Directly targeting ASC by lonidamine alleviates inflammasome-driven diseases.

BACKGROUND: Dysregulated activation of the inflammasome is involved in various human diseases including acute cerebral ischemia, multiple sclerosis and sepsis. Though many inflammasome inhibitors targeting NOD-like receptor protein 3 (NLRP3) have been designed and developed, none of the inhibitors are clinically available. Growing evidence suggests that targeting apoptosis-associated speck-like protein containing a CARD (ASC), the oligomerization of which is the key event for the assembly of inflammasome, may be another promising therapeutic strategy. Lonidamine (LND), a small-molecule inhibitor of glycolysis used as an antineoplastic drug, has been evidenced to have anti-inflammation effects. However, its anti-inflammatory mechanism is still largely unknown. METHODS: Middle cerebral artery occlusion (MCAO), experimental autoimmune encephalomyelitis (EAE) and LPS-induced sepsis mice models were constructed to investigate the therapeutic and anti-inflammasome effects of LND. The inhibition of inflammasome activation and ASC oligomerization by LND was evaluated using western blot (WB), immunofluorescence (IF), quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) in murine bone marrow-derived macrophages (BMDMs). Direct binding of LND with ASC was assessed using molecular mock docking, surface plasmon resonance (SPR), and drug affinity responsive target stability (DARTS). RESULTS: Here, we find that LND strongly attenuates the inflammatory injury in experimental models of inflammasome-associated diseases including autoimmune disease-multiple sclerosis (MS), ischemic stroke and sepsis. Moreover, LND blocks diverse types of inflammasome activation independent of its known targets including hexokinase 2 (HK2). We further reveal that LND directly binds to the inflammasome ligand ASC and inhibits its oligomerization. CONCLUSIONS: Taken together, our results identify LND as a broad-spectrum inflammasome inhibitor by directly targeting ASC, providing a novel candidate drug for the treatment of inflammasome-driven diseases in clinic.

TRIOL Inhibits Rapid Intracellular Acidification and Cerebral Ischemic Injury: The Role of Glutamate in Neuronal Metabolic Reprogramming.

As one of the key injury incidents, tissue acidosis in the brain occurs very quickly within several minutes upon the onset of ischemic stroke. Glutamate, an excitatory amino acid inducing neuronal excitotoxicity, has been reported to trigger the decrease in neuronal intracellular pH (pHi) via modulating proton-related membrane transporters. However, there remains a lack of clarity on the possible role of glutamate in neuronal acidosis via regulating metabolism. Here, we show that 200 µM glutamate treatment quickly promotes glycolysis and inhibits mitochondrial oxidative phosphorylation of primary cultured neurons within 15 min, leading to significant cytosolic lactate accumulation, which contributes to the rapid intracellular acidification and neuronal injury. The reprogramming of neuronal metabolism by glutamate is dependent on adenosine monophosphate-activated protein kinase (AMPK) signaling since the inhibition of AMPK activation by its selective inhibitor compound C significantly reverses these deleterious events in vitro. Moreover, 5α-androst-3ß,5α,6ß-TRIOL (TRIOL), a neuroprotectant we previously reported, can also remarkably reverse intracellular acidification and alleviate neuronal injury through the inhibition of AMPK signaling. Furthermore, TRIOL remarkably reduced the infarct volume and attenuated neurologic impairment in acute ischemic stroke models of middle cerebral artery occlusion in vivo. In summary, we reveal a novel role of glutamate in rapid intracellular acidification injury resulting from glutamate-induced lactate accumulation through AMPK-mediated neuronal reprogramming. Moreover, inhibition of the quick drop in neuronal pHi by TRIOL significantly reduces the cerebral damages, suggesting that it is a promising drug candidate for ischemic stroke.

The Regulatory NOD-Like Receptor NLRC5 Promotes Ganglion Cell Death in Ischemic Retinopathy by Inducing Microglial Pyroptosis.

Retinal ischemia is a common pathological event that can result in retinal ganglion cell (RGC) death and irreversible vision loss. The pathogenic mechanisms linking retinal ischemia to RGC loss and visual deficits are uncertain, which has greatly hampered the development of effective treatments. It is increasingly recognized that pyroptosis of microglia contributes to the indirect inflammatory death of RGCs. In this study, we report a regulatory NOD-like receptor, NOD-, LRR- and CARD-containing 5 (NLRC5), as a key regulator on microglial pyroptosis and the retinal ischemia process. Through an in-depth analysis of our recently published transcriptome data, we found that NLRC5 was significantly up-regulated in retina during ischemia-reperfusion injury, which were further confirmed by subsequent detection of mRNA and protein level. We further found that NLRC5 was upregulated in retinal microglia during ischemia, while NLRC5 knockdown significantly ameliorated retinal ischemic damage and RGC death. Mechanistically, we revealed that knockdown of NLRC5 markedly suppressed gasdermin D (GSDMD) cleavage and activation of interleukin-1ß (IL-1ß) and caspase-3, indicating that NLRC5 promotes both microglial pyroptosis and apoptosis. Notably, we found that NLRC5 directly bound to NLRP3 and NLRC4 in inflammasomes to cooperatively drive microglial pyroptosis and apoptosis mediating retinal ischemic damage. Overall, these findings reveal a previously unidentified key contribution of NLRC5 signaling to microglial pyroptosis under ischemia or hypoxia conditions. This NLRC5-dependent pathway may be a novel therapeutic target for treatment of ischemic retinopathy.

Author Correction: Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys.

Following the publication of our paper (Zhang et al., 2020), it has come to our attention that we erroneously listed two funding sources unrelated to this study in the "ACKNOWLEDGEMENTS" section. Hereby, we wish to update the "ACKNOWLEDGEMENTS" section as a correction.

Pentacyclic triterpene compounds from loquat leaves reduce skin inflammation and epidermal hyperplasia in psoriasis via inhibiting the Th17 cells.

Psoriasis is a refractory inflammatory skin disease affecting 2 %-3 % of the world population, characterized by the infiltration and hyper-proliferation of inflammatory cells and aberrant differentiation of keratinocytes. Targeting the IL-23/ Th17 axis has been well recognized as a promising therapeutic strategy, as the IL-23/ Th17 signal plays a vital role in the pathology of psoriasis. Three pentacyclic triterpene compounds isolated from loquat leaves have been reported with significant inhibitory effects on RORγt transcription activity and Th17 cell differentiation, and excellent performance in preventing lupus nephritis pathogenesis. However, the potential effects of these pentacyclic triterpene compounds on psoriasis remain unknown. In this study, we demonstrated the potent therapeutic effects of these pentacyclic triterpene compounds on psoriasis. These three pentacyclic triterpene compounds significantly alleviated skin inflammation as well as aberrant keratinocyte proliferation in an imiquimod-induced mouse psoriasis model. These compounds also inhibited the infiltration of immune cells and the level of pro-inflammatory cytokine in the dermis, as well as the cells number and changed the cytokine profiling expression of Th17 cells. These compounds could reduce the amount of CD4+ and CD8+ T cells in local lymph node, but not in spleen, which is different from hydrocortisone, the positive control treatment. These results suggest better performance of these compounds than steroids on treating psoriasis with less side effects on the integrated immune system. In summary, our findings uncover the potent therapeutic effects of pentacyclic triterpene compounds on psoriasis, providing potential candidate compounds for drug development.

SMARCB1 Promotes Ubiquitination and Degradation of NR4A3 via Direct Interaction Driven by ROS in Vascular Endothelial Cell Injury.

Nuclear receptor subfamily 4 group A member 3 (NR4A3) protects the vascular endothelial cell (VEC) against hypoxia stress, whose expression is primarily reported to be governed at a transcriptional level. However, the regulation of NR4A3 in the protein level is largely unknown. Here, we report that NR4A3 protein abundance is decreased immensely in VEC injury induced by reoxygenation after oxygen-glucose deprivation (OGD-R), which is significantly blocked by the administration of the antioxidative steroid TRIOL. Moreover, the notable improvement of NR4A3 and the alleviation of pulmonary endothelial barrier hyperpermeability induced by acute hypobaric hypoxia in cynomolgus monkeys are also observed after TRIOL administration. The overproduction of reactive oxygen species (ROS) decreases NR4A3 protein abundance in VEC under OGD-R condition, which is reversed by TRIOL and N-acetylcysteine (NAC). TRIOL dose-dependently increases the NR4A3 protein level by inhibiting ubiquitination and ubiquitin proteasome system- (UPS-) mediated degradation rather than promoting its transcription. Using yeast two-hybrid screening, we further identify the interaction between NR4A3 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), and the DNA-binding domain of NR4A3 is required for this interaction. Knockdown of SMARCB1 reduces ubiquitination and degradation of NR4A3, suggesting the proubiquitylation effect of this interaction which is enhanced by ROS in VEC injury induced by OGD-R. In summary, our study here for the first time reveals a posttranslational regulation in SMARCB1-mediated NR4A3 protein degradation which is driven by ROS, providing further understanding of the impaired regulation of NR4A3-mediated prosurvival pathways under pathological condition in VEC.

Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys.

Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily using non-primate animal models that are genetically distant to humans, thus hindering the development of disease treatment. Here, we report that cynomolgus monkeys ( Macacafascicularis) exposed to acute HH developed human-like HH syndrome involving severe brain injury and abnormal behavior. Transcriptome profiling of white blood cells and brain tissue from monkeys exposed to increasing altitude revealed the central role of the HIF-1 and other novel signaling pathways, such as the vitamin D receptor (VDR) signaling pathway, in co-regulating HH-induced inflammation processes. We also observed profound transcriptomic alterations in brains after exposure to acute HH, including the activation of angiogenesis and impairment of aerobic respiration and protein folding processes, which likely underlie the pathological effects of HH-induced brain injury. Administration of progesterone (PROG) and steroid neuroprotectant 5α-androst-3ß,5,6ß-triol (TRIOL) significantly attenuated brain injuries and rescued the transcriptomic changes induced by acute HH. Functional investigation of the affected genes suggested that these two neuroprotectants protect the brain by targeting different pathways, with PROG enhancing erythropoiesis and TRIOL suppressing glutamate-induced excitotoxicity. Thus, this study advances our understanding of the pathology induced by acute HH and provides potential compounds for the development of neuroprotectant drugs for therapeutic treatment.

Negative regulation of miR-1275 by H3K27me3 is critical for glial induction of glioblastoma cells.

Activation of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway induces glial differentiation of glioblastoma (GBM) cells, but the mechanism by which microRNA (miRNA) regulate this process remains poorly understood. In this study, by performing miRNA genomics and loss- and gain-of-function assays in dibutyryl-cAMP-treated GBM cells, we identified a critical negative regulator, hsa-miR-1275, that modulates a set of genes involved in cancer progression, stem cell maintenance, and cell maturation and differentiation. Additionally, we confirmed that miR-1275 directly and negatively regulates the protein expression of glial fibrillary acidic protein (GFAP), a marker of mature astrocytes. Of note, tri-methyl-histone H3 (Lys27) (H3K27me3), downstream of the PKA/polycomb repressive complex 2 (PRC2) pathway, accounts for the downregulation of miR-1275. Furthermore, decreased miR-1275 expression and induction of GFAP expression were also observed in dibutyryl-cAMP-treated primary cultured GBM cells. In a patient-derived glioma stem cell tumor model, a cAMP elevator and an inhibitor of H3K27me3 methyltransferase inhibited tumor growth, induced differentiation, and reduced expression of miR-1275. In summary, our study shows that epigenetic inhibition of miR-1275 by the cAMP/PKA/PRC2/H3K27me3 pathway mediates glial induction of GBM cells, providing a new mechanism and novel targets for differentiation-inducing therapy.

R406 elicits anti-Warburg effect via Syk-dependent and -independent mechanisms to trigger apoptosis in glioma stem cells.

Given that glioma stem cells (GSCs) play a critical role in the initiation and chemoresistance in glioblastoma multiforme (GBM), targeting GSCs is an attractive strategy to treat GBM. Utilizing an anti-cancer compound library, we identified R406, the active metabolite of a FDA-approved Syk inhibitor for immune thrombocytopenia (ITP), with remarkable cytotoxicity against GSCs but not normal neural stem cells. R406 significantly inhibited neurosphere formation and triggered apoptosis in GSCs. R406 induced a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) and subsequently production of excess ROS in GSCs. R406 also diminished tumor growth and efficiently sensitized gliomas to temozolomide in GSC-initiating xenograft mouse models. Mechanistically, the anti-GSC effect of R406 was due to the disruption of Syk/PI3K signaling in Syk-positive GSCs and PI3K/Akt pathway in Syk-negative GSCs respectively. Overall, these findings not only identify R406 as a promising GSC-targeting agent but also reveal the important role of Syk and PI3K pathways in the regulation of energy metabolism in GSCs.

Marine-Steroid Derivative 5α-Androst-3ß, 5α, 6ß-triol Protects Retinal Ganglion Cells from Ischemia⁻Reperfusion Injury by Activating Nrf2 Pathway.

High intraocular pressure (IOP)-induced retinal ischemia leads to acute glaucoma, which is one of the leading causes of irreversible visual-field loss, characterized by loss of retinal ganglion cells (RGCs) and axonal injury in optic nerves (ONs). Oxidative stress and the inflammatory response play an important role in the ischemic injury of retinal and optic nerves. We focus on 5α-androst-3ß, 5α, 6ß-triol (TRIOL), a synthetic neuroactive derivative of natural marine steroids 24-methylene-cholest-3ß, 5α, 6ß, 19-tetrol and cholestane-3ß, 5α, 6ß-triol, which are two neuroactive polyhydroxysterols isolated from the soft coral Nephthea brassica and the gorgonian Menella kanisa, respectively. We previously demonstrated that TRIOL was a neuroprotective steroid with anti-inflammatory and antioxidative activities. However, the potential role of TRIOL on acute glaucoma and its underlying mechanisms remains unclear. Here, we report TRIOL as a promising neuroprotectant that can protect RGCs and their axons/dendrites from ischemic-reperfusion (I/R) injury in an acute intraocular hypertension (AIH) model. Intravitreal injection of TRIOL significantly alleviated the loss of RGCs and the damage of axons and dendrites in rats and mice with acute glaucoma. As NF-E2-related factor 2 (Nrf2) is one of the most critical regulators in oxidative and inflammatory injury, we further evaluated the effect of TRIOL on Nrf2 knockout mice, and the neuroprotective role of TRIOL on retinal ischemia was not observed in Nrf2 knockout mice, indicating that activation of Nrf2 is responsible for the neuroprotection of TRIOL. Further experiments demonstrated that TRIOL can activate and upregulate Nrf2, along with its downstream hemeoxygenase-1 (HO-1), by negative regulation of Kelch-like ECH (Enoyl-CoA Hydratase) associated Protein-1 (Keap1). In conclusion, our study shed new light on the neuroprotective therapy of retinal ischemia and proposed a promising marine drug candidate, TRIOL, for the therapeutics of acute glaucoma.

A Novel Synthetic Steroid of 2ß,3α,5α-Trihydroxy-androst-6-one Alleviates the Loss of Rat Retinal Ganglion Cells Caused by Acute Intraocular Hypertension via Inhibiting the Inflammatory Activation of Microglia.

Neuroinflammation has been well recognized as a key pathological event in acute glaucoma. The medical therapy of acute glaucoma mainly focuses on lowering intraocular pressure (IOP), while there are still scarce anti-inflammatory agents in the clinical treatment of acute glaucoma. Here we reported that ß,3α,5α-trihydroxy-androst-6-one (sterone), a novel synthetic polyhydric steroid, blocked neuroinflammation mediated by microglia/macrophages and alleviated the loss of retinal ganglion cells (RGCs) caused by acute intraocular hypertension (AIH). The results showed that sterone significantly inhibited the morphological changes, the up-regulation of inflammatory biomarker ionized calcium-binding adapter molecule 1 (Iba-1), and the mRNA increase of proinflammatory tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) in BV2 microglia and RAW264.7 macrophages. Moreover, immunofluorescence and western blotting analysis revealed that sterone markedly abrogated the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB) p65 subunit. Furthermore, sterone significantly suppressed the inflammatory microglial activation and RGCs' reduction caused by retinal ischemia/reperfusion (I/R) injury in a rat AIH model. These results suggest sterone may be a potential candidate in the treatment of acute glaucoma caused by microglial activation-mediated neuroinflammatory injury.

Hexokinase 2-dependent hyperglycolysis driving microglial activation contributes to ischemic brain injury.

Hyperglycolysis, observed within the penumbra zone during brain ischemia, was shown to be detrimental for tissue survival because of lactate accumulation and reactive oxygen species overproduction in clinical and experimental settings. Recently, mounting evidence suggests that glycolytic reprogramming and induced metabolic enzymes can fuel the activation of peripheral immune cells. However, the possible roles and details regarding hyperglycolysis in neuroinflammation during ischemia are relatively poorly understood. Here, we investigated whether overactivated glycolysis could activate microglia and identified the crucial regulators of neuroinflammatory responses in vitro and in vivo. Using BV 2 and primary microglial cultures, we found hyperglycolysis and induction of the key glycolytic enzyme hexokinase 2 (HK2) were essential for microglia-mediated neuroinflammation under hypoxia. Mechanistically, HK2 up-regulation led to accumulated acetyl-coenzyme A, which accounted for the subsequent histone acetylation and transcriptional activation of interleukin (IL)-1ß. The inhibition and selective knockdown of HK2 in vivo significantly protected against ischemic brain injury by suppressing microglial activation and IL-1ß production in male Sprague-Dawley rats subjected to transient middle cerebral artery occlusion (MCAo) surgery. We provide novel insights for HK2 specifically serving as a neuroinflammatory determinant, thus explaining the neurotoxic effect of hyperglycolysis and indicating the possibility of selectively targeting HK2 as a therapeutic strategy in acute ischemic stroke.

Neuroprotectant androst-3ß, 5α, 6ß-triol suppresses TNF-α-induced endothelial adhesion molecules expression and neutrophil adhesion to endothelial cells by attenuation of CYLD-NF-κB pathway.

Neuroinflammation is one of key pathologic element in neurological diseases including stroke, traumatic brain injury, Alzheimer' s Disease, Parkinson's Disease, and multiple sclerosis as well. Up-regulation of endothelial adhesion molecules, which facilitate leukocyte adhesion to the endothelium, is the vital process of endothelial cells mediated neuroinflammation. Androst-3ß, 5α, 6ß-triol (Triol) is a synthetic steroid which has been reported to have neuroprotective effects in hypoxia/re-oxygenation-induced neuronal injury model. In the present study, we firstly investigated whether Triol inhibited the TNF-α-induced inflammatory response in rat brain microvascular endothelial cells (RBMECs). Our data showed that Triol decreased TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and the adhesion of neutrophil to RBMECs. We also found that Triol inhibited TNF-α-induced degradation of IκBα and phosphorylation of NF-κBp65 that are required for NF-κB activation. Furthermore, Triol significantly reversed TNF-α-induced down-expression of CYLD, which is a deubiquitinase that negatively regulates activation of NF-κB. These results suggest that Triol displays an anti-inflammatory effect on TNF-α-induced RBMECs via downregulating of CYLD-NF-κB signaling pathways and might have a potential benefit in therapeutic neuroinflammation related diseases.

The Anti-Warburg Effect Elicited by the cAMP-PGC1α Pathway Drives Differentiation of Glioblastoma Cells into Astrocytes.

Glioblastoma multiforme (GBM) is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP) reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells.

Naturally Existing Oncolytic Virus M1 Is Nonpathogenic for the Nonhuman Primates After Multiple Rounds of Repeated Intravenous Injections.

Cancers figure among the leading causes of morbidity and mortality worldwide. The number of new cases is expected to rise by about 70% over the next 2 decades. Development of novel therapeutic agents is urgently needed for clinical cancer therapy. Alphavirus M1 is a Getah-like virus isolated from China with a genome of positive single-strand RNA. We have previously identified that alphavirus M1 is a naturally existing oncolytic virus with significant anticancer activity against different kinds of cancer (e.g., liver cancer, bladder cancer, and colon cancer). To support the incoming clinical trial of intravenous administration of alphavirus M1 to cancer patients, we assessed the safety of M1 in adult nonhuman primates. We previously presented the genome sequencing data of the cynomolgus macaques (Macaca fascicularis), which was demonstrated as an ideal animal species for virus infection study. Therefore, we chose cynomolgus macaques of either sex for the present safety study of oncolytic virus M1. In the first round of administration, five experimental macaques were intravenously injected with six times of oncolytic virus M1 (1 × 10(9) pfu/dose) in 1 week, compared with five vehicle-injected control animals. The last two rounds of injections were further completed in the following months in the same way as the first round. Body weight, temperature, complete blood count, clinical biochemistries, cytokine profiles, lymphocytes subsets, neutralizing antibody, and clinical symptoms were closely monitored at different time points. Magnetic resonance imaging was also performed to assess the possibility of encephalitis or arthritis. As a result, no clinical, biochemical, immunological, or medical imaging or other pathological evidence of toxicity was found during the whole process of the study. Our results in cynomolgus macaques suggested the safety of intravenous administration of oncolytic virus M1 in cancer patients in the future.