Resveratrol Prevents Vibrio vulnificus-Induced Sepsis by Attenuating Necroptosis.

Objective: This study investigated how the natural phytophenol and potent SIRT1 activator resveratrol (RSV) regulate necroptosis during Vibrio vulnificus (V. vulnificus)-induced sepsis and the potential mechanism. Methods: The effect of RSV on V. vulnificus cytolysin (VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays. Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V. vulnificus-induced sepsis mouse model. Results: RSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells. RSV also inhibited the inflammatory response, had a protective effect on histopathological changes, and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages, lung, spleen, and liver tissues of V. vulnificus-induced septic mice in vivo. Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V. vulnificus-induced septic mice. RSV also improved the survival of V. vulnificus-induced septic mice. Conclusion: Our findings collectively demonstrate that RSV prevented V. vulnificus-induced sepsis by attenuating necroptosis, highlighting its potency in the clinical management of V. vulnificus-induced sepsis.

Mixed lineage kinase domain-like pseudokinase: Conventional (necroptosis) and unconventional (necroptosis-independent) functions and features.

Mixed lineage kinase domain-like pseudokinase (MLKL) is the terminal and indispensable mediator of necroptosis. Necroptosis, also known as programmed cell necrosis, is a caspase-independent cell death mechanism involved in various pathologic and inflammatory processes. Triggering necroptosis could be an alternative approach in treating apoptosis-resistant cancer cells to prevent recurrent disease. In addition to its function in necroptosis, MLKL plays a role as a regulator in many cellular processes independent of necroptosis. A better understanding of the intracellular function of MLKL and its role in various diseases and pathologic conditions is needed to enable discovery of new targeted therapies. Various necroptosis-dependent and independent functions of MLKL are reviewed in this chapter, with a focus on functions of MLKL in necroptosis, autophagy, inflammation, tissue regeneration, and endosomal trafficking.

TREM-1 triggers necroptosis of macrophages through mTOR-dependent mitochondrial fission during acute lung injury.

BACKGROUND: Necroptosis of macrophages is a necessary element in reinforcing intrapulmonary inflammation during acute lung injury (ALI). However, the molecular mechanism that sparks macrophage necroptosis is still unclear. Triggering receptor expressed on myeloid cells-1 (TREM-1) is a pattern recognition receptor expressed broadly on monocytes/macrophages. The influence of TREM-1 on the destiny of macrophages in ALI requires further investigation. METHODS: TREM-1 decoy receptor LR12 was used to evaluate whether the TREM-1 activation induced necroptosis of macrophages in lipopolysaccharide (LPS)-induced ALI in mice. Then we used an agonist anti-TREM-1 Ab (Mab1187) to activate TREM-1 in vitro. Macrophages were treated with GSK872 (a RIPK3 inhibitor), Mdivi-1 (a DRP1 inhibitor), or Rapamycin (an mTOR inhibitor) to investigate whether TREM-1 could induce necroptosis in macrophages, and the mechanism of this process. RESULTS: We first observed that the blockade of TREM-1 attenuated alveolar macrophage (AlvMs) necroptosis in mice with LPS-induced ALI. In vitro, TREM-1 activation induced necroptosis of macrophages. mTOR has been previously linked to macrophage polarization and migration. We discovered that mTOR had a previously unrecognized function in modulating TREM-1-mediated mitochondrial fission, mitophagy, and necroptosis. Moreover, TREM-1 activation promoted DRP1Ser616 phosphorylation through mTOR signaling, which in turn caused surplus mitochondrial fission-mediated necroptosis of macrophages, consequently exacerbating ALI. CONCLUSION: In this study, we reported that TREM-1 acted as a necroptotic stimulus of AlvMs, fueling inflammation and aggravating ALI. We also provided compelling evidence suggesting that mTOR-dependent mitochondrial fission is the underpinning of TREM-1-triggered necroptosis and inflammation. Therefore, regulation of necroptosis by targeting TREM-1 may provide a new therapeutic target for ALI in the future.

Machilin D Promotes Apoptosis and Autophagy, and Inhibits Necroptosis in Human Oral Squamous Cell Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) accounts for about 90% of all head and neck cancers, the prognosis is very poor, and there are no effective targeted therapies. Herein, we isolated Machilin D (Mach), a lignin, from the roots of Saururus chinensis (S. chinensis) and assessed its inhibitory effects on OSCC. Herein, Mach had significant cytotoxicity against human OSCC cells and showed inhibitory effects against cell adhesion, migration, and invasion by inhibiting adhesion molecules, including the FAK/Src pathway. Mach suppressed the PI3K/AKT/mTOR/p70S6K pathway and MAPKs, leading to apoptotic cell death. We investigated other modes of programmed cell death in these cells and found that Mach increased LC3I/II and Beclin1 and decreased p62, leading to autophagosomes, and suppressed the necroptosis-regulatory proteins RIP1 and MLKL. Our findings provide evidence that the inhibitory effects of Mach against human YD-10B OSCC cells are related to the promotion of apoptosis and autophagy and inhibition of necroptosis and are mediated via focal adhesion molecules.

Necroptosis Induced by Delta-Tocotrienol Overcomes Docetaxel Chemoresistance in Prostate Cancer Cells.

Prostate cancer (PCa) represents the fifth cause of cancer death in men. Currently, chemotherapeutic agents for the treatment of cancers, including PCa, mainly inhibit tumor growth by apoptosis induction. However, defects in apoptotic cellular responses frequently lead to drug resistance, which is the main cause of chemotherapy failure. For this reason, trigger non-apoptotic cell death might represent an alternative approach to prevent drug resistance in cancer. Several agents, including natural compounds, have been shown to induce necroptosis in human cancer cells. In this study we evaluated the involvement of necroptosis in anticancer activity of delta-tocotrienol (δ-TT) in PCa cells (DU145 and PC3). Combination therapy is one tool used to overcome therapeutic resistance and drug toxicity. Evaluating the combined effect of δ-TT and docetaxel (DTX), we found that δ-TT potentiates DTX cytotoxicity in DU145 cells. Moreover, δ-TT induces cell death in DU145 cells that have developed DTX resistance (DU-DXR) activating necroptosis. Taken together, obtained data indicate the ability of δ-TT to induce necroptosis in both DU145, PC3 and DU-DXR cell lines. Furthermore, the ability of δ-TT to induce necroptotic cell death may represent a promising therapeutical approach to overcome DTX chemoresistance in PCa.

Necroptosis of macrophage is a key pathological feature in biliary atresia via GDCA/S1PR2/ZBP1/p-MLKL axis.

Biliary atresia (BA) is a severe inflammatory and fibrosing neonatal cholangiopathy disease characterized by progressive obstruction of extrahepatic bile ducts, resulting in cholestasis and progressive hepatic failure. Cholestasis may play an important role in the inflammatory and fibrotic pathological processes, but its specific mechanism is still unclear. Necroptosis mediated by Z-DNA-binding protein 1 (ZBP1)/phosphorylated-mixed lineage kinase domain-like pseudokinase (p-MLKL) is a prominent pathogenic factor in inflammatory and fibrotic diseases, but its function in BA remains unclear. Here, we aim to determine the effect of macrophage necroptosis in the BA pathology, and to explore the specific molecular mechanism. We found that necroptosis existed in BA livers, which was occurred in liver macrophages. Furthermore, this process was mediated by ZBP1/p-MLKL, and the upregulated expression of ZBP1 in BA livers was correlated with liver fibrosis and prognosis. Similarly, in the bile duct ligation (BDL) induced mouse cholestatic liver injury model, macrophage necroptosis mediated by ZBP1/p-MLKL was also observed. In vitro, conjugated bile acid-glycodeoxycholate (GDCA) upregulated ZBP1 expression in mouse bone marrow-derived monocyte/macrophages (BMDMs) through sphingosine 1-phosphate receptor 2 (S1PR2), and the induction of ZBP1 was a prerequisite for the enhanced necroptosis. Finally, after selectively knocking down of macrophage S1pr2 in vivo, ZBP1/p-MLKL-mediated necroptosis was decreased, and further collagen deposition was markedly attenuated in BDL mice. Furthermore, macrophage Zbp1 or Mlkl specific knockdown also alleviated BDL-induced liver injury/fibrosis. In conclusion, GDCA/S1PR2/ZBP1/p-MLKL mediated macrophage necroptosis plays vital role in the pathogenesis of BA liver fibrosis, and targeting this process may represent a potential therapeutic strategy for BA.

Transposable elements activation triggers necroptosis in mouse embryonic stem cells.

Deficiency of the histone H3K9 methyltransferase SETDB1 induces RIPK3-dependent necroptosis in mouse embryonic stem cells (mESCs). However, how necroptosis pathway is activated in this process remains elusive. Here we report that the reactivation of transposable elements (TEs) upon SETDB1 knockout is responsible for the RIPK3 regulation through both cis and trans mechanisms. IAPLTR2_Mm and MMERVK10c-int, both of which are suppressed by SETDB1-dependent H3K9me3, act as enhancer-like cis-regulatory elements and their RIPK3 nearby members enhance RIPK3 expression when SETDB1 is knockout. Moreover, reactivated endogenous retroviruses generate excessive viral mimicry, which promotes necroptosis mainly through Z-DNA-binding protein 1 (ZBP1). These results indicate TEs play an important role in regulating necroptosis.

Canonical cellular stress granules are required for arsenite-induced necroptosis mediated by Z-DNA-binding protein 1.

Cellular stress granules arise in cells subjected to stress and promote cell survival. A cellular protein that localizes to stress granules is Z-DNA-binding protein 1 (ZBP1), which plays a major role in necroptosis, a programmed cell death pathway mediated by the kinase RIPK3. Here, we showed that the stress granule inducer arsenite activated RIPK3-dependent necroptosis. This pathway required ZBP1, which localized to arsenite-induced stress granules. RIPK3 localized to stress granules in the presence of ZBP1, leading to the formation of ZBP1-RIPK3 necrosomes, phosphorylation of the RIPK3 effector MLKL, and execution of necroptosis. Cells that did not form stress granules did not induce necroptosis in response to arsenite. Together, these results show that arsenite induces ZBP1-mediated necroptosis in a manner dependent on stress granule formation.

Single-Cell Sequencing Reveals Necroptosis-Related Prognostic Genes of Glioblastoma.

Background: Glioblastoma (GBM) is one of the most malignant forms of brain cancer, with the extremely lower survival rate. Necroptosis (NCPS) is also one of the most wide types of cell death, and its clinical importance in GBM is not clear. Methods: We first identified necroptotic genes in GBM by single-cell RNA sequencing analysis of our surgical samples and weighted coexpression network analysis (WGNCA) from TCGA GBM data. The cox regression model with least absolute shrinkage and selection operator (LASSO) was used to construct the risk model. Then, KM plot and reactive operation curve (ROC) analysis were used to assess the prediction ability of the model. At last, the infiltrated immune cells and gene mutation profiling were investigated between the high- and low-NCPS groups as well. Result: The risk model including ten necroptosis-related genes was identified as an independent risk factor for the outcome. In addition, we found that the risk model is correlated with the infiltrated immune cells and tumor mutation burden in GBM. NDUFB2 is identified to be a risk gene in GBM with bioinformatical analysis and in vitro experiment validation. Conclusion: This risk model of necroptosis-related genes might provide clinical evidence for GBM interventions.

Identification of a necroptosis-related gene signature as a novel prognostic biomarker of cholangiocarcinoma.

Background: Cholangiocarcinoma (CHOL) is the most prevalent type of malignancy and the second most common form of primary liver cancer, resulting in high rates of morbidity and mortality. Necroptosis is a type of regulated cell death that appears to be involved in the regulation of several aspects of cancer biology, including tumorigenesis, metastasis, and cancer immunity. This study aimed to construct a necroptosis-related gene (NRG) signature to investigate the prognosis of CHOL patients using an integrated bioinformatics analysis. Methods: CHOL patient data were acquired from the Gene Expression Omnibus (GEO) (GSE89748, GSE107943) and The Cancer Genome Atlas (TCGA) databases, with NRGs data from the necroptosis pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Univariate and multivariate regression analyses were performed to establish the NRG signatures. Kaplan-Meier (KM) curves were used to evaluate the prognosis of patients with CHOL. Functional enrichment analysis was performed to identify key NRG-associated biological signaling pathways. We also applied integrative multi-omics analysis to the high- and low-risk score groups. Spearman's rank correlation was used to clarify the relationship between the NRG signature and immune infiltration. Results: 65 differentially expressed (DE) NRGs were screened, five of which were selected to establish the prognostic signature of NRGS based on multivariate Cox regression analysis. We observed that low-risk patients survived significantly longer than high-risk patients. We found that patients with high-risk scores experienced higher immune cell infiltration, drug resistance, and more somatic mutations than patients with low-risk scores. We further found that sensitivities to GW843682X, mitomycin C, rapamycin, and S-trityl-L-cysteine were significantly higher in the low-risk group than in the high-risk group. Finally, we validated the expression of five NRGs in CHOL tissues using the TCGA database, HPA database and our clinical data. Conclusion: These findings demonstrate that the five-NRG prognostic signature for CHOL patients is reasonably accurate and valid, and it may prove to be of considerable value for the treatment and prognosis of CHOL patients in the future.

KW2449 ameliorates collagen-induced arthritis by inhibiting RIPK1-dependent necroptosis.

Objective: Necroptosis has recently been found to be associated with the pathogenesis of many autoimmune diseases, including rheumatoid arthritis (RA). This study was undertaken to explore the role of RIPK1-dependent necroptosis in the pathogenesis of RA and the potential new treatment options. Methods: The plasma levels of receptor-interacting protein kinase 1 (RIPK1) and mixed lineage kinase domain-like pseudokinase (MLKL) in 23 controls and 42 RA patients were detected by ELISA. Collagen-induced arthritis (CIA) rats were treated with KW2449 by gavage for 28 days. Arthritis index score, H&E staining, and Micro-CT analysis were used to evaluate joint inflammation. The levels of RIPK1-dependent necroptosis related proteins and inflammatory cytokines were detected by qRT-PCR, ELISA and Western blot, and the cell death morphology was detected by flow cytometry analysis and high-content imaging analysis. Results: The plasma levels of RIPK1 and MLKL in RA patients were higher than those in healthy people, and were positively correlated with the severity of RA. KW2449 could reduce joint swelling, joint bone destruction, tissue damage, and the plasma levels of inflammatory cytokines in CIA rats. Lipopolysaccharide combined with zVAD (LZ) could induce necroptosis in RAW 264.7 cells, which could be reduced by KW2449. RIPK1-dependent necroptosis related proteins and inflammatory factors increased after LZ induction and decreased after KW2449 treatment or knockdown of RIPK1. Conclusion: These findings suggest that the overexpression of RIPK1 is positively correlated with the severity of RA. KW2449, as a small molecule inhibitor targeting RIPK1, has the potential to be a therapeutic strategy for RA treatment by inhibiting RIPK1-dependent necroptosis.

RIP3-mediated microglial necroptosis promotes neuroinflammation and neurodegeneration in the early stages of diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of blindness that poses significant public health concerns worldwide. Increasing evidence suggests that neuroinflammation plays a key role in the early stages of DR. Microglia, long-lived immune cells in the central nervous system, can become activated in response to pathological insults and contribute to retinal neuroinflammation. However, the molecular mechanisms of microglial activation during the early stages of DR are not fully understood. In this study, we used in vivo and in vitro assays to investigate the role of microglial activation in the early pathogenesis of DR. We found that activated microglia triggered an inflammatory cascade through a process called necroptosis, a newly discovered pathway of regulated cell death. In the diabetic retina, key components of the necroptotic machinery, including RIP1, RIP3, and MLKL, were highly expressed and mainly localized in activated microglia. Knockdown of RIP3 in DR mice reduced microglial necroptosis and decreased pro-inflammatory cytokines. Additionally, blocking necroptosis with the specific inhibitor GSK-872 improved retinal neuroinflammation and neurodegeneration, as well as visual function in diabetic mice. RIP3-mediated necroptosis was activated and contributed to inflammation in BV2 microglia under hyperglycaemic conditions. Our data demonstrate the importance of microglial necroptosis in retinal neuroinflammation related to diabetes and suggest that targeting necroptosis in microglia may be a promising therapeutic strategy for the early stages of DR.

A novel prognostic signature for clear cell renal cell carcinoma constructed using necroptosis-related miRNAs.

BACKGROUND: This work aims to analyze the relationship between necroptosis-related microRNAs (miRNAs) and the prognosis of clear cell renal cell carcinoma (ccRCC). METHODS: The miRNAs expression profiles of ccRCC and normal renal tissues from The Cancer Genome Atlas (TCGA) database were used to construct a matrix of the 13 necroptosis-related miRNAs. Cox regression analysis was used to construct a signature to predict the overall survival of ccRCC patients. The genes targeted by the necroptosis-related miRNAs in the prognostic signature were predicted using miRNA databases. Gene Ontology (Go) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to investigate the genes targeted by the necroptosis-related miRNAs. The expression levels of selected miRNAs in 15 paired samples (of ccRCC tissues and adjacent normal renal tissues) were investigated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). RESULTS: Six necroptosis-related miRNAs were found to differentially expressed between ccRCC and normal renal tissues. A prognostic signature consisting of miR-223-3p, miR-200a-5p, and miR-500a-3p was constructed using Cox regression analysis and risk scores were calculated. Multivariate Cox regression analysis showed that the hazard ratio was 2.0315 (1.2627-3.2685, P = 0.0035), indicating that the risk score of the signature was an independent risk factor. The receiver operating characteristic (ROC) curve showed that the signature has a favorable predictive capacity and the Kaplan-Meier survival analysis indicated that ccRCC patients with higher risk scores had worse prognoses (P < 0.001). The results of the RT-qPCR verified that all three miRNAs used in the signature were differentially expressed between ccRCC and normal tissues (P < 0.05). CONCLUSION: The three necroptosis-related-miRNAs used in this study could be a valuable signature for the prognosis of ccRCC patients. Necroptosis-related miRNAs should be further explored as prognostic indicators for ccRCC.

A novel necroptosis-associated miRNA signature predicting prognosis of endometrial cancer and correlated with immune infiltration.

OBJECTIVE: Necroptosis is a form of programmed cell death identified irrelevant to caspases, which plays an important role in the tumorigenesis and development of cancer. MicroRNAs (miRNAs) are important regulators of both necroptosis and cancer. MATERIALS AND METHODS: Expression of sixteen necroptosis-associated miRNAs were analyzed in 546 endometrial cancer (EC) tissues and 33 paracancerous samples from the Cancer Genome Atlas (TCGA). Cox regression analysis was used to evaluate the correlations between miRNAs and overall survival. MiRNAs risk score (Mrs) and nomogram were established to assess the potential value of necroptosis-related miRNAs on prognosis. Expression of miRNA-148a-3p in endometrial cancer cells and endometrial epithelial cells was detected by quantitative real-time PCR (qRT-PCR). The targets genes of miR-148a-3p were predicted using miRDB, miRTarBase and TargetScan and the prognostic-related genes were screened. Immune infiltration analysis was conducted to explore the potential mechanism of these target genes. RESULTS: We identified fourteen differentially expressed miRNAs and selected seven miRNAs (miR-15a-5p, miR148a-3p, miR-7-5p, miR-141-3p, miR-200a-5p, miR-223-3p, miR-16-5p) for prognostic-model construction. The area under the curve (AUC) of receiver operating characteristic (ROC) curve for 1-, 2- and 5-year survival were 0.678, 0.652 and 0.656 respectively. Multivariate analysis revealed that the Mrs was an independent prognostic factor considering other risk factors (HR = 1.928, 95% CI = 1.072-3.467, P = 0.028). Among these miRNAs, miRNA-148a-3p was up-regulated in cancer tissues and cells, and Kaplan-Meier analysis showed its significance in overall survival (OS). The target genes, DNAJB4 and PRNP, were associated with poor prognosis and correlated with tumor immune infiltration. CONCLUSIONS: Our study constructed a novel necroptosis-associated miRNAs model for prognosis prediction, and DNAJB4 and PRNP may be therapeutic targets for EC.

Identification and Analysis of Necroptosis-Related Genes in COPD by Bioinformatics and Experimental Verification.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous and complex progressive inflammatory disease. Necroptosis is a newly identified type of programmed cell death. However, the role of necroptosis in COPD is unclear. This study aimed to identify necroptosis-related genes in COPD and explore the roles of necroptosis and immune infiltration through bioinformatics. The analysis identified 49 differentially expressed necroptosis-related genes that were primarily engaged in inflammatory immune response pathways. The infiltration of CD8+ T cells and M2 macrophages in COPD lung tissue was relatively reduced, whereas that of M0 macrophages was increased. We identified 10 necroptosis-related hub genes significantly associated with infiltrated immune cells. Furthermore, 7 hub genes, CASP8, IL1B, RIPK1, MLKL, XIAP, TNFRSF1A, and CFLAR, were validated using an external dataset and experimental mice. CFLAR was considered to have the best COPD-diagnosing capability. TF and miRNA interactions with common hub genes were identified. Several related potentially therapeutic molecules for COPD were also identified. The present findings suggest that necroptosis occurs in COPD pathogenesis and is correlated with immune cell infiltration, which indicates that necroptosis may participate in the development of COPD by interacting with the immune response.

FERMT1 Is a Prognostic Marker Involved in Immune Infiltration of Pancreatic Adenocarcinoma Correlating with m6A Modification and Necroptosis.

As an important member of the kindlin family, fermitin family member 1 (FERMT1) can interact with integrin and its aberrant expression involves multiple tumors. However, there are few systematic studies on FERMT1 in pancreatic carcinoma (PAAD). We used several public databases to analyze the expression level and clinicopathological characteristics of FERMT1 in PAAD. Meanwhile, the correlation between FERMT1 expression and diagnostic and prognostic value, methylation, potential biological function, immune infiltration, and sensitivity to chemotherapy drugs in PAAD patients were investigated. FERMT1 was significantly up-regulated in PAAD and correlated with T stage, and histologic grade. High FERMT1 expression was closely connected with poor prognosis and can be used to diagnose PAAD. Moreover, the methylation of six CpG sites of FERMT1 was linked to prognosis, and FERMT1 expression was significantly related to N6-methyladenosine (m6A) modification. Functional enrichment analysis revealed that FERMT1 co-expression genes participated in diverse biological functions including necroptosis. In addition, the expression of FERMT1 was associated with immune cell infiltration and the expression of immune checkpoint molecules. Finally, FERMT1 overexpression may be sensitive to chemotherapy drugs such as Palbociclib, AM-5992 and TAE-226. FERMT1 can serve as a diagnostic and prognostic marker of PAAD, which is connected with immune cell infiltration and the modulation of m6A and necroptosis.

Advances in mechanism and regulation of PANoptosis: Prospects in disease treatment.

PANoptosis, a new research hotspot at the moment, is a cell death pattern in which pyroptosis, apoptosis, and necroptosis all occur in the same cell population. In essence, PANoptosis is a highly coordinated and dynamically balanced programmed inflammatory cell death pathway that combines the main features of pyroptosis, apoptosis, and necroptosis. Many variables, such as infection, injury, or self-defect, may be involved in the occurrence of PANoptosis, with the assembly and activation of the PANoptosome being the most critical. PANoptosis has been linked to the development of multiple systemic diseases in the human body, including infectious diseases, cancer, neurodegenerative diseases, and inflammatory diseases. Therefore, it is necessary to clarify the process of occurrence, the regulatory mechanism of PANoptosis, and its relation to diseases. In this paper, we summarized the differences and relations between PANoptosis and the three types of programmed cell death, and emphatically expounded molecular mechanism and regulatory patterns of PANoptosis, with the expectation of facilitating the application of PANoptosis regulation in disease treatment.

High Glucose-Induced Kidney Injury via Activation of Necroptosis in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is a major microvascular complication of diabetes mellitus (DM) and is closely associated to programmed cell death. However, the complex mechanisms of necroptosis, an alternative cell death pathway, in DKD pathogenesis are yet to be elucidated. This study indicates that necroptosis is involved in DKD induced by high glucose (HG) both in vivo and in vitro. HG intervention led to the activation of RIPK1/RIPK3/MLKL signaling, resulting in renal tissue necroptosis and proinflammatory activation in streptozotocin/high-fat diet- (STZ/HFD-) induced diabetic mice and HG-induced normal rat kidney tubular cells (NRK-52E). We further found that in HG-induced NRK-52E cell, necroptosis might, at least partly, depend on the levels of reactive oxygen species (ROS). Meanwhile, ROS participated in necroptosis via a positive feedback loop involving the RIPK1/RIPK3 pathway. In addition, blocking RIPK1/RIPK3/MLKL signaling by necrostatin-1 (Nec-1), a key inhibitor of RIPK1 in the necroptosis pathway, or antioxidant N-acetylcysteine (NAC), an inhibitor of ROS generation, could effectively protect the kidney against HG-induced damage, decrease the release of proinflammatory cytokines, and rescue renal function in STZ/HFD-induced diabetic mice. Inhibition of RIPK1 effectively decreased the activation of RIPK1-kinase-/NF-κB-dependent inflammation. Collectively, we demonstrated that high glucose induced DKD via renal tubular epithelium necroptosis, and Nec-1 or NAC treatment downregulated the RIPK1/RIPK3/MLKL pathway and finally reduced necroptosis, oxidative stress, and inflammation. Thus, RIPK1 may be a therapeutic target for DKD.

The double-edged functions of necroptosis.

Necroptosis refers to a regulated form of cell death induced by a variety of stimuli. Although it has been implicated in the pathogenesis of many diseases, there is evidence to support that necroptosis is not purely a detrimental process. We propose that necroptosis is a "double-edged sword" in terms of physiology and pathology. On the one hand, necroptosis can trigger an uncontrolled inflammatory cascade response, resulting in severe tissue injury, disease chronicity, and even tumor progression. On the other hand, necroptosis functions as a host defense mechanism, exerting antipathogenic and antitumor effects through its powerful pro-inflammatory properties. Moreover, necroptosis plays an important role during both development and regeneration. Misestimation of the multifaceted features of necroptosis may influence the development of therapeutic approaches targeting necroptosis. In this review, we summarize current knowledge of the pathways involved in necroptosis as well as five important steps that determine its occurrence. The dual role of necroptosis in a variety of physiological and pathological conditions is also highlighted. Future studies and the development of therapeutic strategies targeting necroptosis should fully consider the complicated properties of this type of regulated cell death.

Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells.

Sapoviruses belonging to the genus Sapovirus within the family Caliciviridae are commonly responsible for severe acute gastroenteritis in both humans and animals. Caliciviruses are known to induce intrinsic apoptosis in vitro and in vivo, however, calicivirus-induced necroptosis remains to be fully elucidated. Here, we demonstrate that infection of porcine kidney LLC-PK cells with porcine sapovirus (PSaV) Cowden strain as a representative of caliciviruses induces receptor-interacting protein kinase 1 (RIPK1)-dependent necroptosis and acts as proviral compared to the antiviral function of PSaV-induced apoptosis. Infection of LLC-PK cells with PSaV Cowden strain showed that the interaction of phosphorylated RIPK1 (pRIPK1) with RIPK3 (pRIPK3), mixed lineage kinase domain-like protein (pMLKL) increased in a time-dependent manner, indicating induction of PSaV-induced RIPK1-dependent necroptosis. Interfering of PSaV-infected cells with each necroptotic molecule (RIPK1, RIPK3, or MLKL) by treatment with each specific chemical inhibitor or knockdown with each specific siRNA significantly reduced replication of PSaV but increased apoptosis and cell viability, implying proviral action of PSaV-induced necroptosis. In contrast, treatment of PSaV-infected cells with pan-caspase inhibitor Z-VAD-FMK increased PSaV replication and necroptosis, indicating an antiviral action of PSaV-induced apoptosis. These results suggest that PSaV-induced RIPK1-dependent necroptosis and apoptosis‒which have proviral and antiviral effects, respectively‒counterbalanced each other in virus-infected cells. Our study contributes to understanding the nature of PSaV-induced necroptosis and apoptosis and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections.