RIPK3 Expression Research Articles

Therapeutic contribution of melatonin to the treatment of septic cardiomyopathy: A novel mechanism linking Ripk3-modified mitochondrial performance and endoplasmic reticulum function

The basic pathophysiological mechanisms underlying septic cardiomyopathy have not yet been completely clarified. Disease-specific treatments are lacking, and care is still based on supportive modalities. The aim of our study was to assess the protective effects of melatonin on septic cardiomyopathy, with a focus on the interactions between receptor-interacting protein kinase 3 (Ripk3), the mitochondria, endoplasmic reticulum (ER) and cytoskeletal degradation in cardiomyocytes. Ripk3 expression was increased in heart samples challenged with LPS, followed by myocardial inflammation, cardiac dysfunction, myocardial breakdown and cardiomyocyte death. The melatonin treatment attenuated septic myocardial injury in a comparable manner to the genetic depletion of Ripk3. Molecular investigations revealed that Ripk3 intimately regulated mitochondrial function, ER stress, cytoskeletal homeostasis and cardioprotective signaling pathways. Melatonin-mediated inhibition of Ripk3 improved mitochondrial bioenergetics, reduced mitochondria-initiated oxidative damage, sustained mitochondrial dynamics, ameliorated ER stress, normalized calcium recycling, and activated cardioprotective signaling pathways (including AKT, ERK and AMPK) in cardiomyocytes. Interestingly, Ripk3 overexpression mediated resistance to melatonin therapy following the infection of LPS-treated hearts with an adenovirus expressing Ripk3. Altogether, our findings identify Ripk3 upregulation as a novel risk factor for the development of sepsis-related myocardial injury, and melatonin restores the physiological functions of the mitochondria, ER, contractile cytoskeleton and cardioprotective signaling pathways. Additionally, our data also reveal a new, potentially therapeutic mechanism by which melatonin protects the heart from sepsis-mediated dysfunction, possibly by targeting Ripk3.

Hypothermia Inhibits Cerebral Necroptosis and NOD-Like Receptor Pyrin Domain Containing 3 Pathway in a Swine Model of Cardiac Arrest

BackgroundTargeted temperature management (TTM) is commonly used in hypothermia after cardiopulmonary resuscitation (CPR), and its mechanism to improve cerebral function is complex. This study aimed to investigate the effects of TTM on necroptosis and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome in the brain tissue of pigs after CPR. Materials and methodsVentricular fibrillation was induced, and CPR was performed 10 min later in nine pigs in the normothermia group and nine pigs in the TTM group. The body temperature in the TTM group was dropped to 33°C after CPR and maintained for 24 h, whereas in the normothermia group, it was maintained at 38°C. Before CPR and at 30 h after CPR, serum neuron-specific enolase and S-100β were measured. At 30 h after CPR, pigs were euthanized, and brain tissues were collected for measurement of receptor-interacting protein kinase (RIPIK) 1, RIPK3, mixed lineage kinase domain-like (MLKL), NLRP3, cysteinyl aspartate-specific proteinase (caspase)-1, interleukin (IL)-1β, and IL-18. ResultsSerum neuron-specific enolase and S-100β were increased significantly (P < 0.05) in the two CPR-treated groups compared with the sham group and more obviously in the normothermia group. In addition, the expression of RIPK3, phosphorylated MLKL, and NLRP3 in brain tissues was increased. The expression of RIPK3, phosphorylated MLKL, NLRP3, and caspase-1 as well as the levels of IL-1β and IL-18 were lower (P < 0.05) in the TTM group compared with the normothermia group. ConclusionsNecroptosis and the NLRP3 pathway were activated after CPR. TTM may attenuate postresuscitation brain injury through the regulation of necroptosis and the NLRP3 pathway.

MicroRNA-325-3p protects the heart after myocardial infarction by inhibiting RIPK3 and programmed necrosis in mice

BackgroundReceptor-interacting serine-threonine kinase 3 (RIPK3)-mediated necroptosis has been implicated in the progression of myocardial infarction (MI), but the underlying mechanisms, particularly whether microRNAs (miRNAs) are involved, remain largely unknown.ResultsA microarray analysis was used to screen for miR-325-3p expression in myocardial tissues from MI mice, and the expression was confirmed with qRT-PCR. The levels of myocardial enzymes were measured using commercial kits, and an echocardiography system was utilized for the detection of cardiac function parameters. The pathological features and infarction sizes of cardiac tissues were examined using H&E, TCC and Masson’s trichrome staining, and the amount of cell apoptosis was determined using an in situ TUNEL assay. Cardiomyocytes were isolated and then subjected to hypoxia induction in vitro. The expression of the RIPK1, RIPK3 and phosphorylated MLKL (p-MLKL) proteins was measured using a Western blot. The mouse cardiomyocyte cell viability was analyzed by an MTT assay. The mRNA target of miR-325-3p was predicted using TargetScan v7.2 and then validated using a dual-luciferase reporter assay. The overexpression of miR-325-3p evidently decreased the expression levels of lactate dehydrogenase (LDH), phosphocreatine kinase (CK), superoxide dismutase (SOD) and malondialdehyde (MDA), inhibited left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD), and promoted left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVES). In addition, miR-325-3p overexpression attenuated the degree of injury to the cardiac tissue, decreased the infarct sizes and downregulated the expression of the necrosis-related proteins RIPK1, RIPK3 and p-MLKL.ConclusionsThe RIPK1/RIPK3/p-MLKL axis-induced necroptosis that occurred during MI was mediated by a miRNA module, miR-325-3p, which can effectively ameliorate the symptoms of MI by suppressing the expression of RIPK3.

High glucose-induced apoptosis and necroptosis in podocytes is regulated by UCHL1 via RIPK1/RIPK3 pathway

Diabetic nephrology (DN) is attributed largely to the depletion of podocytes, which is closely associated to apoptosis. However, the complex mechanism of podocyte loss in DN pathogenesis remains unclear. Recently, necroptosis has emerged as an important cell death model in many pathological conditions, which is regulated through RIPK1/RIPK3 pathway. In addition, necroptosis was found to share several upstream signaling pathways with apoptosis. Therefore, it was speculated that both apoptosis and necroptosis may occur in podocytes during the process of podocyte injury in DN. Herein, necroptosis and apoptosis were shown to be involved in podocyte injury induced by high glucose (HG), both in vitro and in vivo, with a high level of positive signaling markers RIPK1 (298.4 ± 17.35), cleaved caspase 3 (497.1 ± 23.09), RIPK3 (108.4 ± 14.92), and MLKL (470.4 ± 15.73) than the control groups. Scaning electron microscopy examination revealed the morphological characteristics of necroptotic and apoptotic cells, which differed remarkably. z-VAD-fmk, a pan-inhibitor of apoptosis, could block apoptosis and enhance necroptosis. Furthermore, UCHL1 was found to play a major role in promoting podocyte necroptosis by regulating the ubiquitination state of the RIPK1/RIPK3 pathway. The half-life of RIPK1 and RIPK3 proteins reduced and the expression of RIPK1, RIPK3, and MLKL decreased significantly after the knockdown of UCHL1. It was shown that UCHL1 exerted a more regulatory response to necroptosis. These data suggested that necroptosis may have more effect on the loss of podocytes than apoptosis in DN with the regulation of UCHL1. Thus, inhibiting UCHL1 to downregulate the RIPK1/RIPK3 pathway may be a novel strategy to protect the podocytes in DN patients.

Role of docosahexaenoic acid in enhancement of docetaxel action in patient-derived breast cancer xenografts.

The objective of this study was to investigate if DHA dietary supplementation enhances the anticancer actions of docetaxel (TXT) in two different drug resistant triple negative breast cancer (TNBC) patient-derived xenografts (PDX)s. In two experiments, female NSG mice bearing TNBC PDXs were randomized to one of two nutritionally adequate diets (20% w/w): control (0% DHA), or DHA (3.9% w/w of total fat) and injected with 0 or 5mg/kg TXT, twice weekly for 6weeks (n = 8 per group). Treatment response was determined by significant differences in tumor weight, and apoptotic, proliferation and cell cycle markers at endpoint. Mice bearing MAXF574 xenografts fed DHA diet and treated with TXT had a 57% reduction in tumor weight compared to mice fed control diet (P < 0.004), a 64% reduction compared to control + TXT (P < 0.01) and a 34% reduction compared to DHA with no TXT (P < 0.04). DHA + TXT reduced MAXF401 xenografts growth compared to control and control + TXT (by 43% and 34%, respectively, P < 0.05). In both xenografts, DHA + TXT resulted in a higher expression of proapoptotic proteins Ripk1 and Bid, lower expression of proliferation marker Ki67 and anti-apoptotic proteins Bcl-2 and Parp, and a greater increase in cell cycle arrest as measured by decreased Survivin expression when compared to control + TXT mice (P < 0.05). This work is the first to confirm that DHA supplementation during chemotherapy treatment improves TXT action in two PDX models of TNBC. The results suggest that decreases in tumor size occurred via changes in apoptosis, cell proliferation, and cell cycle pathways.

Upregulated necroptosis-pathway-associated genes are unfavorable prognostic markers in low-grade glioma and glioblastoma multiforme.

BackgroundGlioma accounts for 70% of primary brain malignancies in adults with unfavorable prognoses. In the past decades, much efforts have been invested to identify better biomarkers for predicting prognoses. Recently, necroptosis has been reported as a specialized pathway of programmed necrosis. Moreover, regulators of necroptosis-pathway-associated genes (RIPK1, RIPK3 and MLKL) were reported to be related to prognoses of many types of tumors. However, the prognostic value of these genes in diffuse glioma including low-grade glioma (LGG) and glioblastoma multiforme (GBM) remains unknown.MethodsAn online tool-Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) was used to analyze different expression of necroptosis-pathway-associated genes between tumor and normal tissue, correlation between RIPK1, RIPK3 and MLKL, the relationship between necroptosis-pathway-associated genes and prognosis [overall survival (OS) and disease-free survival (DFS)] in LGG and GBM. The median expression of RIPK1, RIPK3 and MLKL was used to divide patients into high- versus low-expression group. All graphic presentations were drawn by Gepia database.ResultsExpression of RIPK1 and RIPK3 were significantly higher in tumor tissue of GBM as compared with normal tissue. A moderate correlation between MLKL and RIPK3 was demonstrated in both LGG (R =0.79) and GBM (R =0.79). In LGG, higher expression of RIPK1, RIPK3, and MLKL were associated with poor OS and DFS with HR values of 2.2, 2, 1.9 for OS and 1.7, 1.8, 1.6 for DFS, respectively. In GBM, only a higher expression of MLKL was associated with worse OS and DFS with HR values of 1.5 and 1.6, respectively.ConclusionsRegulators of necroptosis-pathway-associated genes appear to have a potential to serve as biomarkers of prognosis in both LGG and GBM.

PS1327 PRO‐NECROPTOTIC RIPK3 AS A STAGE‐DEPENDENT MARKER IN THE BONE MARROW OF PATIENTS WITH MDS AND CMML

Background:Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro‐inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high‐risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro‐inflammatory processes trigger necroptosis.Aims:The aim of this project is to identify pro‐necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation.Methods:We therefore analyzed the impact of pro‐necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS‐MLD, MDS‐RS‐SLD and MDS‐RS‐MLD), 5 patients with late MDS (MDS‐EB1 and MDS‐EB2), 5 patients with CMML‐1, 7 patients with CMML‐2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age‐matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitors for 72 h. Short term viability analysis by flow cytometry were performed and long‐term analysis using growth factor enriched methylcellulose.Results:Expression of RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML‐1, CMML‐2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression.Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitorsfor 72 h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10–14 days colony forming capacity was significantly increased in low‐risk MDS patients after inhibition of pro‐necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.Summary/Conclusion:We conclude that RIPK3 plays a critical stage‐dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3‐Expression. We further hypothesize that pro‐inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

Genome-wide DNA methylation and RNA expression profiles identified RIPK3 as a differentially methylated gene in Chlamydia pneumoniae infection lung carcinoma patients in China.

Aim: To explore the relationship between Chlamydia pneumonia (Cpn) infection and lung cancer using integrative methylome and transcriptome analyses.Methods: Twelve primary lung cancer patients who were positive for Cpn and twelve patients who were negative were selected for demographic, clinicopathological, and lifestyle matching. Genomic DNA and RNA were extracted and DNA methylation and mRNA levels were detected using the Infinium Human Methylation 450 Beadchip array and mRNA + lncRNA Human Gene Expression Microarray. We identified differentially expressed methylation and genes profiles.Results: Integrative analysis revealed an inverse correlation between differentially expressed genes and DNA methylation. Cpn-related lung cancer methylated genes (target genes) were introduced into the gene ontology and KEGG, PID, BioCarta, Reactome, BioCyc and PANTHER enrichment analyses using a q-value cutoff of 0.05 to identify potentially functional methylation of abnormal genes associated with Cpn infection. Gene sets enrichment analysis was evaluated according to MsigDB. Levels of differentially expressed methylated sites were quantitatively verified. The promoter methylation sites of 62 genes were inversely related to expression levels. According to the quantitative analysis of DNA methylation, the methylation level of the RIPK3 promoter region was significantly different between Cpn-positive cancerous and adjacent tissues, but not between Cpn-negative cancerous and adjacent tissues.Conclusion: Hypomethylation of the RIPK3 promoter region increases RIPK3 expression, leading to regulated programmed necrosis and activation of NF-κB transcription factors, which may contribute to the development and progression of Cpn-related lung cancer.

FKBP12 mediates necroptosis by initiating RIPK1-RIPK3-MLKL signal transduction in response to TNF receptor 1 ligation.

Necroptosis is a regulated form of necrotic cell death that is mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed-lineage kinase domain-like protein (MLKL), which mediates necroptotic signal transduction induced by tumor necrosis factor (TNF). Although many target proteins for necroptosis have been identified, no report had indicated that FK506-binding protein 12 (FKBP12, also known as FKBP1A), an endogenous protein that regulates protein folding and conformation alteration, is involved in mediating necroptosis. In this study, we found that FKBP12 acts as a novel target protein in mediating necroptosis and the related systemic inflammatory response syndrome triggered by TNF. The mechanistic study discovered that FKBP12 is essential for initiating necrosome formation and RIPK1-RIPK3-MLKL signaling pathway activation in response to TNF receptor 1 ligation. In addition, FKBP12 is indispensable for RIPK1 and RIPK3 expression and subsequent spontaneous phosphorylation, which are essential processes for initial necrosome formation and necroptotic signal transduction; therefore, FKBP12 may target RIPK1 and RIPK3 to mediate necroptosis in vitro and in vivo Collectively, our data demonstrate that FKBP12 could be a potential therapeutic target for the clinical treatment of necroptosis-associated diseases.

Furosine, a Maillard Reaction Product, Triggers Necroptosis in Hepatocytes by Regulating the RIPK1/RIPK3/MLKL Pathway.

As one of the typical Maillard reaction products, furosine has been widely reported in a variety of heat-processed food. Though furosine was shown to be toxic on organs, its toxicity mechanism is still unclear. The present study aimed to investigate the toxicity mechanism of furosine in liver tissue. An intragastric gavage mice model (42-day administration, 0.1/0.25/0.5 g/kg of furosine per day) and a mice primary hepatocyte model were employed to investigate the toxicity mechanism of furosine on mice liver tissue. A metabonomics analysis of mice liver, serum, and red blood cells (RBC) was performed. The special metabolic mediator of furosine, lysophosphatidylcholine 18:0 (LPC (18:0)) was identified. Then, the effect of the upstream gene phospholipase A2 gamma (PLA2-3) on LPC (18:0), as well as the effect of furosine (100 mg/L) on the receptor-interacting serine/threonine-protein kinase (RIPK)1/RIPK3/mixed lineage kinase domain-like protein (MLKL) pathway and inflammatory factors, was determined in liver tissue and primary hepatocytes. PLA2-3 was found to regulate the level of LPC (18:0) and activate the expression of RIPK1, RIPK3, P-MLKL, and of the inflammatory factors including tumor necrosis factor α (TNF-α) and interleukin (IL-1β), both in liver tissue and in primary hepatocytes. Upon treatment with furosine, the upstream sensor PLA2-3 activated the RIPK1/RIPK3/MLKL necroptosis pathway and caused inflammation by regulating the expression of LPC (18:0), which further caused liver damage.

144 Expression and significance of RIPK3 in the cutaneous squamous cell carcinoma

Background: The cutaneous squamous cell carcinoma is a malignant tumor that occurs in the epidermis or accessory cells. It accounts for about 20% of all non-melanoma skin cancers and is the second most common skin malignancy. RIPK3, a member of the receptor-interacting protein kinase family, is a TNFR1-regulated transcription factor that plays an important role in cellular stress of various factors and may be involved in regulating cell cycle, inhibiting apoptosis, and regulating programmed cell death, etc. But the role of RIPK3 in the cSCC has not been reported. Objective: To compare the expression of RIPK3 in normal skin tissues and cSCC lesions, and to analyze the clinical significance of RIPK3 in cSCC. Materials and Methods: 30 cases of normal skin tissue and 30 cases of cSCC lesions were collected. The expression of RIPK3 protein was detected by immunohistochemical SP method. Cutaneous squamous cell carcinoma cell lines SCL-1, HSC-1 and A431 cells were cultured, and the primary keratinocytes HEKa cells were used as the control group. The expression of RIPK3 was detected by western blot and qRT-PCR. Results: In normal skin tissues, RIPK3 protein was mainly expressed in the basal layer of the epidermis and the lower part of the spinous layer. The positive expression rate was 60%, while in the cSCC lesions, the expression was significantly decreased. The positive expression rate was 0%. The positive expression rate and expression intensity of RIPK3 were statistically significant (*** P<0.001, vs. Ctrl). Western blot and qRT-PCR results showed that RIPK3 expression was decreased in SCL-1, HSC-1 and A431 cells compared with the control group (*** P<0.001, vs. Ctrl). Conclusion: The expression of RIPK3 protein in cSCC lesions and cells is significantly lower than that in control. It can be seen that the abnormal expression of RIPK3 may play a role in the development of cutaneous squamous cell carcinoma.

RIPK1 and death receptor signaling drive biliary damage and early liver tumorigenesis in mice with chronic hepatobiliary injury.

Hepatocyte apoptosis is intrinsically linked to chronic liver disease and hepatocarcinogenesis. Conversely, necroptosis of hepatocytes and other liver cell types and its relevance for liver disease is debated. Using liver parenchymal cell (LPC)-specific TGF-beta-activated kinase 1 (TAK1)-deficient (TAK1LPC-KO) mice, which exhibit spontaneous hepatocellular and biliary damage, hepatitis, and early hepatocarcinogenesis, we have investigated the contribution of apoptosis and necroptosis in hepatocyte and cholangiocyte death and their impact on liver disease progression. Here, we provide in vivo evidence showing that TAK1-deficient cholangiocytes undergo spontaneous necroptosis induced primarily by TNFR1 and dependent on RIPK1 kinase activity, RIPK3, and NEMO. In contrast, TAK1-deficient hepatocytes die by FADD-dependent apoptosis, which is not significantly inhibited by LPC-specific RIPK1 deficiency, inhibition of RIPK1 kinase activity, RIPK3 deficiency or combined LPC-specific deletion of TNFR1, TRAILR, and Fas. Accordingly, normal mouse cholangiocytes can undergo necroptosis, while primary hepatocytes are resistant to it and die exclusively by apoptosis upon treatment with cell death-inducing stimuli in vitro, likely due to the differential expression of RIPK3. Interestingly, the genetic modifications that conferred protection from biliary damage also prevented the spontaneous lethality that was often observed in TAK1LPC-KO mice. In the presence of chronic hepatocyte apoptosis, preventing biliary damage delayed but did not avert hepatocarcinogenesis. On the contrary, inhibition of hepatocyte apoptosis fully prevented liver tumorigenesis even in mice with extensive biliary damage. Altogether, our results suggest that using RIPK1 kinase activity inhibitors could be therapeutically useful for cholestatic liver disease patients.

RIPK3 expression as a potential predictive and prognostic marker in metastatic colon cancer.

Colorectal cancer is one of the primary causes of cancer-related deaths and 5-fluorouracil (5-FU) therapy remains the cornerstone of treatment in these patients. Resistance to 5-FU represents a major obstacle; therefore, finding new predictive and prognostic markers is crucial for improvement of patient outcomes. Recently a new type of programmed cell death was discovered-necroptosis, which depends on receptor interacting protein 3 (RIPK3). Preclinical data showed that necroptotic cell death is an important effector mechanism of 5-FU-mediated anticancer activity. To investigate the predictive and prognostic performance of RIPK3 expression in primary tumors. Colon cancer patients (n=74) with metastatic stage were included in this retrospective study and all were treated with first-line 5-FU based chemotherapy. Immunohistochemical staining was performed. The progression free survival for the low expression group of RIPK3 was 5.6 months (95% CI, 4.4-6.8) vs 8.4 months (95% CI, 6.4-10.3) of the group with high expression (p=0.02). Moreover, patients with high expression of RIPK3 were associated with lower risk of disease progression HR 0.61 (95% CI, 0.38-0.97; p=0.044). Patients with high expression levels of RIPK3 also had significantly longer mean overall survival (OS) of 29.3 months (95% CI, 20.8-37.8) as compared with those with low expression: 18.5 months (95% CI, 15.06-21.9) (p= 0.036). In addition, univariate analysis showed that high level of RIPK3 expression was associated with a longer OS HR 0.59 (95% CI, 0.35-0.98; p=0.044). This study suggests that expression of RIPK3 in primary tumors of metastatic colon cancer patients should be further investigated for its potential as a promising predictive and prognostic marker.

Role of necroptosis in intestinal injury induced by autologous orthotopic liver transplantation in rats

Objective To evaluate the role of necroptosis in intestinal injury induced by autologous orthotopic liver transplantation (AOLT) in rats. Methods Twenty-four SPF adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 3 groups (n=8 each) using a random number table method: sham operation group (group S), AOLT group (group T) and necroptosis inhibitor necrostatin-1 group (group N). Necrostatin-1 1.0 mg/kg and the equal volume of dimethyl sulfoxide (DMSO) were intraperitoneally injected at 30 min before surgery in N and T groups, respectively.Blood samples were collected from the inferior vena cava at 6 h after opening the portal vein (at 6 h after the end of surgery in group S) for determination of serum diamine oxidase (DAO), D-lactic acid (D-LA) and intestinal fatty acid binding protein (I-FABP) concentrations by enzyme-linked immunosorbent assay.Rats were sacrificed after blood sampling, and the intestine was removed for examination of the pathological changes (with a light microscope) and for determination of malondialdehyde (MDA) contents and superoxide dismutase (SOD) activities (using a spectrophotometer), and the expression of receptor-interacting protein kinase-1 (RIPK1), RIPK3 and mixed lineage kinase domain-like protein (MLKL) in intestinal tissues (by Western blot). Intestinal damage was assessed and scored using Chiu′s scoring system. Results Compared with group S, the serum DAO, D-LA and I-FABP concentrations, MDA content and Chiu′s score were significantly increased, SOD activity was decreased, and the expression of RIPK1, RIPK3 and MLKL was up-regulated in group T (P<0.05). Compared with group T, the serum DAO, D-LA and I-FABP concentrations, MDA content and Chiu′s score were significantly decreased, the SOD activity was increased, and the expression of RIPK1, RIPK3 and MLKL was down-regulated in group N (P<0.05). Conclusion Necroptosis is involved in the pathophysiological process of intestinal injury induced by AOLT in rats. Key words: Liver transplantation; Intestine; Necrosis

Induction of two independent immunological cell death signaling following hemoglobinuria -induced acute kidney injury: In vivo study

The main important clinical signs in acute kidney injury (AKI) after sever Hemiscorpius lepturus envenomation in patients is associated with proteinuria, hemolysis and hemoglobinuria. Unfortunately, our limited knowledge of molecular cell death mechanism in H. lepturus induced AKI restricts the development of desirable therapeutics. So, in the present study, the potential role of necroptosis and ferroptosis in H. lepturus induced AKI were investigated in male albino mice. The animals were administrated by SC injection of venom (1, 2.5, 5 and 10 mg/kg) based on LD50 determination. After 1 and 7 days, urinalysis, stereological assessments and gene expression of Ngal, Tnf-α, Tlr-4, Ripk3, Mlkl and Acsl4 were evaluated by real time PCR. Our data revealed that upregulation of renal Ngal expression is associated with the gene over expression of Tnf-α, Tlr-4, Ripk3 and Mlkl in venom treated kidneys. We observed that the Malondialdehyde (MDA) level was increased in dose-dependent manner similar to Acsl4 gene over expression suggesting a main role of ferroptosis in hemoglobinuria mediated AKI following envenomation. Moreover, transcriptional enhancement of Tlr-4and Tnf-α receptor can cause phosphorylation of Ripk3-Mlkl complex, collapse of membrane potential and DAMPs release which intensified the inflammation cytokines in kidney. Taken together, it supposes co-existence of two separate pathways of regulated necrosis and inflammatory environment provides a promising outlook in prevention and management of hemoglobinuria induced AKI following envenomation in clinical practice.

Wnt signaling induces neurite outgrowth in mouse retinal ganglion cells

Wingless-type (Wnt) signaling pathways mediate axonal growth and remodeling in the embryonic optic nerve, brain and spinal cord. Recent studies demonstrated that the canonical Wnt/β-catenin signaling pathway also induces axonal regeneration after injury in the optic nerve of adult animals. However, the molecular mechanisms of Wnt-mediated axonal growth are not well understood. Additionally, because Wnt signaling is stimulated in neurons as well as neighboring non-neuronal cells, the cell type(s) responsible for Wnt-induced axonal regeneration are not known. The objectives of this study were to investigate potential mechanisms and target cells of Wnt3a stimulated neurite growth using primary retinal ganglion cell (RGC) cultures. We demonstrated that Wnt3a ligand induced dose-dependent increases in average neurite length and number of neurites in RGCs. QPCR analysis of candidate mediators showed that Wnt3a-dependent neurite growth was associated with lower expression of Ripk1 and Ripk3 genes. Additionally, inhibiting Ripk1 signaling with Necrostatin-1s led to increased neurite number per cell but not increased neurite length. Therefore, Ripk signaling may be involved in mediating the effects of Wnt3a on neurite number but Ripk activity does not seem to be required for Wnt3a-dependent regulation of neurite length. This study shows that RGCs are direct cellular targets of Wnt3a-induced axonal growth, and we identified a novel association between Wnt signaling and Rip kinases in neurite formation.

Fine particulate matter (PM2.5) enhances airway hyperresponsiveness (AHR) by inducing necroptosis in BALB/c mice

ObjectiveTo observe the effects of prolonged exposure to high concentrations of PM2.5 on the trachea and lungs of mice and to determine whether the damages to the trachea and lung are induced by necroptosis. MethodsSix- to eight-week-old female Balb/C mice of PM group were restrained in an animal restraining device using a nose-only “PM2.5 online enrichment system” for 8 weeks, in Shijiazhuang, Hebei, China. Anti -Fas group was exposed to PM2.5 inhalation and anti-Fas treatment via intranasal instillation. The mice in the control group inhaled filtered clean air. PM2.5 sample was collected and analyzed. Airway Hyperresponsiveness (AHR) was tested. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for Hematoxylin and eosin (HE) staining, electron microscopy, cellular inflammation, cytokines, Tunel, Fas, RIPK3 and MLKL expression. ResultsCompared to the other two groups, PM group displayed significantly increased AHR, neutrophils in BALF, significant bronchitis and alveolar epithelial hyperplasia and inflammation and necroptosis which were indicated by increased TUNEL, Fas, RIPK3 and MLKL measure. ConclusionOur findings suggest that PM2.5 can enhance AHR and these changes are induced by necroptosis-related inflammation.

TAK1 suppresses RIPK1-dependent cell death and is associated with disease progression in melanoma.

Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies as well as immunotherapies frequently remain inefficient. Alternative means to treat melanoma, in particular through the induction of programmed cell death modalities such as apoptosis or necroptosis, therefore still need to be explored. Here, we report that melanoma cell lines expressing notable amounts of RIPK1, RIPK3 and MLKL, the key players of necroptosis signal transduction, fail to execute necroptotic cell death. Interestingly, the activity of transforming growth factor β-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5Z)-7-Oxozeaenol, deletion of MAP3K7 or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNFα or TRAIL based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5Z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Correspondingly, elevated expression of TAK1 correlates with reduced disease free survival in patients diagnosed with primary melanoma. Overall, our results therefore demonstrate that TAK1 suppresses the susceptibility to RIPK1-dependent cell death and that high expression of TAK1 indicates an increased risk for disease progression in melanoma.

Effect of ulinastatin on programmed necrosis in hippocampal neurons in a rat model of global cerebral ischemia-reperfusion

Objective To evaluate the effect of ulinastatin on programmed necrosis in hippocampal neurons in a rat model of global cerebral ischemia-reperfusion (I/R). Methods Forty-eight clean-grade healthy adult male Sprague-Dawley rats, aged 8 weeks, weighing 280-320 g, were divided into 3 groups(n=16 each)using a random number table method: sham operation group(Sham group), global cerebral I/R group(I/R group)and ulinastatin group(UT group). Global cerebral I/R was produced by 4-vessel occlusion method in chloral hydrate-anesthetized rats in I/R and UT groups. Ulinastatin 100 000 U/kg was injected via the tail vein at the onset of ischemia in group UT, and the equal volume of normal saline was given instead in Sham and I/R groups.Neurological deficit score(NDS)was estimated at 6, 12 and 24 h of reperfusion.Animals were sacrificed at 24 h of reperfusion, brains were removed and the hippocampi were obtained for examination of pathological changes (with a light microscope) and for determination of the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity (by spectrophotometry), and expression of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) in hippocampal tissues (by Western blot). Results Compared with Sham group, the NDS was significantly increased at each time point, the MDA content was increased, the SOD activity was decreased, and the expression of RIPK1, RIPK3 and MLKL was up-regulated in I/R and UT groups (P<0.05). Compared with I/R group, the NDS was significantly decreased at each time point, the MDA content was decreased, the SOD activity was increased, and the expression of RIPK1, RIPK3 and MLKL was down-regulated in UT group (P<0.05). The pathological changes of hippocampi were significantly attenuated in UT group when compared with I/R group. Conclusion The mechanism by which ulinastatin ameliorates global cerebral I/R injury is related to inhibiting programmed necrosis in hippocampal neurons of rats. Key words: Trypsin inhibitors; Reperfusion injury; Hippocampus; Neurons; Necrosis

Receptor interacting protein kinases-dependent necroptosis as a new, potent mechanism for elimination of the endothelial cells during luteolysis in cow

Necroptosis is an alternative form of programmed cell death regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent. In the present study, to clarify if necroptosis in luteal endothelial cells (LECs) participates and contributes for bovine luteolysis, we investigated RIPK1 and RIPK3 localization in luteal tissue and their expression in cultured LECs after treatment with selected immune factors - mediators of luteolytic action of prostaglandin F2α (PGF). In addition, effects of tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM), and/or nitric oxide donor - NONOate (100 μM) on viability and CASP3 activity in the cultured LECs were investigated. Furthermore, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 μM) on RIPKs and CASPs expression, were evaluated. Localization of RIPK1 and RIPK3 protein in the cultured LECs were determined. In cultured LECs, expression of RIPKs mRNA were up-regulated by TNF + IFNG at 12 h, and by PGF (1 μM) or NONOate at 24 h, respectively (P < 0.05). Although NONOate decreased cell viability, it prevented TNF + IFNG-stimulated CASP3 activity in cultured LECs. Nec-1 prevented TNF + IFNG-induced RIPK1 and CASP3 mRNA expression at 12 h and prevented RIPK3 mRNA expression. These findings suggest that RIPKs-dependent necroptosis which are induced by TNF + IFNG, PGF or NO could be potent mechanism responsible for LECs cell death and disappearance of luteal capillaries in regressing bovine CL.