Discovery and optimization of 3-(indolin-5-yloxy)pyridin-2-amine derivatives as potent necroptosis inhibitors.

Necroptosis is a form of regulated necrotic cell death and has been confirmed to play pivotal roles in the pathogenesis of multiple autoimmune diseases such as rheumatoid arthritis (RA) and psoriasis. The development of necroptosis inhibitors may offer a promising therapeutic strategy for the treatment of these autoimmune diseases. Herein, starting from the in-house hit compound 1, we systematically performed structural optimization to discover potent necroptosis inhibitors with good pharmacokinetic profiles. The resulting compound 33 was a potent necroptosis inhibitor for both human I2.1 cells (IC50 < 0.2 nM) and murine Hepa1-6 cells (IC50 < 5 nM). Further target identification revealed that compound 33 was an inhibitor of receptor interacting protein kinase 1 (RIPK1) with favorable selectivity. In addition, compound 33 also exhibited favorable pharmacokinetic profiles (T1/2 = 1.32 h, AUC = 1157 ng·h/mL) in Sprague-Dawley rats. Molecular docking and molecular dynamics simulations confirmed that compound 33 could bind to RIPK1 with high affinity. In silico ADMET analysis demonstrated that compound 33 possesses good drug-likeness profiles. Collectively, compound 33 is a promising candidate for antinecroptotic drug discovery.

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.

Necroptosis and Its Involvement in Various Diseases.

Necroptosis is a regulated form of cell death involved in the development of various pathological conditions. In contrast to apoptosis, plasma membrane rupture (PMR) occurs in cells in the relatively early stage of necroptosis; therefore, necroptosis induces a strong inflammatory response. Stimuli, including tumor necrosis factor (TNF), interferon (IFN)α/ß, lipopolysaccharide, polyI:C, and viral infection, induce the formation of necrosomes that lead to membrane rupture and the release of intracellular contents, termed danger-associated molecular patterns (DAMPs). DAMPs are the collective term for molecules that normally reside in the cytoplasm or nucleus in living cells without inducing inflammation but induce strong inflammatory responses when released outside cells. Recent studies have provided a better understanding of the mechanisms underlying PMR and the release of DAMPs. Moreover, necroptosis is involved in various pathological conditions, and mutations in necroptosis-related genes can cause hereditary autoinflammatory syndromes. Thus, manipulating necroptosis signaling pathways may be useful for treating diseases involving necroptosis.

RIP3 in Necroptosis: Underlying Contributions to Traumatic Brain Injury.

Traumatic brain injury (TBI) is a global public safety issue that poses a threat to death, characterized by high fatality rates, severe injuries and low recovery rates. There is growing evidence that necroptosis regulates the pathophysiological processes of a variety of diseases, particularly those affecting the central nervous system. Thus, moderate necroptosis inhibition may be helpful in the management of TBI. Receptor-interacting protein kinase (RIP) 3 is a key mediator in the necroptosis, and its absence helps restore the microenvironment at the injured site and improve cognitive impairment after TBI. In this report, we review different domains of RIP3, multiple analyses of necroptosis, and associations between necroptosis and TBI, RIP3, RIP1, and mixed lineage kinase domain-like. Next, we elucidate the potential involvement of RIP3 in TBI and highlight how RIP3 deficiency enhances neuronal function.

Synthesis and evaluation of a novel PET ligand, a GSK'963 analog, aiming at autoradiography and imaging of the receptor interacting protein kinase 1 in the brain.

BACKGROUND: Receptor interacting protein kinase 1 (RIPK1) is a serine/threonine kinase, which regulates programmed cell death and inflammation. Recently, the involvement of RIPK1 in the pathophysiology of Alzheimer's disease (AD) has been reported; RIPK1 is involved in microglia's phenotypic transition to their dysfunctional states, and it is highly expressed in the neurons and microglia in the postmortem brains in AD patients. They prompt neurodegeneration leading to accumulations of pathological proteins in AD. Therefore, regulation of RIPK1 could be a potential therapeutic target for the treatment of AD, and in vivo imaging of RIPK1 may become a useful modality in studies of drug discovery and pathophysiology of AD. The purpose of this study was to develop a suitable radioligand for positron emission tomography (PET) imaging of RIPK1. RESULTS: (S)-2,2-dimethyl-1-(5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)propan-1-one (GSK'963) has a high affinity, selectivity for RIPK1, and favorable physiochemical properties based on its chemical structure. In this study, since 11C-labeling (half-life: 20.4 min) GSK'963 retaining its structure requiring the Grignard reaction of tert-butylmagnesium halides and [11C]carbon dioxide was anticipated to give a low yield, we decided instead to 11C-label a GSK'963 analog ((S)-2,2-dimethyl-1-(5-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)propan-1-one, GG502), which has a high RIPK1 inhibitory activity equivalent to that of the original compound GSK'963. Thus, we successfully 11C-labeled GG502 using a Pd-mediated cross-coupling reaction in favorable yields (3.6 ± 1.9%) and radiochemical purities (> 96%), and molar activity (47-115 GBq/µmol). On autoradiography, radioactivity accumulation was observed for [11C]GG502 and decreased by non-radioactive GG502 in the mouse spleen and human brain, indicating the possibility of specific binding of this ligand to RIPK1. On brain PET imaging in a rhesus monkey, [11C]GG502 showed a good brain permeability (peak standardized uptake value (SUV) ~3.0), although there was no clear evidence of specific binding of [11C]GG502. On brain PET imaging in acute inflammation model rats, [11C]GG502 also showed a good brain permeability, and no significant increased uptake was observed in the lipopolysaccharide-treated side of striatum. On metabolite analysis in rats at 30 min after administration of [11C]GG502, ~55% and ~10% of radioactivity was from unmetabolized [11C]GG502 in the brain and the plasma, respectively. CONCLUSIONS: We synthesized and evaluated a 11C-labeled PET ligand based on the methylated analog of GSK'963 for imaging of RIPK1 in the brain. Although in autoradiography of the resulting [11C]GG502 indicated the possibility of specific binding, the actual PET imaging failed to detect any evidence of specific binding to RIPK1 despite its good brain permeability. Further development of radioligands with a higher binding affinity for RIPK1 in vivo and more stable metabolite profiles compared with the current compound may be required.

Clinicopathological and prognosis significance of RIPK1 in patients with cervical squamous cell carcinoma: a retrospective cohort study.

Background: Cervical cancer is one of the most common types of carcinoma in women and has high morbidity and mortality rates worldwide. Recurrent and metastatic disease remains difficult to treat. Receptor interacting protein kinase 1 (RIPK1) is a key molecule in mediating apoptosis, necroptosis, and inflammatory pathways downstream of death receptors and pattern recognition receptors. This study sought to explore the clinicopathological and prognosis significance of RIPK1 expression in cervical squamous cell carcinoma (CSCC). Methods: The data of 100 CSCC patients who underwent curative surgery from 2019 to 2020 were retrospectively included in this study. We collected the clinicopathological information of the patients and detected RIPK1 protein expression using immunohistochemistry. The Chi-square test and a 1-way analysis of variance were used to make comparisons between groups stratified by RIPK1 expression. A Pearson linear correlation analysis was used to evaluate the association between RIPK1 expression and the clinicopathological characteristics of the patients. A Cox regression analysis and Kaplan-Meier curves were used to analyze overall survival (OS) and progression-free survival (PFS). A multivariable regression analysis was conducted to identify the risk factors for an impaired prognosis in CSCC. Results: RIPK1 was found to be overexpressed in the CSCC tissues. RIPK1 expression was significantly associated with age, the preoperative serum squamous cell carcinoma antigen (SCC-Ag) level, lymph node metastasis, invasion depth, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor size, PFS, and OS (P<0.05). The PFS and OS differed significantly among patients with RIPK1 expression (P<0.05). The multivariate analysis showed that RIPK1 was not independent risk factors for PFS and OS in CSCC patients (P>0.05). Conclusions: The expression of RIPK1 was significantly upregulated in CSCC and was associated with the clinicopathological features of CSCC. RIPK1 might serve as a novel marker that can be used to predict the prognosis of CSCC patients and as a biological target for the treatment of CSCC.

TAK1 Reduces Surgery-induced Overactivation of RIPK1 to Relieve Neuroinflammation and Cognitive Dysfunction in Aged Rats.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a common clinical complication in elderly patients, but its underlying mechanism remains unclear. Receptor-interacting protein kinase 1 (RIPK1), a key molecule mediating necroptosis and regulated by transforming growth factor ß-activated kinase 1 (TAK1), was reported to be associated with cognitive impairment in several neurodegenerative diseases. This study was conducted to investigate the possible role of TAK1/RIPK1 signalling in POCD development following surgery in rats. METHODS: Young (2-month-old) and old (24-month-old) Sprague-Dawley rats were subjected to splenectomy under isoflurane anaesthesia. The young rats were treated with the TAK1 inhibitor takinib or the RIPK1 inhibitor necrostatin-1 (Nec-1) before surgery, and old rats received adeno-associated virus (AAV)-TAK1 before surgery. The open field test and contextual fear conditioning test were conducted on postoperative day 3. The changes in TNF-α, pro-IL-1ß, AP-1, NF-κB p65, pRIPK1, pTAK1 and TAK1 expression and astrocyte and microglia activation in the hippocampus were assessed. RESULTS: Old rats had low TAK1 expression and were more susceptible to surgery-induced POCD and neuroinflammation than young rats. TAK1 inhibition exacerbated surgery-induced pRIPK1 expression, neuroinflammation and cognitive dysfunction in young rats, and this effect was reversed by a RIPK1 inhibitor. Conversely, genetic TAK1 overexpression attenuated surgery-induced pRIPK1 expression, neuroinflammation and cognitive dysfunction in old rats. CONCLUSION: Ageing-related decreases in TAK1 expression may contribute to surgery-induced RIPK1 overactivation, resulting in neuroinflammation and cognitive impairment in old rats.

O-GlcNAcylation of RIPK1 rescues red blood cells from necroptosis.

Necroptosis is a type of cell death with excessive inflammation and organ damage in various human diseases. Although abnormal necroptosis is common in patients with neurodegenerative, cardiovascular, and infectious diseases, the mechanisms by which O-GlcNAcylation contributes to the regulation of necroptotic cell death are poorly understood. In this study, we reveal that O-GlcNAcylation of RIPK1 (receptor-interacting protein kinase1) was decreased in erythrocytes of the mouse injected with lipopolysaccharide, resulting in the acceleration of erythrocyte necroptosis through increased formation of RIPK1-RIPK3 complex. Mechanistically, we discovered that O-GlcNAcylation of RIPK1 at serine 331 in human (corresponding to serine 332 in mouse) inhibits phosphorylation of RIPK1 at serine 166, which is necessary for the necroptotic activity of RIPK1 and suppresses the formation of the RIPK1-RIPK3 complex in Ripk1 -/- MEFs. Thus, our study demonstrates that RIPK1 O-GlcNAcylation serves as a checkpoint to suppress necroptotic signaling in erythrocytes.

Necroptosis has a crucial role in the development of chronic periodontitis.

Background and aim: Periodontitis is a non-communicable chronic inflammatory disease that affects the entire periodontium and its severe types cause irreparable destruction. The purpose of this study was to determine the type of cell death in chronic periodontitis (CP) with the expression of receptor-interacting protein kinase (RIPK) type1 and RIPK3 genes. Materials and methods: This cross-sectional study was carried out from September 2019 to 2020. The samples (38 participants) were divided into two groups: 20 recently diagnosed CP patients and 18 healthy individuals. Participants' data was collected in the periodontology Department, Dental school, Mashhad University of Medical Sciences and sent to the Immunology Lab for assessment of RIPK1 and RIPK3 expressions using quantitative real time-PCR. Results: The study sample consisted of 30 females (78.9%) and 8 males (21.1%) with a mean age of 34 ± 5 years. The expression of the genes of interest in CPs exhibited an opposite pattern. Although, RIPK3 gene expression was significantly greater in CP patients compared to the control group (P = 0.024), the expression of RIPK1 decreased (p < 0.001). Moreover, no significant correlation was observed between age and gender with these molecules in CPs. Conclusion: The RIPK3 selectively contributes to necroptosis, therefore, it seems that RIPK3-mediated necroptosis is involved in chronic periodontitis. RIPK1 also participates in necroptosis, but mostly in apoptosis. Therefore, necroptosis as an unprogrammed inflammatory cell death induced by pathogenic damages seems to be another mechanism complicated in periodontitis and could be used as a novel target for CP therapy.

RIPK1-Induced A1 Reactive Astrocytes in Brain in MPTP-Treated Murine Model of Parkinson's Disease.

Neuroinflammation is one of the hallmarks of Parkinson's disease, including the massive activation of microglia and astrocytes and the release of inflammatory factors. Receptor-interacting protein kinase 1 (RIPK1) is reported to mediate cell death and inflammatory signaling, and is markedly elevated in the brain in PD mouse models. Here, we aim to explore the role of RIPK1 in regulating the neuroinflammation of PD. C57BL/6J mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 20 mg/kg four times/day), followed by necrostatin-1 treatment (Nec-1, RIPK1 inhibitor; 1.65 mg/kg once daily for seven days. Notably, the first Nec-1 was given 12 h before MPTP modeling). Behavioral tests indicated that inhibition of RIPK1 greatly relieved motor dysfunction and anxiety-like behaviors of PD mice. It also increased striatal TH expression, rescue the loss of dopaminergic neurons, and reduce activation of astrocytes in the striatum of PD mice. Furthermore, inhibition of RIPK1 expression reduced A1 astrocytes' relative gene expression (CFB, H2-T23) and inflammatory cytokine or chemokine production (CCL2, TNF-α, IL-1ß) in the striatum of PD mice. Collectively, inhibition of RIPK1 expression can provide neuroprotection to PD mice, probably through inhibition of the astrocyte A1 phenotype, and thus RIPK1 might be an important target in PD treatment.

Dermatophagoides farinae Extract Induces Interleukin 33-Mediated Atopic Skin Inflammation via Activation of RIP1.

Receptor-interacting protein kinase (RIP) family 1 signaling has complex effects on inflammatory processes and cell death, but little is known concerning allergic skin diseases. We examined the role of RIP1 in Dermatophagoides farinae extract (DFE)-induced atopic dermatitis (AD)-like skin inflammation. RIP1 phosphorylation was increased in HKCs treated with DFE. Nectostatin-1, a selective and potent allosteric inhibitor of RIP1, inhibited AD-like skin inflammation and the expression of histamine, total IgE, DFE-specific IgE, IL-4, IL-5, and IL-13 in an AD-like mouse model. The expression of RIP1 was increased in ear skin tissue from a DFE-induced mouse model with AD-like skin lesions and in the lesional skin of AD patients with high house dust mite sensitization. The expression of IL-33 was down-regulated after RIP1 inhibition, and the levels of IL-33 were increased by over-expression of RIP1 in keratinocytes stimulated with DFE. Nectostatin-1 reduced IL-33 expression in vitro and in the DFE-induced mouse model. These results suggest that RIP1 can be one of the mediators that regulate IL-33-mediated atopic skin inflammation by house dust mites.

Discovery of Sibiriline derivatives as novel receptor-interacting protein kinase 1 inhibitors.

Receptor-interacting protein kinase 1 (RIPK1), a vital protein of the necroptosis pathway, plays a pivotal role in various inflammatory diseases. Sibiriline has been reported to be a potent ATP-competitive RIPK1 inhibitor, but its anti-necroptotic effects are limited. A series of structural analogues of Sibiriline were synthesized and evaluated for their anti-necroptotic activity. Comprehensive structure-activity relationship (SAR) was performed to left azaindole and right substituents of benzene of Sibiriline, respectively. The optimal compound KWCN-41, specifically inhibiting cell necroptosis but not apoptosis, protects cell survival by blocking the necroptotic pathway, which inhibits the phosphorylation of essential proteins of the necroptosis. It also prevented the development of inflammation and reduced the level of inflammatory factors in mice. KWCN-41 is expected to be a lead compound for further studies in inflammatory diseases.

Silencing RIPK1/mTORC1 signalling attenuated the inflammation and oxidative stress in diabetic cardiomyopathy.

BACKGROUND: Diabetes cardiomyopathy (DCM) is one of the major risk factors for the heart failure of the diabetic patients. RIPK1 maybe participate in the regulation of the oxidative stress and inflammation during DCM. METHODS: H&E and Masson staining were utilized to assess the inflammation and fibrosis in myocardial tissues. CCK-8 and TUNEL staining were utilized to analyze cell viability and apoptosis, respectively. SOD activity and MDA content were detected utilizing the kits. The formation of autophagosomes was measured by immunofluorescence assay. RESULTS: RIPK1 and RPTOR (a component of mTORC1) expression and oxidative stress level were upregulated, but autophagy was decreased in the myocardial tissues of DCM rat characterized by the high body weight and blood glucose, abnormal cardiac function, myocardial inflammation and fibrosis. High glucose (HG) treatment resulted in cell viability and autophagy level decreasing, inflammatory cytokines expression increasing and oxidative stress increasing in cardiac fibroblasts (CFs). Meanwhile, RIPK1 and RPTOR expression also was increased in HG-treated cells. HG-induced CFs apoptosis, inflammation, oxidative stress and the inhibition of HG to cell viability and autophagy was partly reversed by the inhibitor of RIPK1 and mTORC1. CONCLUSION: Overall, RIPK1/mTORC1 signalling suppression improved HG-induced apoptosis, inflammation and oxidative stress through activation autophagy. These data provided a reliable evidence that RIPK1 may be a potential target for DCM therapeutic.

Effects and mechanisms of wogonin on airway inflammation in rats with chronic obstructive pulmonary disease / 中国药房

OBJECTIVE To study the effects and potential mechanism of wogonin （Wog） on airway inflammation in rats with chronic obstructive pulmonary disease （COPD）. METHODS Eighty-four rats were randomly divided into control group， model group， Wog low-dose and high-dose groups （intragastric administration of 50， 100 mg/kg）， aminophylline group （positive control， intragastric administration of 2.3 mg/kg）， recombinant rat receptor-interacting protein kinase 1 [rRIPK1， receptor-interacting protein kinase 1 （RIPK1） activator] group （tail vein injection of 8 µg/kg）， and Wog high-dose+rRIPK1 group （intragastric administration of Wog 100 mg/kg+tail vein injection of rRIPK 8 µg/kg）， with 12 rats in each group. Except for control group， COPD model of other groups was induced by smoking combined with tracheal injection of lipopolysaccharide. Twenty-four hours after successful modeling， the rats were administered once a day for 4 weeks. The changes of peak inspiratory flow （PIF）， peak expiratory flow （PEF） and minute ventilation （MV），forced expiratory volume in one second（FEV1）/forced vital capacity（FVC） were measured after the last medication； the serum levels of interleukin 1β（IL-1β）， IL-6 and tumor necrosis factor-α （TNF-α） were measured by ELISA； the pathological changes of lung tissue in rats were observed； the apoptotic rate of pulmonary epithelial cells was detected. mRNA expressions of RIPK1， RIPK3 and mixed lineage kinase domain-like protein （MLKL）， and protein expressions of RIPK1， RIPK3 and p-MLKL were all detected in lung tissue of rats. RESULTS Compared with control group， PIF， PEF， MV and FEV1/FVC of model group were decreased significantly （P＜0.05）， while the levels of IL-1β， IL-6 and TNF- α were increased significantly （P＜0.05）； there was a large number of inflammatory cells infiltration in the lung tissue and bronchialwall thickening in model group； the apoptotic rate of pulmonary epithelial cells，mRNA expressions of RIPK1， RIPK3 and MLKL， protein expressions of RIPK1， RIPK3 and p-MLKL were increased significantly （P＜0.05）. Compared with model group， above indexes of rats were improved significantly in Wog low-dose and high-dose groups （P＜0.05）， and pathological injuries were alleviated significantly. The corresponding indexes of rats were worsened in rRIPK1 group （P＜0.05）， and pathological damage had further worsened. rRIPK1 significantly attenuated the inhibitory effect of high-dose Wog on airway inflammation and RIPK1/RIPK3/ MLKL pathway in COPD rats （P＜0.05）. CONCLUSIONS Wog may improve airway inflammation in COPD rats by inhibiting RIPK1/RIPK3/MLKL signal pathway.

Necroptosis in inflammatory bowel disease: A potential effective target. / ç‚Žç—‡æ€§è‚ ç— ä¸­çš„åæ­»æ€§å‡‹äº¡ï¼šä¸€ä¸ªæ½œåœ¨çš„æ²»ç–—é¶ç‚¹ï¼ˆè‹±æ–‡ï¼‰.

The morbidity of inflammatory bowel diseases (IBD) is rising rapidly but no curative therapies to prevent its recurrence. Cell death is crucial to maintaining homeostasis. Necroptosis is a newly identified programmed cell death and its roles played in IBD need to be explored. Necroptosis is mediated by receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), which resulted in cell swelling, plasma membrane rupture, intracellular content leaking, and eventually cell death as well as the promotion of inflammation. Studies have found that inhibiting necroptosis alleviated IBD in animal models and IBD patients with an increased level of necroptosis in inflammatory tissues, indicating that necroptosis is related to the pathogenesis of IBD. However, due to the complexity in regulation of necroptosis and the involvement of multiple functions of relevant signaling molecules, the specific mechanism remains elusive. Necroptosis may play a vital regulatory role in the pathogenesis of IBD, which provides a new idea and method for further exploring the therapeutic target of IBD.

A novel gut-restricted RIPK1 inhibitor, SZ-15, ameliorates DSS-induced ulcerative colitis.

As a key mediator of cell death and inflammation, receptor-interacting protein kinase 1 (RIPK1) responds to a broad set of inflammatory and pro-death stimuli in human diseases. Inhibitors targeting RIPK1 are being investigated for the treatment of a wide range of human diseases, including ulcerative colitis. In the present study, we designed, synthesized, and investigated the anti-necroptosis and RIPK1-inhibition effects of SZ-15-a symmetrical high-molecular-weight (>500 Da) compound. SZ-15 effectively inhibited necroptosis in U937 and HT-29 cells at concentrations of 1 nM and 10 nM, respectively, and SZ-15 at a concentration of 10 nM almost completely blocked RIPK1, RIPK3, and mixed-lineage kinase domain-like (MLKL) protein phosphorylation induced by necrosis inducers. SZ-15 suppressed the pro-necroptosis function of RIPK1 by downregulating the mRNA expression of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. The activities of SZ-15 were effectively restricted to the gut: The percent recovery of the parent form of SZ-15 in mouse feces was 85.75%. Nevertheless, SZ-15 was effectively absorbed and detected in colon tissues after 1 h at a concentration of 3335 ± 868 ng/g, indicating that membrane permeability was maintained. SZ-15 alleviated dextran sulfate sodium (DSS)-induced ulcerative colitis in vivo by decreasing TNF-α, IL-1ß, IL-22, and IL-6 mRNA expression in colonic tissues. Our preclinical study describes a novel gut-restricted RIPK1 inhibitor that shows great potential for use in the clinical treatment of ulcerative colitis.

RIPK1 in Liver Parenchymal Cells Limits Murine Hepatitis during Acute CCl4-Induced Liver Injury.

Some life-threatening acute hepatitis originates from drug-induced liver injury (DILI). Carbon tetrachloride (CCl4)-induced acute liver injury in mice is the widely used model of choice to study acute DILI, which pathogenesis involves a complex interplay of oxidative stress, necrosis, and apoptosis. Since the receptor interacting protein kinase-1 (RIPK1) is able to direct cell fate towards survival or death, it may potentially affect the pathological process of xenobiotic-induced liver damage. Two different mouse lines, either deficient for Ripk1 specifically in liver parenchymal cells (Ripk1LPC-KO) or for the kinase activity of RIPK1 (Ripk1K45A, kinase dead), plus their respective wild-type littermates (Ripk1fl/fl, Ripk1wt/wt), were exposed to single toxic doses of CCl4. This exposure led in similar injury in Ripk1K45A mice and their littermate controls. However, Ripk1LPC-KO mice developed more severe symptoms with massive hepatocyte apoptosis as compared to their littermate controls. A pretreatment with a TNF-α receptor decoy exacerbated liver apoptosis in both Ripk1fl/fl and Ripk1LPC-KO mice. Besides, a FasL antagonist promoted hepatocyte apoptosis in Ripk1fl/fl mice but reduced it in Ripk1LPC-KO mice. Thus, the scaffolding properties of RIPK1 protect hepatocytes from apoptosis during CCl4 intoxication. TNF-α and FasL emerged as factors promoting hepatocyte survival. These protective effects appeared to be independent of RIPK1, at least in part, for TNF-α, but dependent on RIPK1 for FasL. These new data complete the deciphering of the molecular mechanisms involved in DILI in the context of research on their prevention or cure.

Piperlongumine increases the sensitivity of bladder cancer to cisplatin by mitochondrial ROS.

BACKGROUND: The development of cisplatin resistance often results in cisplatin inefficacy in advanced or recurrent bladder cancer. However, effective treatment strategies for cisplatin resistance have not been well established. METHODS: Gene expression was measured by qRT-PCR and Western blotting. CCK-8 assay was performed to detect cell survival. The number of apoptotic cells was determined using the Annexin V-PI double-staining assay. The level of reactive oxygen species (ROS) was measured using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye, and the ATP level was detected using an ATP measurement kit. RESULTS: The expression of receptor-interacting protein kinase 1 (RIPK1), a key regulator of necroptosis, gradually decreased during cisplatin resistance. We first used piperlongumine (PL) in combination with cisplatin to act on cisplatin-resistant BC cells and found that PL-induced activation of RIPK1 increased the sensitivity of T24 resistant cells to cisplatin treatment. Furthermore, we revealed that PL killed T24 cisplatin-resistant cells by triggering necroptosis, because cell death could be rescued by the mixed lineage kinase domain-like (MLKL) protein inhibitor necrotic sulfonamide or MLKL siRNA, but could not be suppressed by the apoptosis inhibitor z-VAD. We further explored the specific mechanism and found that PL activated RIPK1 to induce necroptosis in cisplatin-resistant cells by stimulating mitochondrial fission to produce excessive ROS. CONCLUSIONS: Our results demonstrated the role of RIPK1 in cisplatin-resistant cells and the sensitization effect of the natural drug PL on bladder cancer. These may provide a new treatment strategy for overcoming cisplatin resistance in bladder cancer.

Protective effect of necrosulfonamide on rat pulmonary ischemia-reperfusion injury via inhibition of necroptosis.

BACKGROUND: Necroptosis plays an important role in cell death during pulmonary ischemia-reperfusion injury (IRI). We hypothesized that therapy with necrosulfonamide (NSA), a mixed-lineage kinase domain-like protein inhibitor, would attenuate lung IRI. METHODS: Rats were assigned at random into the sham operation group (n = 6), vehicle group (n = 8), or NSA group (n = 8). In the NSA and vehicle groups, the animals were heparinized and underwent left thoracotomy, and the left hilum was clamped for 90 minutes, followed by reperfusion for 120 minutes. NSA (0.5 mg/body) and a solvent were administered i.p. in the NSA group and the vehicle group, respectively. The sham group underwent 210 minutes of perfusion without ischemia. After reperfusion, arterial blood gas analysis, physiologic data, lung wet-to-dry weight ratio, histologic changes, and cytokine levels were assessed. Fluorescence double immunostaining was performed to evaluate necroptosis and apoptosis. RESULTS: Arterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) was better, dynamic compliance was higher, and mean airway pressure and lung edema were lower in the NSA group compared with the vehicle group. Moreover, in the NSA group, lung injury was significantly alleviated, and the mean number of necroptotic cells (55.3 ± 4.06 vs 78.2 ± 6.87; P = .024), but not of apoptotic cells (P = .084), was significantly reduced compared with the vehicle group. Interleukin (IL)-1ß and IL-6 levels were significantly lower with NSA administration. CONCLUSIONS: In a rat model, our results suggest that NSA may have a potential protective role in lung IRI through the inhibition of necroptosis.

Necroptosis in inflammatory bowel disease: A potential effective target / 中南大学学报(医学版)

The morbidity of inflammatory bowel diseases (IBD) is rising rapidly but no curative therapies to prevent its recurrence. Cell death is crucial to maintaining homeostasis. Necroptosis is a newly identified programmed cell death and its roles played in IBD need to be explored. Necroptosis is mediated by receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), which resulted in cell swelling, plasma membrane rupture, intracellular content leaking, and eventually cell death as well as the promotion of inflammation. Studies have found that inhibiting necroptosis alleviated IBD in animal models and IBD patients with an increased level of necroptosis in inflammatory tissues, indicating that necroptosis is related to the pathogenesis of IBD. However, due to the complexity in regulation of necroptosis and the involvement of multiple functions of relevant signaling molecules, the specific mechanism remains elusive. Necroptosis may play a vital regulatory role in the pathogenesis of IBD, which provides a new idea and method for further exploring the therapeutic target of IBD.