The brain protection of MLKL inhibitor necrosulfonamide against focal ischemia/reperfusion injury associating with blocking the nucleus and nuclear envelope translocation of MLKL and RIP3K.

Mixed lineage kinase like protein (MLKL) is a key mediator of necroptosis. While previous studies highlighted the important role of MLKL as one of the central regulators of brain damage against acute ischemic neuronal injury, how the activation of MLKL mediates brain injuries and cell death remains unclear, especially in astrocytes. In a transient middle cerebral artery occlusion (tMCAO) rat model in vivo, and an oxygen-glucose deprivation and reoxygenation (OGD/Re) injury model in both primary cultured astrocytes and human astrocytes, we show that necrosulfonamide (NSA), a MLKL specific inhibitor, reduces infarction volume and improves neurological deficits in tMCAO-treated rats. In addition, NSA treatment, as well as RIP1K inhibitor Nec-1 or RIP3K inhibitor GSK-872 treatment, decreases the OGD/Re-induced leakage of LDH in both primary cultured astrocytes and human astrocytes. NSA treatment also reduces the number of propidium iodide (PI)-positive cells, and prevents the upregulation of necroptotic biomarkers such as MLKL/p-MLKL, RIP3K/p-RIP3K, and RIP1K/p-RIP1K in ischemic penumbra of cerebral cortex in tMCAO-treated rats or in OGD/Re-treated human astrocytes. Importantly, NSA treatment blocks both the nucleus and nuclear envelope localization of MLKL/p-MLKL and RIP3K/p-RIP3K in ischemic cerebral cortex induced by tMCAO. Similarly, Co-immunoprecipitation assay shows that NSA treatment decreases tMCAO- or OGD/Re- induced increased combination of MLKL and RIP3K in nuclear envelope of ischemic penumbra of cerebral cortex or of primary cultured astrocytes, respectively. RIP3K inhibitor GSK-872 also reduces tMCAO-induced increased combination of MLKL and RIP3K in nuclear envelope of ischemic penumbra of cerebral cortex. These data suggest NSA exerts protective effects against focal ischemia/reperfusion injury via inhibiting astrocytic necroptosis through preventing the upregulation of necroptotic kinases as well as blocking both the nucleus and nuclear envelope co-localization of p-MLKL and p-RIP3K. The translocation of p-MLKL, along with p-RIP3K, to the nuclear envelope and the nucleus may play a crucial role in MLKL-mediated necroptosis under ischemic conditions.

RIPK1 inhibition contributes to lysosomal membrane stabilization in ischemic astrocytes via a lysosomal Hsp70.1B-dependent mechanism.

Receptor-interacting protein kinase 1 (RIPK1) contributes to necroptosis. Our previous study showed that pharmacological or genetic inhibition of RIPK1 protects against ischemic stroke-induced astrocyte injury. In this study, we investigated the molecular mechanisms underlying RIPK1-mediated astrocyte injury in vitro and in vivo. Primary cultured astrocytes were transfected with lentiviruses and then subjected to oxygen and glucose deprivation (OGD). In a rat model of permanent middle cerebral artery occlusion (pMCAO), lentiviruses carrying shRNA targeting RIPK1 or shRNA targeting heat shock protein 70.1B (Hsp70.1B) were injected into the lateral ventricles 5 days before pMCAO was established. We showed that RIPK1 knockdown protected against OGD-induced astrocyte damage, blocked the OGD-mediated increase in lysosomal membrane permeability in astrocytes, and inhibited the pMCAO-induced increase in astrocyte lysosome numbers in the ischemic cerebral cortex; these results suggested that RIPK1 contributed to the lysosomal injury in ischemic astrocytes. We revealed that RIPK1 knockdown upregulated the protein levels of Hsp70.1B and increased the colocalization of Lamp1 and Hsp70.1B in ischemic astrocytes. Hsp70.1B knockdown exacerbated pMCAO-induced brain injury, decreased lysosomal membrane integrity and blocked the protective effects of the RIPK1-specific inhibitor necrostatin-1 on lysosomal membranes. On the other hand, RIPK1 knockdown further exacerbated the pMCAO- or OGD-induced decreases in the levels of Hsp90 and the binding of Hsp90 to heat shock transcription factor-1 (Hsf1) in the cytoplasm, and RIPK1 knockdown promoted the nuclear translocation of Hsf1 in ischemic astrocytes, resulting in increased Hsp70.1B mRNA expression. These results suggest that inhibition of RIPK1 protects ischemic astrocytes by stabilizing lysosomal membranes via the upregulation of lysosomal Hsp70.1B; the mechanism underlying these effects involves decreased Hsp90 protein levels, increased Hsf1 nuclear translocation and increased Hsp70.1B mRNA expression.

Nickel/Copper Cooperative Catalysis Decarbonylative Heteroarylation of Aryl Anhydrides with Benzoxazoles via C-O/C-H Coupling.

A new strategy for the synthesis of 2-arylbenzoxazole derivatives via nickel-/copper-catalyzed decarbonylative heteroarylation of aryl anhydrides via C-O/C-H coupling has been developed. The reaction is promoted by a user-friendly, inexpensive, and air- and moisture-stable Ni precatalyst. A variety of 2-arylbenzoxazole derivatives have been successfully synthesized and have good functional group tolerance in this process, which afforded products in moderate-to-excellent yields.

Valproic Acid Inhibits Glial Scar Formation after Ischemic Stroke.

INTRODUCTION: Cerebral ischemia induces reactive proliferation of astrocytes (astrogliosis) and glial scar formation. As a physical and biochemical barrier, the glial scar not only hinders spontaneous axonal regeneration and neuronal repair but also deteriorates the neuroinflammation in the recovery phase of ischemic stroke. OBJECTIVES: Previous studies have shown the neuroprotective effects of the valproic acid (2-n-propylpentanoic acid, VPA) against ischemic stroke, but its effects on the ischemia-induced formation of astrogliosis and glial scar are still unknown. As targeting astrogliosis has become a therapeutic strategy for ischemic stroke, this study was designed to determine whether VPA can inhibit the ischemic stroke-induced glial scar formation and to explore its molecular mechanisms. METHODS: Glial scar formation was induced by an ischemia-reperfusion (I/R) model in vivo and an oxygen and glucose deprivation (OGD)-reoxygenation (OGD/Re) model in vitro. Animals were treated with an intraperitoneal injection of VPA (250 mg/kg/day) for 28 days, and the ischemic stroke-related behaviors were assessed. RESULTS: Four weeks of VPA treatment could markedly reduce the brain atrophy volume and improve the behavioral deficits in rats' I/R injury model. The results showed that VPA administrated upon reperfusion or 1 day post-reperfusion could also decrease the expression of the glial scar makers such as glial fibrillary acidic protein, neurocan, and phosphacan in the peri-infarct region after I/R. Consistent with the in vivo data, VPA treatment showed a protective effect against OGD/Re-induced astrocytic cell death in the in vitro model and also decreased the expression of GFAP, neurocan, and phosphacan. Further studies revealed that VPA significantly upregulated the expression of acetylated histone 3, acetylated histone 4, and heat-shock protein 70.1B in the OGD/Re-induced glial scar formation model. CONCLUSION: VPA produces neuroprotective effects and inhibits the glial scar formation during the recovery period of ischemic stroke via inhibition of histone deacetylase and induction of Hsp70.1B.

Necroptotic kinases are involved in the reduction of depression-induced astrocytes and fluoxetine's inhibitory effects on necroptotic kinases.

The role of astrocytes in major depressive disorder has received great attention. Increasing evidence indicates that decreased astrocyte numbers in the hippocampus may be associated with depression, but the role of necroptosis in depression is unknown. Here, in a chronic unpredictable mild stress (CUMS) mouse model and a corticosterone (Cort)-induced human astrocyte injury model in vitro, we found that mice treated with chronic unpredictable mild stress for 3-5 weeks presented depressive-like behaviors and reduced body weight gain, accompanied by a reduction in astrocytes and a decrease in astrocytic brain-derived neurotropic factors (BDNF), by activation of necroptotic kinases, including RIPK1 (receptor-interacting protein kinase 1)/p-RIPK1, RIPK3 (receptor-interacting protein kinase 3)/p-RIPK3 and MLKL (mixed lineage kinase domain-like protein)/p-MLKL, and by upregulation of inflammatory cytokines in astrocytes of the mouse hippocampus. In contrast, necroptotic kinase inhibitors suppressed Cort-induced necroptotic kinase activation, reduced astrocytes, astrocytic necroptosis and dysfunction, and decreased Cort-mediated inflammatory cytokines in astrocytes. Treatment with fluoxetine (FLX) for 5 weeks improved chronic unpredictable mild stress-induced mouse depressive-like behaviors; simultaneously, fluoxetine inhibited depression-induced necroptotic kinase activation, reversed the reduction in astrocytes and astrocytic necroptosis and dysfunction, decreased inflammatory cytokines and upregulated brain-derived neurotropic factors and 5-HT1A levels. Furthermore, fluoxetine had no direct inhibitory effect on receptor-interacting protein kinase 1 phosphorylation. The combined administration of fluoxetine and necroptotic kinase inhibitors further reduced corticosterone-induced astrocyte injury. In conclusion, the reduction in astrocytes caused by depressive-like models in vivo and in vitro may be associated with the activation of necroptotic kinases and astrocytic necroptosis, and fluoxetine exerts an antidepressive effect by indirectly inhibiting receptor-interacting protein kinase 1-mediated astrocytic necroptosis.

Nickel-Catalyzed Selective Decarbonylation of α-Amino Acid Thioester: Aminomethylation of Mercaptans.

The nickel-catalyzed aminomethylation of mercaptans has been disclosed that offers efficient and expedient access to synthesize α-aminosulfides. The intramolecular fragment coupling shows excellent chemoselectivity. This transformation shows good functional-group compatibility, tolerates a wide range of electron-withdrawing, electron-neutral, and electron-donating substituents in this process, and can serve as a powerful synthetic tool for the synthesis of α-aminosulfides at a gram scale. Thus, the newly developed methodology enables a facile route for C-S bond formation in a straightforward fashion.

The Key Regulator of Necroptosis, RIP1 Kinase, Contributes to the Formation of Astrogliosis and Glial Scar in Ischemic Stroke.

Necroptosis initiation relies on the receptor-interacting protein 1 kinase (RIP1K). We recently reported that genetic and pharmacological inhibition of RIP1K produces protection against ischemic stroke-induced astrocytic injury. However, the role of RIP1K in ischemic stroke-induced formation of astrogliosis and glial scar remains unknown. Here, in a transient middle cerebral artery occlusion (tMCAO) rat model and an oxygen and glucose deprivation and reoxygenation (OGD/Re)-induced astrocytic injury model, we show that RIP1K was significantly elevated in the reactive astrocytes. Knockdown of RIP1K or delayed administration of RIP1K inhibitor Nec-1 down-regulated the glial scar markers, improved ischemic stroke-induced necrotic morphology and neurologic deficits, and reduced the volume of brain atrophy. Moreover, knockdown of RIP1K attenuated astrocytic cell death and proliferation and promoted neuronal axonal generation in a neuron and astrocyte co-culture system. Both vascular endothelial growth factor D (VEGF-D) and its receptor VEGFR-3 were elevated in the reactive astrocytes; simultaneously, VEGF-D was increased in the medium of astrocytes exposed to OGD/Re. Knockdown of RIP1K down-regulated VEGF-D gene and protein levels in the reactive astrocytes. Treatment with 400 ng/ml recombinant VEGF-D induced the formation of glial scar; conversely, the inhibitor of VEGFR-3 suppressed OGD/Re-induced glial scar formation. RIP3K and MLKL may be involved in glial scar formation. Taken together, these results suggest that RIP1K participates in the formation of astrogliosis and glial scar via impairment of normal astrocyte responses and enhancing the astrocytic VEGF-D/VEGFR-3 signaling pathways. Inhibition of RIP1K promotes the brain functional recovery partially via suppressing the formation of astrogliosis and glial scar.

Chemical structure and antioxidant activity of a polysaccharide from Siraitia grosvenorii.

A novel polysaccharide from Siraitia grosvenorii residues (SGP, molecular weight 1.93 × 103 KDa) was isolated and purified. SGP was composed of α-L-Arabinose, α-D-Mannose, α-d-Glucose, α-D-Galactose, Glucuronic acid, and Galacturonic acid with the ratio of 1: 1.92: 3.98: 7.63: 1.85: 7.34. The backbone of SGP was consist of galactoses and linked by α-(1,4)-glycosidic bond. The branch chains including α-1,6 linked glucose branch, α-1,6 linked mannose branch, α-1,3 linked galactose branch and arabinose branched (α-L-Ara(1→). The results of bioactivity experiments suggested that SGP had antioxidant in vitro, especially on scavenging DPPH radicals. Besides, SGP resulted in the decrease of ROS and the percentage of apoptotic and necrotic cells in a dose-dependent manner in H2O2 oxide injury PC12 cells. This research could help to develop the potential value and utilization of Siraitia grosvenorii.

Synthesis of Biaryls via Decarbonylative Nickel-Catalyzed Suzuki-Miyaura Cross-Coupling of Aryl Anhydrides.

Transition metal-catalyzed cross-couplings have been widely employed in the synthesis of many important molecules in synthetic chemistry for the construction of diverse C-C bonds. Conventional cross-coupling reactions require active electrophilic coupling partners, such as organohalides or sulfonates, which are not environmentally friendly enough. Herein, we disclose the first nickel-catalyzed Suzuki-Miyaura cross-coupling of aryl anhydrides and arylboronic acids for the synthesis of biaryls in a decarbonylation manner. The reaction tolerates a wide range of electron-withdrawing, electron-neutral, and electron-donating substituents in this process.

Inhibition of GSK3ß and RIP1K Attenuates Glial Scar Formation Induced by Ischemic Stroke via Reduction of Inflammatory Cytokine Production.

In the chronic phase following ischemic stroke, glial scars can prevent axonal regeneration and the intensification of inflammation. The protective effect of inhibition of glycogen synthase kinase-3ß (GSK3ß) or receptor-interacting protein 1 kinase (RIP1K) on ischemic stroke has been previously reported. The current study examined the effects of RIP1K and GSK3ß on ischemic stroke-induced glial scar formation. To investigate this, we used an in vivo model of ischemic stroke based on middle cerebral artery occlusion for 90 min followed by reperfusion for 7 d, and an in vitro model in primary cultured astrocytes involving oxygen and glucose deprivation for 6 h followed by reoxygenation for 24 h. Both in vivo and in vitro, we found that SB216763, a GSK3ß inhibitor, and necrostatin-1 (Nec-1), a RIP1K inhibitor, decreased levels of glial scar markers, including glial fibrillary acidic protein (GFAP), neurocan, and phosphacan. SB216763 and Nec-1 also decreased levels of inflammatory related cytokines, including interleukin-6 (IL-6), interleukin-1 ß (IL-1ß), and tumor necrosis factor-α (TNF-α). However, only Nec-1 increased the level of interleukin-1 receptor antagonist. Concurrent neutralization of TNF-α, IL-1ß, and IL-6 with their antibodies provided better reduction in oxygen and glucose deprivation-induced increases in scar markers than obtained with separate use of each antibody. Further investigations showed that SB216763 reduced the levels of necroptosis-related proteins, including RIP1K, p-RIP1K, RIP3K, p-RIP3K, mixed lineage kinase domain-like protein (MLKL), and p-MLKL, while Nec-1 decreased the expression of p-GSK3ß. Compared with Nec-1 (10 µM) and SB216763 (1 µM) alone, Nec-1 and SB216763 in combination reduced levels of GFAP, neurocan, and inflammatory-related cytokines. In conclusion, inhibition of GSK3ß or RIP1K reduced glial scar formation induced by ischemic stroke. The underlying mechanisms might be at least, partially related to reducing levels of inflammatory-related cytokines and to blocking an interaction between GSK3ß- and RIP1K-mediated pathways.

Analysis the effect of miR-141-3p/HMGB1 in LPS-induced mucus production and the apoptosis in nasal epithelial cells.

Allergic rhinitis (AR) is an allergic disease characterized by immunoglobulin E (IgE)-mediated type I hypersensitivity disorder. In the current study, we illuminated the potential roles of microRNA-141-3p (miR-141-3p) in lipopolysaccharide (LPS)-induced mucus production and the apoptosis in nasal epithelial cells (NECs). We demonstrated that miR-141-3p was significantly downregulated in nasal tissues from patients with AR and LPS-treated NECs. Upregulation of miR-141-3p decreased the level of mucin 5AC (MUC5AC) in LPS-treated NECs and induced NECs apoptosis. High Mobility Group Box 1 (HMGB1) was proved as a target of miR-141-3p and miR-141-3p negatively regulated its expression. In addition, we observed that HMGB1 was overexpressed in nasal mucosal tissues from patients with AR and LPS-treated NECs. Finally, we proved that miR-141-3p decreased the level of MUAC5AC in LPS-treated NECs through regulating HMGB1. In conclusion, miR-141-3p inhibited LPS-induced MUAC5AC production and the apoptosis of LPS-treated NECs by targeting HMGB1.

Chemical structure and effects of antioxidation and against α-glucosidase of natural polysaccharide from Glycyrrhiza inflata Batalin.

GIBP, a homogeneous polysaccharide extracted from Glycyrrhiza inflata Batalin with a molecular weight 1.96 × 103 kDa, had a triple helix structure, smooth and sheet-like structure. Comprehensive analysis showed that the main chain of GIBP was composed of α-D-1,4 linked glucose, branch points were composed of α-D-1,3,6 and α-D-1,2,3,6 linked glucoses, and side chains were composed of α-D-1,3 and ß-D-1,6 linked galactose, ß-L-1,2 linked arabinose, α-D-1,3 and ß-D-1,3 mannose. The scavenging abilities of GIBP (3 mg/mL) against DPPH radical, OH radical, O2- radical and ABTS were 50.75 ± 0.13% and 52.32 ± 0.13, 25.84 ± 0.35% and 44.57 ± 0.15% and it also demonstrated an obvious dose-effect relationship. The inhibitory activity of α-glucosidase showed that the inhibitory effect of GIBP was enhanced with the increase of concentration. When the concentration reached 6 mg/mL, the inhibition rate of α-glucosidase activity reached 64.77%. And the ka, kd and KD were 6.472 × 104 1/Ms., 2.934 × 10-3 1/s and 4.534 × 10-8 M.

Coordination of Autophagy and Other Cellular Activities.

Conventionally, autophagy (=self-eating) is thought to be a catabolic cellular process that is responsible for regulating cell homeostasis. However, the newly evidence have expanded the range of the impact of autophagy in biology. Autophagy interplays with endocytosis through shared factors such as phosphatidylinositol 3 kinase complex (PI(3)K complex), autophagy associated gene (Atg), and lysosome. Autophagy and phagocytosis orchestrate in maintaining homeostasis, in MHC class II antigen processing, in the removal of pathogens, in cell death, immunity, and inflammation. There are numerous cross talks of autophagy with biosynthetic processes such as conventional and unconventional secretion of biologically active cargo and trafficking of integral membrane proteins, as well as the exosome secretion. There are also links between autophagy and trafficking events from plasma membrane, including lateral plasma membrane proteins connexins, cell connections, and ciliogenesis.

Forging C-S Bonds through Nickel-Catalyzed Aryl Anhydrides with Thiophenols: Decarbonylation or Decarbonylation Accompanied by Decarboxylation.

A nickel-catalyzed decarbonylation or decarbonylation accompanied by decarboxylation cross-coupling reaction of aryl anhydrides with thiophenols as coupling partners was disclosed. This method is promoted by a commercially available, moisture-stable, and inexpensive nickel(II) precatalyst. The process can tolerate a variety of functional groups using ubiquitous aryl anhydrides as cross-coupling precursors to produce thioethers in moderate to excellent yields.

One-Pot Synthesis of N-Imidoyl-(1 H)-indoles via Palladium-Catalyzed Oxidative Insertion Domino Reaction with Isocyanide and Arylboronic Acid.

Efficient one-pot synthesis of N-imidoyl-(1 H)-indoles has been described, which is achieved by the palladium-catalyzed oxidative insertion of 2-(phenylethynyl)aniline, arylboronic acid, and isonitrile. This method provides a new way to synthesize N-imidoyl-(1 H)-indoles, which has a wide substrate scope, good functional group tolerance, and mild reaction condition.

CID1067700, a late endosome GTPase Rab7 receptor antagonist, attenuates brain atrophy, improves neurologic deficits and inhibits reactive astrogliosis in rat ischemic stroke.

Increasing evidence suggests that Ras-related in brain 7 (Rab7), an endosome-localized small GTPase contributes to cerebral ischemic brain injury. In the present study, we investigated the role of Rab7 in ischemic stroke-induced formation of astrogliosis and glial scar. Rats were subjected to transient middle cerebral artery occlusion (tMCAO); the rats were injected with the Rab7 receptor antagonist CID1067700 (CID). Primary astrocytes were subjected to an oxygen and glucose deprivation and reoxygenation (OGD/Re) procedure; CID was added to the cell culture media. We found that Rab7 was significantly elevated over time in both the in vivo and in vitro astrocytic injury models, and administration of CID significantly down-regulated the glial scar markers such as glial fibillary acidic protein (GFAP), neurocan and phosphacan. Moreover, administration of CID significantly attenuated the brain atrophy and improved neurologic deficits in tMCAO rats, and protected astrocytes against OGD/Re-induced injury. Further, CID downregulated the protein levels of Lamp1 and active cathepsin B in astrocytes after OGD/Re or tMCAO injury; CID inhibited the co-localization of cathepsin B and Rab7, Lamp1 and Rab7; CID decreased OGD/Re-induced increase in lysosomal membrane permeability and blocked OGD/Re-induced release of cathepsin B from the lysosome into the cytoplasm in astrocytes. Taken together, these results suggest that Rab7 is involved in ischemic stroke-induced formation of astrogliosis and glial scar. CID administration attenuates brain atrophy and improves neurologic deficits and inhibits astrogliosis and glial scar formation after ischemic stroke via reducing the activation and release of cathepsin B from the lysosome into the cytoplasm.

Neuroprotective Effects of DTIO, A Novel Analog of Nec-1, in Acute and Chronic Stages After Ischemic Stroke.

Receptor-interacting protein 1 kinase (RIP1K) plays a key role in necroptosis. Necrostatin-1 (Nec-1), a specific inhibitor of RIP1K, provides neuroprotection against ischemic brain injury, associating with inhibition of inflammation. Recently, our group synthesized a novel analog of Nec-1, 5-(3',5'-dimethoxybenzal)-2-thio-imidazole-4-ketone (DTIO). The present study investigated the effect of DTIO on ischemic stroke-induced brain injury in both acute and chronic phase and its underlying mechanism. In vivo, DTIO treatment reduced infarct volume and improved neurological deficits in the acute phase after permanent middle cerebral artery occlusion (pMCAO) and it also attenuated brain atrophy and promoted brain functional recovery in the chronic phase post-cerebral ischemia/reperfusion (I/R). In vitro, DTIO treatment decreased lactate dehydrogenase (LDH) leakage and necrotic cell death in the oxygen and glucose deprivation (OGD) or oxygen and glucose deprivation and reoxygenation (OGD/R)-induced neuronal or astrocytic cell injury. Simultaneously, DTIO suppressed the production and release of inflammatory cytokines, and reduced the formation of glial scar. Homology modeling analysis illustrated that DTIO had an ability of binding to RIP1K. Furthermore, immunoprecipitation analysis showed that DTIO inhibited the phosphorylation of RIP1K and decreased the interaction between the RIP1K and RIP3K. In addition, knockdown of RIP1K had neuroprotective effects and inhibited the release of proinflammatory cytokines, but didn't have a significant effect on DTIO-mediated neuroprotection. In conclusion, DTIO has protective effects on acute ischemic stroke and promotes functional recovery during chronic phase, associating with protecting ischemic neurons and astrocytes, inhibiting inflammation, and lessening the glial scar formation via inhibiting of the RIP1K.

Palladium-Catalyzed Oxidative Three-Component Coupling of Anthranilamides with Isocyanides and Arylboronic Acids: Access to 2,3-Disubstituted Quinazolinones.

A novel palladium-catalyzed oxidative three-component coupling of easily accessible N-substituted anthranilamides with isocyanides and arylboronic acids is achieved. This protocol offers an alternative approach toward 2,3-disubstituted quinazolinones with a wide substrate scope and good functional group tolerance.

Cu-Mediated Stereoselective [4+2] Annulation between N-Hydroxybenzimidoyl Cyanide and Norbornene.

A Cu-mediated stereoselective [4+2] annulation between N-hydroxybenzimidoyl cyanides and norbornene (NBE) has been developed for the synthesis of 4 H-1,2-oxazin-4-ones. The reaction proceeds through sequentially forming C-O/C-C bonds. The advantage of this reaction includes high stereoselectivity, excellent yields, as well as simple and mild reaction conditions. A total of 26 examples are presented along with some control experiments.

RIP1K Contributes to Neuronal and Astrocytic Cell Death in Ischemic Stroke via Activating Autophagic-lysosomal Pathway.

Although the receptor-interacting protein 1 kinase (RIP1K)-regulated necroptosis can be evoked by cerebral ischemia, the effects of RIP1K in mediating neuronal and astrocytic cell death and the underlying mechanisms remain poorly understood. This study evaluates the contribution of RIP1K to ischemic stroke-induced neuronal and astrocytic cell death, and the activation of autophagic-lysosomal pathway. Using an in vitro oxygen and glucose deprivation (OGD) in primary cultured neurons or astrocytes and a permanent middle cerebral artery occlusion (pMCAO) model in rats or mice, we observed the role of RIP1K in the ischemic neuronal and astrocytic cell death and the underlying mechanisms by pharmacological or genetic inhibition of RIP1K. pMCAO or OGD condition led to an increase in RIP1K, RIP3K and RIP1K-RIP3K complex. RIP1K knockdown or necrostatin-1 (Nec-1, a specific inhibitor of RIP1K) treatment reduced infarct volume, improved neurological deficits, increased microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) levels, and attenuated neuronal or astrocytic necrotic cell death in the ischemic cortex. RIP1K knockdown decreased RIP1K-RIP3K complex formation, light chain 3 II (LC3II) and active cathepsin B levels and lysosomal membrane permeability (LMP). Furthermore, a combination of Nec-1 and an inhibitor of autophagy or cathepsin B produced an enhancement of protective effect on neuronal or astrocytic cell death. RIP1K-mediated necroptosis may play important roles in ischemia-induced neuronal and astrocytic cell death through the activation of autophagic-lysosomal pathway.