentage of live neurons was 66.6% ± 6.2% on day 3 and 62.3% ± 9.8% on day 27, which was significantly higher than 55.5% ± 4.8% in stenosis group. Ischemic postconditioning markedly improved the live neurons to 92.5% ± 6.7% on day 3 and 88.6% ± 9.1% on day 27. Further study showed that, neuronal death caused by relief of stenosis is associated with increased oxidative stress and enhanced inflammatory response, and the protection of ischemic postconditioning is related to inhibition of oxidative stress and suppression of inflammatory response.

Anhelation due to formation of tuberculomas at the medulla oblongata during chemotherapy of tuberculous meningitis.

Formation of tuberculoma is a rare response of neurotuberculosis in patients regularly and adequately treated with anti-tuberculous drugs. We report a 13-year-old girl with two tuberculomas which formed in the dorsal part of the medulla oblongata during chemotherapy for tuberculous meningitis. The tuberculomas were both removed via a suboccipital midline approach and were demonstrated by pathological findings but the girl died of cardiac arrest that was thought to be caused by postoperative medulla oblongata oedema. In combination with a literature review, we discuss the clinical features and treatment options of brainstem tuberculomas.

Sellar Chordoma Presenting as Pseudo-macroprolactinoma with Unilateral Third Cranial Nerve Palsy.

We described a 61-year-old female with a sellarchordoma, which presented as pseudo-macroprolactinoma with unilateral third cranial nerve palsy. Physical examination revealed that her right upper lid could not be raised by itself, right eyeball movement limited to the abduction direction, right pupil dilated to 4.5 mm with negative reaction to light, and hemianopsia in bitemporal sides. CT scanning showed a hyperdense lesion at sellar region without bone destruction. Magnetic resonance imaging (MRI) revealed the tumor was 2.3 cm×1.8 cm×2.6 cm, with iso-intensity on T1WI, hyper-intensity on T2WI and heterogeneous enhancement on contrast imaging. Endocrine examination showed her serum prolactin level increased to 1,031.49 mIU/ml. The tumor was sub-totally resected via pterional craniotomy under microscope and was histologically proven to be a chordoma. Postoperatively, she recovered uneventfully but ptosis and hemianopsia remained at the 6th month.

Pink1/Parkin-Mediated Mitophagy Regulated the Apoptosis of Dendritic Cells in Sepsis.

Dendritic cells (DCs) are vital antigen-presenting cells (APCs) in the immune system, whose apoptosis is closely related to the development of sepsis. Mitophagy is one of the necessary forms of selective autophagy that removes damaged or dysfunctional mitochondria to regulate immunity and inflammation. However, its effect on the apoptosis of DC in sepsis remains unknown. Here, we showed that sepsis activated the apoptosis and mitophagy of DC, and mitophagy had an anti-apoptotic effect on sepsis-induced DC apoptosis. In this study, we used cecal ligation and puncture (CLP) to simulate the pathophysiological state of sepsis. Apoptosis and mitophagy of DC were significantly enhanced in CLP mice compared with controls, and in the Pink1-KO (Pink1-knockout) mice CLP model, the level of apoptosis in DC was further increased while the level of mitophagy was decreased. In addition, more severe mitochondrial dysfunction was exhibited in DC of Pink1-KO mice CLP model compared to wild-type (WT) mice. The results suggest that Pink1/Parkin-mediated mitophagy is activated during sepsis and has an anti-apoptotic effect on DC, which regulates immune functions.

Proteasome inhibitor MG-132 induces C6 glioma cell apoptosis via oxidative stress.

AIM: Proteasome inhibitors have been found to suppress glioma cell proliferation and induce apoptosis, but the mechanisms are not fully elucidated. In this study we investigated the mechanisms underlying the apoptosis induced by the proteasome inhibitor MG-132 in glioma cells. METHODS: C6 glioma cells were used. MTT assay was used to analyze cell proliferation. Proteasome activity was assayed using Succinyl-LLVY-AMC, and intracellular ROS level was evaluated with the redox-sensitive dye DCFH-DA. Apoptosis was detected using fluorescence and transmission electron microscopy as well as flow cytometry. The expression of apoptosis-related proteins was investigated using Western blot analysis. RESULTS: MG-132 inhibited C6 glioma cell proliferation in a time- and dose-dependent manner (the IC(50) value at 24 h was 18.5 µmol/L). MG-132 (18.5 µmol/L) suppressed the proteasome activity by about 70% at 3 h. It induced apoptosis via down-regulation of antiapoptotic proteins Bcl-2 and XIAP, up-regulation of pro-apoptotic protein Bax and caspase-3, and production of cleaved C-terminal 85 kDa PARP). It also caused a more than 5-fold increase of reactive oxygen species. Tiron (1 mmol/L) effectively blocked oxidative stress induced by MG-132 (18.5 µmol/L), attenuated proliferation inhibition and apoptosis in C6 glioma cells, and reversed the expression pattern of apoptosis-related proteins. CONCLUSION: MG-132 induced apoptosis of C6 glioma cells via the oxidative stress.

Pilomyxoid astrocytoma in cerebellum.

Pilomyxoid astrocytoma is a new identified variant type of pilocytic astrocytoma, and typically locates in the hypothalamic and chiasmatic region. Herein, we reported a nine-year-old boy with pilomyxoid astrocytoma in the cerebellum. MRI scanning showed a tumor involved the cerebellar vermis, tonsil, the forth ventricle and brainstem. It was homogeneous isointensity on T1WI, relative hyper-intensity on T2WI, hyper-intensity on fluid attenuated inversion recovery (FLAIR) images, and uniform enhancement on contrast T1WI. The tumor was sub-totally removed and was proved histologically to be pilomyxoid astrocytoma. Follow-up at the 5th month, MRI showed the residual tumor enlarged at the brainstem. The patient survived 10 months after the operation, and finally died of respiration failure resulting from brainstem dysfunction.

Neurological Complications of Veno-Arterial Extracorporeal Membrane Oxygenation: A Retrospective Case-Control Study.

Background: To explore the epidemiology, clinical features, risk indicators, and long-term outcomes of neurological complications caused by veno-arterial extracorporeal membrane oxygenation (V-A ECMO). Methods: We retrospectively analyzed 60 adult patients who underwent V-A ECMO support in our unit from February 2012 to August 2020. These patients were separated into the neurological complications group (NC group) and the non-neurological complications group (nNC group). The differences in basic data and ECMO data between the two groups were compared. The data of long-term neurological prognosis were collected by telephone follow-up. Results: Thirty-nine patients (65.0%) had neurological complications. There were significant differences between the two groups in terms of median age, hypertension, median blood urea nitrogen, median troponin I (TNI), median lactic acid, pre-ECMO percutaneous coronary intervention, continuous renal replacement therapy (CRRT), median Sequential Organ Failure Assessment score, median Acute Physiology and Chronic Health Evaluation II score, median peak inspiratory pressure, median positive end expiratory pressure, and median fresh frozen plasma (P < 0.05). The median Intensive Care Unit length of stay (ICU LOS), 28-day mortality, median post-ECMO vasoactive inotropic score, non-pulsate perfusion (NP), and median ECMO duration of the NC group were significantly higher than those of the nNC group (P < 0.05). Furthermore, multiple logistic regression analysis revealed that TNI (P = 0.043), CRRT (P = 0.047), and continuous NP > 12 h (P = 0.043) were independent risk indicators for neurological complications in patients undergoing ECMO. Forty-four patients (73.3%) survived after discharge, and 38 patients (63.3%) had Cerebral Performance Category score of 1-2. And there were significant differences between the two groups in long-term neurological outcomes after discharge for 6 months (P < 0.05). Conclusion: The incidence of neurological complications was higher in patients undergoing V-A ECMO and was closely related to adverse outcomes (including ICU LOS and 28-day mortality). TNI, CRRT, and continuous NP > 12 h were independent risk indicators for predicting neurological complications in ECMO supporting patients. And the neurological complications of patients during ECMO support had significant adverse effect on long-term surviving and neurological outcomes of patients after discharge for 6 months.

Ischemic post-conditioning: a feasible preventive method for cerebral hyperperfusion syndrome secondary to revascularization.

Cerebral hyperperfusion syndrome is a serious complication of revascularization after carotid endarterectomy, extracranial-intracranial bypass surgery, or stent placement. The mechanisms underlying cerebral hyper-perfusion syndrome are mainly ascribed to the dysfunction of cerebral auto-regulation to blood flow and injury of the endothelia in blood vessels. Although blood pressure control is often used to prevent cerebral hyper-perfusion after revascularization, cases of cerebral hyperperfusion are still reported in the literature. Current animal studies have shown that ischemic post-conditioning has protective effects on brain tissue, and further studies also showed that it had positive effects on human brachial artery and heart. Therefore, we propose the hypothesis that ischemic post-conditioning could be used to prevent cerebral hyperperfusion syndrome following revascularization. Although the mechanism of ischemic post-conditioning and its clinical application should be further investigated, it has shown its potential effectiveness as a feasible method for prevention of cerebral hyperperfusion syndrome.

Two subtypes of meningiomas with different imaging co-existed at sphenoid ridge.

Intracranial multiple meningiomas are not uncommon, but multiple meningiomas consisting of different subtypes are rare. Here, we describe an adult male patient with two meningiomas located at sphenoid ridge, with different features on MRI and CTA. Histological examination revealed that one was fibrous meningioma and the other was psammomatous meningioma.

Sestrin2 protects dendritic cells against endoplasmic reticulum stress-related apoptosis induced by high mobility group box-1 protein.

Sestrin2 (SESN2) is a highly evolutionary conserved protein and involved in different cellular responses to various stresses. However, the potential function of SESN2 in immune system remains unclear. The present study was designed to test whether dendritic cells (DCs) could express SESN2, and investigate the underlying molecular mechanism as well as its potential significance. Herein, we firstly reported that SESN2 was expressed in DCs after high mobility group box-1 protein (HMGB1) stimulation and the apoptosis of DCs was obviously increased when SESN2 gene silenced by siRNA. Cells undergone SESN2-knockdown promoted endoplasmic reticulum (ER) stress (ERS)-related cell death, markedly exacerbated ER disruption as well as the formation of dilated and aggregated structures, and they significantly aggravated the extent of ERS response. Conversely, overexpressing SESN2 DCs markedly decreased apoptotic rates and attenuated HMGB1-induced ER morphology fragment together with inhibition of ERS-related protein translation. Furthermore, sesn2-/--deficient mice manifested increased DC apoptosis and aggravated ERS extent in septic model. These results indicate that SESN2 appears to be a potential regulator to inhibit apoptotic ERS signaling that exerts a protective effect on apoptosis of DCs in the setting of septic challenge.

Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

AIM: The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. METHODS: The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. RESULTS: MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. CONCLUSION: Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis.

Chromatinolysis refers to enzymatic degradation of nuclear DNA and is regarded as one of the crucial events leading to cell death. Mixed-lineage kinase domain-like protein (MLKL) has been identified as a key executor of necroptosis, but it remains unclear whether MLKL contributes to necroptosis via regulation of chromatinolysis. In this study, we find that shikonin induces MLKL activation and chromatinolysis in glioma cells in vitro and in vivo, which are accompanied with nuclear translocation of AIF and γ-H2AX formation. In vitro studies reveal that inhibition of MLKL with its specific inhibitor NSA or knockdown of MLKL with siRNA abrogates shikonin-induced glioma cell necroptosis, as well as chromatinolysis. Mechanistically, activated MLKL targets mitochondria and triggers excessive generation of mitochondrial superoxide, which promotes AIF translocation into nucleus via causing mitochondrial depolarization and aggravates γ-H2AX formation via improving intracellular accumulation of ROS. Inhibition of nuclear level of AIF by knockdown of AIF with siRNA or mitigation of γ-H2AX formation by suppressing ROS with antioxidant NAC effectively prevents shikonin-induced chromatinolysis. Then, we found that RIP3 accounts for shikonin-induced activation of MLKL, and activated MLKL reversely up-regulates the protein level of CYLD and promotes the activation of RIP1 and RIP3. Taken together, our data suggest that MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis, and shikonin induces a positive feedback between MLKL and its upstream signals RIP1 and RIP3.

Endoplasmic reticulum stress regulates oxygen-glucose deprivation-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS.

AIMS: Endoplasmic reticulum (ER) stress has been demonstrated to regulate neuronal death caused by ischemic insults via activation of apoptosis, but it still remains unclear whether ER stress participates in regulation of parthanatos, a new type of programmed cell death characterized by PARP-1 overactivation and intracellular accumulation of PAR polymer. METHODS: we used oxygen-glucose deprivation (OGD) and human SH-SY5Y cells to simulate neuronal damage caused by ischemia. RESULTS: Oxygen-glucose deprivation induced time-dependent death in SH-SY5Y cells, which was accompanied with upregulation of PARP-1, accumulation of PAR polymer, decline of mitochondrial membrane potentials and nuclear translocation of AIF. Pharmacological inhibition of PARP-1 with its specific inhibitor 3AB rescued OGD-induced cell death, as well as prevented PAR polymer accumulation, mitochondrial depolarization, and AIF translocation into nucleus. Similar results could be found when PARP-1 was genetically knocked down with SiRNA. These indicated that OGD triggered parthanatos in SH-SY5Y cells. Then, we found inhibition of overproduction of ROS with antioxidant NAC attenuated obviously OGD-induced parthanatos in SH-SY5Y cells, suggesting ROS regulated OGD-induced parthanatos. Additionally, OGD also induced upregulation of ER stress-related proteins. Mitigation of ER stress with chemical chaperone 4-PBA or trehalose suppressed significantly OGD-induced overproduction of ROS, PARP-1 upregulation, PAR polymer accumulation, and nuclear accumulation of AIF, and cell death in SH-SY5Y cells. CONCLUSION: Endoplasmic reticulum stress regulates OGD-induced parthanatos in human SH-SY5Y cells via improvement of intracellular ROS.

RIP1 and RIP3 contribute to shikonin-induced glycolysis suppression in glioma cells via increase of intracellular hydrogen peroxide.

RIP1 and RIP3 are necroptosis initiators, but their roles in regulation of glycolysis remain elusive. In this study, we found shikonin activated RIP1 and RIP3 in glioma cells in vitro and in vivo, which was accompanied with glycolysis suppression. Further investigation revealed that shikonin-induced decreases of glucose-6-phosphate and pyruvate and downregulation of HK II and PKM2 were significantly prevented when RIP1 or RIP3 was pharmacologically inhibited or genetically knocked down with SiRNA. Moreover, shikonin also triggered accumulation of intracellular H2O2 and depletion of GSH and cysteine. Mitigation of intracellular H2O2 via supplement of GSH reversed shikonin-induced glycolysis suppression. The role of intracellular H2O2 in regulation of glycolysis suppression was further confirmed in the cells treated with exogenous H2O2. Notably, inhibition of RIP1 or RIP3 prevented intracellular H2O2 accumulation, which was correlated with preventing shikonin-induced downregulation of x-CT and depletion of GSH and cysteine. In addition, supplement of pyruvate effectively inhibited shikonin- or exogenous H2O2-induced accumulation of intracellular H2O2 and glioma cell death. Taken together, we demonstrated in this study that RIP1 and RIP3 contributed to shikonin-induced glycolysis suppression via increasing intracellular H2O2.