Brazilin inhibits bladder cancer by promoting cell necroptosis.

Brazilin possesses anticancer effects, but the mechanisms are poorly understood. This study investigated the mechanisms of brazilin-induced cell death in the T24 human bladder cancer cell line. Low serum cell culture and the lactate dehydrogenase assay were used to confirm the antitumor effect of brazilin. Annexin V and propidium iodide double staining, transmission electron microscopy, fluo-3-AM assay for Ca2+ mobilization and caspase activity assay were performed to identify the type of cell death after brazilin treatment. Mitochondria membrane potentials were measured using JC-1. Quantitative real-time polymerase chain reaction and western blot analyses were performed to verify the expression of the necroptosis-related genes and proteins receptor interacting protein 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL). The results showed that brazilin induced necrosis in T24 cells and upregulated the mRNA and protein levels of RIP1, RIP3 and MLKL and Ca2+ influx. The necroptosis-mediated cell death was rescued by the necroptosis inhibitor necrostatin-1 (Nec-1), but not by the apoptosis inhibitor z-VAD-fmk. Brazilin repressed caspase 8 expression and decreased the mitochondrial membrane potentials; both effects were partially reversed by Nec-1. Brazilin induced physiological and morphological changes in T24 cells and RIP1/RIP3/MLKL-mediated necroptosis might be involved. In conclusion, the results confirm the involvement of necroptosis in brazilin-induced cell death and suggest that brazilin could be explored as an anticancer agent against bladder cancer.

Necrostatin-1 attenuates Caspase-1-dependent pyroptosis induced by the RIPK1/ZBP1 pathway in ventilator-induced lung injury.

BACKGROUND: Ventilator-induced lung injury (VILI) is a complex pathophysiological process leading to acute respiratory distress syndrome (ARDS) and poor outcomes in affected patients. As a form of programmed cell death, pyroptosis is proposed to play an important role in the development of ARDS. Here we investigated whether treating mice with the specific RIPK1 inhibitor Necrostatin-1 (Nec-1) before mechanical ventilation could inhibit pyroptosis and alleviate lung injury in a mouse model. METHODOLOGYS: Anesthetized C57BL/6J mice received a transtracheal injection of Nec-1 (5 mg/kg) or vehicle (DMSO) 30 min before the experiment which was ventilated for up to 4 h. Lung damage was assessed macroscopically and histologically with oedema measured as the wet/dry ratio of lung tissues. The release of inflammatory mediators into bronchoalveolar lavage fluid (BALF) was assessed by ELISA measurements of TNF-α,interleukin-1ß (IL-1ß), and IL-6. The expression of RIPK1, ZBP1, caspase-1, and activated (cleaved) caspase-1 were analyzed using western blot and immunohistochemistry, and the levels of gasdermin-D (GSDMD) and IL-1ß were analyzed by immunofluorescence staining. RESULTS: High tidal ventilation produced time-dependent inflammation and lung injury in mice which could be significantly reduced by pretreatment with Nec-1. Notably, Nec-1 reduced the expression of key pyroptosis mediator proteins in lung tissues exposed to mechanical ventilation, including caspase-1, cleaved caspase-1, and GSDMD together with inhibiting the release of inflammatory cytokines. CONCLUSION: Nec-1 pretreatment alleviates pulmonary inflammatory responses and protects the lung from mechanical ventilation damage. The beneficial effects were mediated at least in part by inhibiting caspase-1-dependent pyroptosis through the RIPK1/ZBP1 pathway.

RIP3 knockdown inhibits necroptosis of human intestinal epithelial cells via TLR4/MyD88/NF-κB signaling and ameliorates murine colitis.

BACKGROUND: Ulcerative colitis (UC) is a common inflammatory bowel disease, during which cell necroptosis plays key roles in driving inflammation initiation and aggravation. Previous studies reported Receptor Interacting Protein Kinase 3 (RIP3)-mediated necroptosis in multiple diseases, and RIP3 protein in Paneth cells significantly enriched in the intestines of both humans and mice. Therefore, we hypothesized targeting RIP3 to inhibit necroptosis may depress UC. METHODS: We classified clinical UC samples according to the modified Truelove & Witts criterion. The expression of RIP3 was measured by western blot and immunohistochemistry. Cell proliferation and apoptosis were analyzed by MTT assay and flow cytometry. ROS production and the secretion of inflammatory cytokines were measured by DCFH-DA probe and ELISA assay. TLR4/MyD88/NF-κB signaling pathway was analyzed by western blot. We established experimental colitis model in RIP3 knockout and wild-type mice and disease activity index (DAI) score was calculated. The expression and distribution of tight junction protein were analyzed by immunofluorescence. The ratio of CD4+Foxp3+ T cells in the spleen was detected by flow cytometry. Oxidative damage of mouse colon was assessed by detecting the levels of SOD, MDA and MPO. Data were analyzed by one-way ANOVA or student's t test. RESULTS: The expression of RIP3 in human colon is positively associated with the severity of UC. RIP3 inhibitor GSK872 or RIP3 knockdown reverses the inhibitory effect of TNF-α on proliferation and the promoting effect of TNF-α on apoptosis and necrosis in human intestinal epithelial cells. In addition, RIP3 deficiency inhibits the secretion of inflammatory cytokines (IL-16, IL-17 and IFN-γ) and ROS production induced by TNF-α. In vivo, RIP3 inhibitor Nec-1 effectively improves DSS-induced colitis in mice. In mechanism, RIP3 depression could upregulate the proportion of CD4+Foxp3+ immunosuppressive Treg cells in the spleen while suppressed TLR4/MyD88/NF-κB signaling pathway and ROS generation, and all these anti-inflammation factors together suppress the secretion of inflammatory cytokines and necroptosis of intestinal epithelial cells. CONCLUSIONS: This study preliminarily explored the regulating mechanism of RIP3 on UC, and Nec-1 may be a promising drug to alleviate the inflammation and necroptosis of the colon in UC patients.

Determination of Novel Highly Effective Necrostatin Nec-1s in Rat Plasma by High Performance Liquid Chromatography Hyphenated with Quadrupole-Time-of-Flight Mass Spectrometry.

Necrostatins have been shown to retard necroptosis, a programmed necrotic-like cell death, which has been shown to underlie pathophysiology of various diseases. Nec-1s, a novel highly effective necrostatin, overcomes some drawbacks of former necrostatin analogues. The determination of Nec-1s in biological system, however, has not been carried out so far. Therefore, this study was undertaken to optimize and validate the HPLC-DAD-Q-TOF method for the assessment of Nec-1s levels in the plasma what is the necessity for designing its proper dosing regimen for in vivo studies. Benefits of the proposed analytical protocol include: (i) simple sample preparation (precipitation of plasma proteins, evaporation of acetonitrile, reconstitution in mobile phase), (ii) fast, selective and sensitive analysis due to a highly orthogonal LC-MS system providing less than 8 min analysis time, (iii) detection of Nec-1s without any matrix interferences, and quantitation of very low concentration levels of Nec-1s (LLOQ ~ 20 ng/mL), (iv) high reliability of Nec-1s determination with precision and accuracy values meeting the FDA criteria for biomedical analysis. The proposed analytical protocol is suitable for routine use in relevant biological studies, and, in this work, it was successfully applied for monitoring of Nec-1s plasma levels in rats providing reproducible and consistent results. Based on pharmacokinetic features, which can also be assessed due to the results of this study, there will be efforts to perform both acute and chronic in vivo studies and potential clinical safety studies first.

Necrostatin-1 rescues mice from lethal irradiation.

There is an emerging need in new medical products that can mitigate and/or treat the short- and long-term consequences of radiation exposure after a radiological or nuclear terroristic event. The direct effects of ionizing radiation are realized primarily via apoptotic death pathways in rapidly proliferating cells within the initial 1-2days after the exposure. However later in the course of the radiation disease necrotic cell death may ensue via direct and indirect pathways from increased generation of pro-inflammatory cytokines. Here we evaluated radiomitigative potential of necrostatin-1 after total body irradiation (TBI) and the contribution of necroptosis to cell death induced by radiation. Circulating TNFα levels were increased starting on d1 after TBI and associated with increased plasmalemma permeability in ileum of irradiated mice. Necrostatin-1 given iv. 48h after 9.5Gy TBI attenuated radiation-induced receptor interacting protein kinase 3 (RIPK3) serine phosphorylation in ileum and improved survival vs. vehicle. Utilizing apoptosis resistant cytochrome c(-/-) cells, we showed that radiation can induce necroptosis, which is attenuated by RNAi knock down of RIPK1 and RIPK3 or by treatment with necrostatin-1 or -1s whereas 1-methyl-L-tryptophan, an indoleamine-2,3-dioxygenase inhibitor, did not exhibit radiomitigative effect. This suggests that the beneficial effect of necrostatin-1 is likely through inhibition of RIPK1-mediated necroptotic pathway. Overall, our data indicate that necroptosis, a form of programmed necrosis, may play a significant role in cell death contributing to radiation disease and mortality. This study provides a proof of principle that necrostatin-1 and perhaps other RIPK1 inhibitors are promising therapeutic agents for radiomitigation after TBI.

RIPK1/RIPK3/MLKL-mediated necroptosis contributes to compression-induced rat nucleus pulposus cells death.

The aim of this study was to systematically investigate the role of necroptosis in compression-induced rat nucleus pulposus (NP) cells death, as well as explore the underlying mechanisms involved. Rat NP cells underwent various periods of exposure to 1.0 MPa pressure. Cell viability and cell death were quantified by using cell counting kit-8 (CCK-8), and Calcein-AM/propidium iodine (PI) staining respectively. Necroptosis-associated target molecules receptor-interacing protein kinase 1 (RIPK1), phosphorylated RIPK1 (pRIPK1), receptor-interacing protein kinase 3 (RIPK3), phosphorylated RIPK3 (pRIPK3) and mixed lineage kinase domain-like (MLKL) were analyzed by Western-blot and RT-PCR. NP cells were also examined for morphological and ultrastructural changes, which can indicate necroptosis. To indirectly establish the presence of necroptosis, the RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 inhibitor GSK'872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) were utilized. The results established necroptosis was taking place in NP cells. The level of necroptosis increased in a time-dependent manner, and this effect was reduced by Nec-1 in vitro. Additionally, NP cells death were significantly attenuated following treatment with Nec-1, GSK'872 or NSA. SiRNA-induced knockdown of RIPK3 or MLKL increased cell survival rate, while knockdown of RIPK1 resulted in a decreased cell survival rate. In summary, RIPK1/RIPK3/MLKL-mediated necroptosis may play an important role in NP cells death induced by continuous mechanical stress. Treatment strategies which aim to regulate necroptosis may prove beneficial, by both reducing NP cells death and slowing IVD degeneration.

Involvement of necroptosis, a newly recognized cell death type, in steroid-induced osteonecrosis in a rabbit model.

We investigated the role of programmed necrosis (necroptosis), a newly recognized form of cell necrosis that has been implicated in the development of steroid-induced osteonecrosis. We used an osteonecrosis model in which 30 Japanese white rabbits each weighing 3.5kg were injected once with methylprednisolone at 20 mg/kg body weight into the right gluteal muscle. Ten animals killed 14 days thereafter were designated as S14d groups, while another 10 animals injected with necroptosis, a specific inhibitor of necrostatin-1 i.v. at 1.65mg/kg on the same day as the steroid were also killed on the 14th day and designated as SN14d group. As a control, 10 animals injected only with physiological saline were studied as N group. After the animals were sacrificed the bilateral femoral bone was examined histopathologically and the presence of osteonecrosis determined. Furthermore, animals subjected to the same treatment and killed on the 3rd day after drug administration were set up as S3d group and SN3d group, and Western blotting of Receptor-interacting protein ( RIP ) 1 and RIP3 in femoral bone performed. The osteonecrosis rate was 70% in S14d group, and 0% in both N and SN groups. In 2 of 10 animals in SN group fatty marrow was found. On Western blotting significantly increased expression of both RIP1 and RIP3 was noted in S3d group, confirming that Nec-1 was suppressed. Necroptosis mediated by RIP1 and RIP3 expression was thought to be implicated in the development of steroid-induced osteonecrosis. Also, by suppressing expression of RIP1 and 3 with the administration of Nec-1 the osteonecrosis rate was significantly decreased. These results suggest that necroptosis may have potential as a novel target for both elucidating the mechanisms underlying steroid-induced osteonecrosis and establishing more effective prophylactic countermeasures.

Necrostatin-1 alleviates reperfusion injury following acute myocardial infarction in pigs.

BACKGROUND: In rodents, it has previously been shown that necrostatin-1 (Nec-1) inhibits RIP1, a central regulator of programmed necrosis, thereby decreasing cell death and reducing infarct size (IS) after ischaemia/reperfusion (I/R) injury. To address unanswered questions on feasibility and efficacy of Nec-1 in a large animal model, we assessed the effects of Nec-1 in a porcine I/R model, relevant to human disease. MATERIALS AND METHODS: In Dalland landrace pigs (69 ± 3 kg), I/R injury was induced by a 75-min surgical ligation of the left circumflex coronary artery (LCx). Ten minutes prior to reperfusion, pigs were randomly allocated to different Nec-1 doses (1.0 mg/kg or 3.3 mg/kg) or vehicle treatment (control, CTRL). Functional endpoints and immunohistological analyses were performed 24 h after reperfusion. RESULTS: Nec-1 3.3 mg/kg significantly reduced IS (n = 6; 24.4 ± 15.6%) compared to Nec-1 1.0 mg/kg (n = 5; 54.8 ± 16.9%) or CTRLs (n = 6; 62.1 ± 26.6%; P = 0.016). In line, LV ejection fraction (LVEF) was significantly higher in Nec-1 3.3 mg/kg, copared to Nec-1 1.0 mg/kg or CTRL treated animals (50.0 ± 12.0% vs. 32.5 ± 12.9% vs. 31.9 ± 6.6%, respectively, P = 0.015). Hemodynamically, a preserved contractility was observed [end-systolic volume at 100 mmHg (ESV100 )] at 24-h follow-up (87.6 ± 17.3 mL vs. 74.5 ± 41.1 mL vs. 56.8 ± 11.8 mL, respectively, P = 0.032), reflecting improved cardiac function. CONCLUSIONS: In the pig model of I/R injury, intravenous administration of Nec-1 prior to reperfusion was an effective and above all practical therapeutic strategy that significantly reduced IS and preserved left ventricular function. These data highlight the potential of cardioprotection as a promising adjuvant therapy in the setting of early reperfusion following I/R injury.

RIP1 kinase inactivation protects against LPS-induced acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) is characterized by lung tissue oedema and inflammatory cell infiltration, with limited therapeutic interventions available. Receptor-interacting protein kinase 1 (RIPK1), a critical regulator of cell death and inflammation implicated in many diseases, is not fully understood in the context of ARDS. In this study, we employed RIP1 kinase-inactivated (Rip1K45A/K45A) mice and two distinct RIPK1 inhibitors to investigate the contributions of RIP1 kinase activity in lipopolysaccharide (LPS)-induced ARDS pathology. Our results indicated that RIPK1 kinase inactivation, achieved through both genetic and chemical approaches, significantly attenuated LPS-induced ARDS pathology, as demonstrated by reduced polymorphonuclear neutrophil percentage (PMN%) in alveolar lavage fluid, expression of inflammatory and fibrosis-related factors in lung tissues, as well as histological examination. Results by tunnel staining and qRT-PCR analysis indicated that RIPK1 kinase activity played a role in regulating cell apoptosis and inflammation induced by LPS administration in lung tissue. In summary, employing both pharmacological and genetic approaches, this study demonstrated that targeted RIPK1 kinase inactivation attenuates the pathological phenotype induced by LPS inhalation in an ARDS mouse model. This study enhances our understanding of the therapeutic potential of RIPK1 kinase modulation in ARDS, providing insights for the pathogenesis of ARDS.

Antagonistic effect of selenium on programmed necrosis of testicular Leydig cells caused by cadmium through endoplasmic reticulum stress in chicken.

Cadmium (Cd) is a widely distributed environmental contaminant that is highly toxic to animals and humans. However, detailed reports on Cd-induced programmed necrosis have not been seen in chicken testicular Leydig cells. Selenium (Se) is a trace element in the human body that has cytoprotective effects in a variety of pathological damages caused by heavy metals. This study investigated the potential mechanisms of Cd-induced programmed cell necrosis and the antagonistic effect of Se on Cd toxicity. Chicken testis Leydig cells were divided into six groups, namely, control, Se (5 µmol/L Na2SeO3), Cd (20 µmol/L CdCl2), Se + Cd (5 µmol/L Na2SeO3 and 20 µmol/L CdCl2), 4-phenylbutyric acid (4-PBA) + Cd (10 mmol/L 4-phenylbutyric acid and 20 µmol/L CdCl2), and Necrostatin-1 (Nec-1) + Cd (60 µmol/L Necrostatin-1 and 20 µmol/L CdCl2). The results showed that Cd exposure decreased the activity of CAT, GSH-Px, and SOD and the concentration of GSH, and increased the concentration of MDA and the content of ROS. Relative mRNA and protein expression of GRP78, PERK, ATF6, IRE1, CHOP, and JNK increased in the Cd group, and mRNA and protein expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 significantly increased in the Cd group, while Caspase-8 mRNA and protein expression significantly decreased. The abnormal expression of endoplasmic reticulum stress-related proteins was significantly reduced by 4-PBA pretreatment; the increased expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 caused by Cd toxicity was alleviated; and the expression of caspase-8 was upregulated. Conversely, the increased mRNA and protein expression of endoplasmic reticulum stress marker genes (GRP78, ATF6, PERK, IRE1, CHOP, JNK) caused by Cd was not affected after pretreatment with Nec-1. We also found that these Cd-induced changes were significantly attenuated in the Se + Cd group. We clarified that Cd can cause programmed necrosis of chicken testicular Leydig cells through endoplasmic reticulum stress, and Se can antagonize Cd-induced programmed necrosis of chicken testicular Leydig cells.

Necroptosis Blockade Potentiates the Neuroprotective Effect of Hypothermia in Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal encephalopathy (NE) caused by hypoxia-ischemia (HI) affects around 1 per 1000 term newborns and is the leading cause of acquired brain injury and neurodisability. Despite the use of hypothermia (HT) as a standard of care, the incidence of NE and its devastating outcomes remains a major issue. Ongoing research surrounding add-on neuroprotective strategies against NE is important as HT effects are limited, leaving 50% of treated patients with neurological sequelae. Little is known about the interaction between necroptotic blockade and HT in neonatal HI. Using a preclinical Lewis rat model of term human NE induced by HI, we showed a neuroprotective effect of Necrostatin-1 (Nec-1: a compound blocking necroptosis) in combination with HT. The beneficial effect of Nec-1 added to HT against NE injuries was observed at the mechanistic level on both pMLKL and TNF-α, and at the anatomical level on brain volume loss visualized by magnetic resonance imaging (MRI). HT alone showed no effect on activated necroptotic effectors and did not preserve the brain MRI volume. This study opens new avenues of research to understand better the specific cell death mechanisms of brain injuries as well as the potential use of new therapeutics targeting the necroptosis pathway.

Phylogenetic Analyses of Some Key Genes Provide Information on Pollinator Attraction in Solanaceae.

Floral syndromes are known by the conserved morphological traits in flowers associated with pollinator attraction, such as corolla shape and color, aroma emission and composition, and rewards, especially the nectar volume and sugar concentration. Here, we employed a phylogenetic approach to investigate sequences of genes enrolled in the biosynthetic pathways responsible for some phenotypes that are attractive to pollinators in Solanaceae genomes. We included genes involved in visible color, UV-light response, scent emission, and nectar production to test the hypothesis that these essential genes have evolved by convergence under pollinator selection. Our results refuted this hypothesis as all four studied genes recovered the species' phylogenetic relationships, even though some sites were positively selected. We found differences in protein motifs among genera in Solanaceae that were not necessarily associated with the same floral syndrome. Although it has had a crucial role in plant diversification, the plant-pollinator interaction is complex and still needs further investigation, with genes evolving not only under the influence of pollinators, but by the sum of several evolutionary forces along the speciation process in Solanaceae.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors Necrostatin-1 (Nec-1) and 7-Cl-O-Nec-1 (Nec-1s) are potent inhibitors of NAD(P)H: Quinone oxidoreductase 1 (NQO1).

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has been identified as a critical mediator of cell death (necroptosis and apoptosis) and inflammation. Necrostatin-1 (Nec-1) and 7-Cl-O-Nec-1 (Nec-1s) are widely used as selective small-molecule inhibitors of RIPK1 in various culture cells and disease models. NAD(P)H: quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes the reduction and detoxification of quinones and other organic compounds. Here, we showed that Nec-1 and Nec-1s could bind and inhibit NQO1 activity. Similar to dicoumarol, the specific inhibitor of NQO1, both Nec-1 and Nec-1s significantly suppress NQO1-dependent cell death. However, dicoumarol failed to reverse necroptosis induced by TNFα/BV6/Z-VAD-FMK (TBZ) in HT29 cells. These findings suggest that besides RIPK1, NQO1 might be another target for Nec-1 and Nec-1s and provide new insights for the interpretation of Nec-1-based experimental results.

RIPK1 inhibitor ameliorates the MPP+/MPTP-induced Parkinson's disease through the ASK1/JNK signalling pathway.

Receptor-interacting protein kinase 1 (RIPK1) is up-regulated in patients with neurodegenerative diseases. Our study aimed to explore the underlying mechanisms that involved in the neurotoxic function of RIPK1 in Parkinson's disease (PD). MPP+/MPTP-induced PD cellular and mice models were used in this study. The results showed that RIPK1 was high expressed and activated in MPP+-treated SH-SY5Y cells and MPTP-induced PD mice. Overexpression of RIPK1 facilitated cell apoptosis, necrosis, inflammation response, ROS production and mitochondrial dysfunction in MPP+- treated SH-SY5Y cells, while the RIPK1 inhibitor Nec-1s has an opposite effect. In addition, the Apoptosis-signaling kinase-1 (ASK1)/c-Jun N-terminal kinase (JNK) signalling pathway was activated during the overexpression of RIPK1, and inhibiting the ASK1/JNK signal by the ASK1 inhibitor partially reversed the decline of cell viability, the increase of cell apoptosis, necrosis and inflammation induced by RIPK1 overexpression in MPP+-treated SH-SY5Y cells. Further studies suggested that the inhibition of RIPK1 by Nec-1s largely alleviated the behavioural impairment in PD mice. Hence, our study indicated that the RIPK1 inhibitor Nec-1s has neuroprotective effects against PD through inactivating the ASK1/JNK signalling pathway.

Nec-1 attenuates inflammation and cytotoxicity induced by high glucose on THP-1 derived macrophages through RIP1.

OBJECTIVES: This research aimed to study whether necrostain-1 (Nec-1) could alleviate inflammatory injury induced by high glucose upon THP-1 derived macrophages through RIP1. DESIGN: Firstly, THP-1 derived macrophages were incubated with 5.5 mM glucose (normal glucose, NG), 25 mM glucose (high glucose, HG), and mannitol as the high osmotic pressure group (5.5 mM glucose+19.5 mM mannitol) for 24, 48, and 72 h respectively. TNF-α, IL-1ß, IL-6, and IL-8 levels were measured by ELISA. Secondly, macrophages were exposed to NG, HG, or HG plus 5 µM necrostatin-1 (Nec-1) for 72 h. mRNA expression of inflammatory cytokine was measured by RT-PCR, and protein levels of inflammatory cytokines and LDH leakage were determined by ELISA. RIP1 expression was determined by RT-PCR and WB. Thirdly, macrophages were transfected with si-RIP1 or negative control (si-NC). Wild type and RIP1-silenced macrophages were incubated with NG or HG, and TNF-α, IL-1ß, IL-6, IL-8, and LDH levels were measured again by ELISA. RESULTS: 1) TNF-α, IL-1ß, IL-6, and IL-8 levels were elevated in the HG group, as compared with that the NG group. Inflammation remained unchanged in the mannitol group. 2) Inflammatory response and LDH levels in the HG plus Nec-1 group were remarkably lower than in the HG group. 3) Inflammatory injury in the si-NC group was more severe than in the si-RIP1 group. CONCLUSIONS: Current results indicated that Nec-1 could alleviate HG-caused inflammatory injury on THP-1 derived macrophages by regulating RIP1. These findings could help cast light on the relationships between diabetes and periodontitis.

Nec-1 Attenuates Neurotoxicity Induced by Titanium Dioxide Nanomaterials on Sh-Sy5y Cells Through RIP1.

Titanium dioxide nanomaterials are applied in numerous fields due to their splendid physicochemical characteristics, which in turn poses a potential threat to human health. Recently, numerous in vivo studies have revealed that titanium dioxide nanoparticles (TNPs) can be transported into animal brains after exposure through various routes. Absorbed TNPs can accumulate in the brain and may disturb neuronal cells, leading to brain dysfunction. In vitro studies verified the neurotoxicity of TNPs. The mechanisms underlying the neurotoxicity of TNPs remains unclear. Whether necroptosis is involved in the neurotoxicity of TNPs is unknown. Therefore, we performed an in vitro study and found that TNPs induced inflammatory injury in SH-SY5Y cells in a dose-dependent way, which was mitigated by necrostatin-1 (Nec-1) pretreatment. Since receptor-interacting protein kinase 1 (RIP1) is reported to be the target of Nec-1, we silenced it by siRNA. We exposed mutant and wild-type cells to TNPs and assessed inflammatory injury. Silencing RIP1 expression inhibited inflammatory injury induced by TNPs exposure. Taken together, Nec-1 ameliorates the neurotoxicity of TNPs through RIP1. However, more studies should be performed to comprehensively assess the correlation between the neurotoxicity of TNPs and RIP1.

Necrostatin-1 Attenuates Renal Ischemia and Reperfusion Injury via Meditation of HIF-1α/mir-26a/TRPC6/PARP1 Signaling.

Necroptosis, oxidative stress, and inflammation are major contributors to the pathogenesis of ischemic acute kidney injury. Necrostatin-1 (Nec-1), an inhibitor of the kinase domain of receptor-interacting protein kinase-1 (RIP1), has been reported to regulate renal ischemia and reperfusion (I/R) injury; however, its underlying mechanism of action remains unclear. HK-2 cells were used to create an in vitro I/R model, in which the cells were subjected to hypoxia, followed by 2, 6, and 12 h of reoxygenation. For the in vivo study, a rat model of renal I/R was established in which samples of rat blood serum and kidney tissue were harvested after reperfusion to assess renal function and detect histological changes. Cell viability and necroptosis were analyzed using the Cell Counting Kit (CCK)-8 assay and flow cytometry, respectively. The expression levels of molecules associated with necroptosis, oxidative stress, and inflammation were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to confirm the relevant downstream signaling pathway. We found that pretreatment with Nec-1 significantly decreased hypoxia-inducible factor-1α (HIF-1α) and miR-26a expression, as well as the levels of factors associated with necroptosis (RIP1, RIP3, and Sirtuin-2), oxidative stress (malondialdehyde [MDA], NADP+/NADPH ratio), and inflammation (interleukin [IL]-1ß, IL-10, and tumor necrosis factor alpha [TNF-α]) in I/R injury cells and the rat model. However, these effects could be reversed by miR-26a overexpression or TRPC6 knockdown. Mechanistic studies demonstrated that HIF-1α directly binds to the promoter region of miR-26a, and that TRPC6 is a potential target gene for miR-26a. Our findings indicate that Nec-1 can effectively protect against renal I/R injury by inhibiting necroptosis, oxidative stress, and inflammation, and may exert its effects through mediation of the HIF-1α/miR-26a/TRPC6/PARP1 signaling pathway.

Arabidopsis mutant dnd2 exhibits increased auxin and abscisic acid content and reduced stomatal conductance.

Arabidopsis thaliana cyclic nucleotide-gated ion channel gene 4 (AtCNGC4) loss-of-function mutant dnd2 exhibits elevated accumulation of salicylic acid (SA), dwarfed morphology, reduced hypersensitive response (HR), altered disease resistance and spontaneous lesions on plant leaves. An orthologous barley mutant, nec1, has been reported to over-accumulate indole-3-acetic acid (IAA) and to exhibit changes in stomatal regulation in response to exogenous auxin. Here we show that the Arabidopsis dnd2 over-accumulates both IAA and abscisic acid (ABA) and displays related phenotypic and physiological changes, such as, reduced stomatal size, higher stomatal density and stomatal index. dnd2 showed increased salt tolerance in root growth assay and significantly reduced stomatal conductance, while maintaining near wt reaction in stomatal conductance upon external application of ABA, and probably consequently increased drought stress tolerance. Introduction of both sid2-1 and fmo1 into dnd2 background resulting in removal of SA did not alter stomatal conductance. Hence, the closed stomata of dnd2 is probably a result of increased ABA levels and not increased SA levels. The triple dnd2sid2abi1-1 mutant exhibited intermediate stomatal conductance compared to dnd2 and abi1-1 (ABA insensitive, open stomata), while the response to external ABA was as in abi1-1 suggesting that reduced stomatal conductance in dnd2 is not due to impaired ABA signaling. In conclusion, Arabidopsis dnd2 mutant exhibited ABA overaccumulation and stomatal phenotypes, which may contribute to the observed improvement in drought stress resistance. Thus, Arabidopsis dnd2 mutant may serve as a model for studying crosstalk between biotic and abiotic stress and hormonal response in plants.

Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling.

Pharmacological studies have indirectly shown that necroptosis participates in ischemic neuronal death. However, its mechanism has yet to be elucidated in the ischemic brain. TNFα-triggered RIPK1 kinase activation could initiate RIPK3/MLKL-mediated necroptosis under inhibition of caspase-8. In the present study, we performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia in rats and used immunoblotting and immunostaining combined with pharmacological analysis to study the mechanism of necroptosis in ischemic brains. In the ipsilateral hemisphere, we found that ischemia induced the increase of (i) RIPK1 phosphorylation at the Ser166 residue (p-RIPK1), representing active RIPK1 kinase and (ii) the number of cells that were double stained with P-RIPK1 (Ser166) (p-RIPK1+) and TUNEL, a label of DNA double-strand breaks, indicating cell death. Furthermore, ischemia induced activation of downstream signaling factors of RIPK1, RIPK3 and MLKL, as well as the formation of mature interleukin-1ß (IL-1ß). Treatment with necrostatin-1 (Nec-1), an inhibitor of necroptosis, significantly decreased ischemia-induced increase of p-RIPK1 expression and p-RIPK1+ neurons, which showed protection from brain damage. Meanwhile, Nec-1 reduced RIPK3, MLKL and p-MLKL expression levels and mature IL-1ß formation in Nec-1 treated ischemic brains. Our results clearly demonstrated that phosphorylation of RIPK1 at the Ser166 residue was involved in the pathogenesis of necroptosis in the brains after ischemic injury. Nec-1 treatment protected brains against ischemic necroptosis by reducing the activation of RIPK1 and inhibiting its downstream signaling pathways. These results provide direct in vivo evidence that phosphorylated RIPK1 (Ser 166) plays an important role in the initiation of RIPK3/MLKL-dependent necroptosis in the pathogenesis of ischemic stroke in the rodent brain.

MCC1019, a selective inhibitor of the Polo-box domain of Polo-like kinase 1 as novel, potent anticancer candidate.

Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest-a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.