ABSTRACT
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.
ABSTRACT
Background Programmed necrosis is closely related to the occurrence and development of neurodegenerative diseases, but whether lead causes programmed cell necrosis has not been reported. Objective This experiment is designed to probe into the function of programmed necrosis and the effect of its inhibitor on lead-induced microglia (BV2 cell) injury. Methods The BV2 cells at logarithmic growth phase were treated with 0, 1, 5, 10, 25, 50, 100, and 200 μmol·L−1 lead acetate for 12, 24, 36, and 48 h, respectively, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to determine cell viability. After treatment with 0, 25, 50, and 100 μmol·L−1 lead acetate for 24 h, enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to determine the expressions of tumor necrosis factor-α (TNF-α), receptor-interacting protein kinase 3 (RIPK3), receptor-interacting protein kinase 1 (RIPK1), and mixed lineage kinase domain-like protein (MLKL) in the cells, and the effect of RIPK1 inhibitor Nec-1 pretreatment on lead-induced BV2 cell injury . Results The BV2 cell viability decreased with higher lead concentration (r12 h=−0.995, r24 h=−0.984, r36 h=−0.983, r48 h=−0.981, all P<0.01) and time extension (only for 5 μmol·L−1 lead acetate, r=−0.994, P<0.01). Compared with the control group, the BV2 cell viability decreased at the same exposure time when the concentration of lead was above 10 μmol·L−1 (P<0.01). Compared with the control group, the expressions of RIPK1 and MLKL were increased in the 25, 50, and 100 μmol·L−1 lead groups (P<0.05 or 0.01), accompanied by an increase in the contents of inflammatory cytokine TNF-α, especially in the 100 μmol·L−1 lead group, the increment was the highest (P<0.01). The expression levels of p-RIPK1 and p-MLKL in BV2 cells were both increased when the concentration of lead acetate was above 50 μmol·L−1 (P<0.01). In addition, pretreatment with Nec-1 increased the cell viability rate and decreased the necrosis and late apoptosis rate of BV2 cells exposed to lead compared with corresponding lead exposure groups (P<0.05). Conclusions Lead can reduce BV2 cell viability, increase necrosis rate, and up-regulate the expressions of RIPK1, RIPK3, amd MLKL, and the phosphorylation levels of RIPK1 and MLKL. The RIPK1 inhibitor Nec-1 has an intervention effect on lead-induced damage in BV2 cells, indicating that programmed necrosis may play a role in lead neurotoxicity.
ABSTRACT
Objective:To explore the level of expression, clinical significance of receptor-interacting protein kinase-3(RIPK3) in the bronchoalveolar alveolar lavage fluid (BALF) of mycoplasma pneumoniae pneumonia(MPP) in children and the relationship between RIPK3 and various cytokines.Methods:Using a case-control study, 30 refractory mycoplasma pneumoniae pneumonia children treated in Children′s Hospital of Xuzhou Medical University from February 2019 to February 2021 were selected as the MRPP group, 35 mycoplasma pneumoniae pneumonia children as the RPP group.Meanwhile, 20 sex- and age-matched hospitalized children with bronchial foreign body were selected as the case-control group.In all subjects, protein levels of RIPK3 and mixed lineage kinase domain-like protein(MLKL) were determined by Western Blot.Meanwhile, levels of IL-6, IL-1β and TNF-α were determined by enzyme linked immuno sorbent assay(ELISA). Compare levels of different parameters between the three groups and analyze the correlation between levels of RIPK3 and MLKL, L-6, IL-1β, TNF-α in the BALF of MPP children using Spearman rank correlation analysis.Results:There were statistically significant differences in the levels of RIPK3, MLKL, IL-6, IL-1β and TNF-α in BALF between RMPP group, MPP group and control group (all P<0.001). Pairwise comparisons also showed statistical differences, and the RMPP group was the highest, followed by MPP group, and the control group was the lowest.The level of RIPK3 in BALF of MPP children was positively correlated with MLKL, IL-6, IL-1β and TNF-α ( r=0.711, 0.676, 0.725, 0.651, P<0.001). Linear regression analysis shows: MLKL=0.432×RIPK3. Conclusion:RIPK3 may be involved in the occurrence and development of MPP in children, and is closely related to cytokines such as IL-6, IL-1β and TNF-α.
ABSTRACT
Osteoarthritis (OA) is the most common chronic disabling joint disease, and currently there is no effective treatment for the cause. Necroptosis plays a key role in many diseases, and receptor-interacting protein kinase 3 (RIP3) is a key regulator during necroptosis process. Studies have shown that the expression level of RIP3 was significantly upregulated in human and mouse OA degenerative cartilage tissues, suggesting the occurrence of necroptosis. However, the specific pathophysiological role of RIP3 in cartilage is still unclear. This study intends to sequence and analyze the transcriptome of chondrocytes before and after RIP3 overexpression, and explore the specific functional mechanism of RIP3 in OA pathogenesis. RNA sequencing results showed that overexpression of RIP3 induced upregulation of 244 genes and downregulation of 277 genes in chondrocytes. Sixteen candidate target genes were screened out by constructing gene co-expression network for further verification at mRNA level, and the results suggested that RIP3 had the most significant inductive effect on the expression of phosphoinositide-3kinase, regulatory subunit 5 (Pik3r5), integrin subunit beta 3 (Itgb3) and MYB proto-oncogene like 2 (Mybl2). Results from CCK-8 and lactate dehydrogenase activity analysis showed that silencing the expression of Itgb3 by siRNA significantly rescued chondrocyte viability decline and necroptosis induced by RIP3, and it also inhibited the upregulating effect of RIP3 on the expression of catabolism-related genes Mmp1, Mmp13 and Il6, as well as the downregulating effect of RIP3 on the expression of anabolism-related genes Acan, Col2a1 and Sox9. This study has demonstrated that RIP3 promotes chondrocyte necrosis and cartilage matrix metabolism disorders by upregulating the expression of Itgb3 in chondrocytes, and ultimately leads to cartilage degeneration. These findings provided potential novel targets for the clinical treatment of OA, and further clarified the pathophysiological significance of necroptosis.
ABSTRACT
Background@#Necroptosis plays an important role in human atherosclerosis and atheroma development. Since receptor interacting protein kinase-3 (RIP3) acts as a key mediator of necroptosis, this study aimed to explore its relationship between plasma RIP3 levels and coronary artery disease (CAD) and discover a potential new biomarker for screening CAD subtypes and severity.@*Methods@#A total of 318 patients with CAD who had coronary angiography and 166 controls in Peking Union Medical College Hospital from September 2017 to January 2018 were enrolled in this study. Patients with CAD were divided into three subgroups: patients with stable coronary artery disease (SCAD), patients with unstable angina (UA), and patients with myocardial infarction (MI). The severity of atherosclerosis was determined by Gensini score (GSS). Logistic regression was used to determine the relationship between plasma RIP3 levels and CAD. The correlation between plasma RIP3 and GSS was calculated using multiple linear regression models.@*Results@#Overall, plasma RIP3 levels were significantly higher than serum RIP3 levels. Plasma RIP3 levels in patients with CAD were significantly higher than those in controls. Plasma RIP3 levels were strongly associated with CAD (odds ratio: 6.00, 95% confidence interval 3.04–11.81; P < 0.001). Plasma RIP3 levels increased linearly from controls to patients with SCAD, then patients with UA, and finally to patients with MI. We found a significantly positive correlation between proportion of cases of acute coronary syndrome in subjects and their plasma RIP3 level quartile. Plasma RIP3 levels were also associated with GSS (B 0.027; standard error 0.012; P < 0.05).@*Conclusions@#Plasma RIP3 levels were independently associated with CAD. Plasma RIP3 levels could potentially supplement clinical assessment to screen CAD and determine CAD severity.
ABSTRACT
Objective:To explore the kinase activity of novel receptor interacting protein kinase 3 (RIPK3) mutants. Methods:The four amino acids (Q84WDF87) of RIPK3 were mutated respectively and these mutants were co-transfected with mixed lineage kinase domain like pseudokinase (MLKL) into HEK293T cells. The auto-phosphorylation of these mutants at S232 and phosphorylation of MLKL at S345 were detected by Western blotting. The interaction between RIPK3 and MLKL was tested by co-immunoprecipitation. The oligomerization of MLKL was detected by non-reducing gel. Results:The kinase activities of RIPK3ΔQ84, RIPK3ΔW85 and RIPK3ΔD86 were effectively decreased. Nevertheless, the kinase activities of RIPK3Q84A/RIPK3Q84E, RIPK3W85Y and RIPK3D86A/RIPK3D86Y did not change markedly. The auto-phosphorylation of RIPK3W85A at S232 was decreased without affecting phosphorylation and oligomerization of MLKL. Conclusion:The amino acid site Q84, W85 or D86 plays a critical role in RIPK3 kinase activity. The kinase activity of RIPK3W85A is decreased, but it does not affect MLKL.
ABSTRACT
Objective To explore the effects of bone marrow mesenchymal stem cells (MSCs) transplantation on receptor-interacting protein kinase 1 (RIP1) and RIP3 in rat brain after cardiac arrest (CA).Methods Sprague Dawley (SD) rats were randomly (random number) divided into sham group (n=8),CA group (n=8) and MSCs group (n=8).Animals were subjected to asphyxial cardiac arrest and followed by cardiopulmonary resuscitation (CPR).In MSCs group or CA group,animals received intravenous injection of 1 × 106 MSCs in 0.5 mL phosphate buffer solution (PBS) or 0.5 mL PBS alone at 1 h after successful resuscitation.Neurological deficit scores (NDS) were assessed at 3 d after CPR.Donor MSCs in brain were detected under a fluorescent microscope.HE staining of brain tissue was performed to observe necrotic neurons.Western blot analysis was performed to measure the levels of RIP1 and RIP3 in brain.Multiple comparisons were made by analysis of variance or Kruskal-Wallis H test.Results At 3 d after CPR,MSCs group demonstrated higher NDS than CA group [72.5(71.5,73.2) vs.63.0(62.5,64.1),Z=3.376,P=0.001].DAPI-labeled MSCs were primarily observed in the cerebral cortex.The percentage of necrotic neurons in MSCs group was significantly lower than that in CA group [(29.6±5.9)％ vs.(57.2±6.4)％,t=8.922,P＜0.01].The levels of RIP1 and RIP3 expression in brain in MSCs group were significantly lower than those in CA group [RIP1:0.227(0.193,0.243) vs.0.599(0.535,0.629),Z=3.151,P=0.001;RIP3:0.217(0.203,0.274) vs.0.543(0.533,0.555),Z=3.361,P=0.001].Conclusion MSCs transplantation improves neurological function after CPR from CA in rats likely associated with inhibiting necroptosis.
ABSTRACT
Objective To investigate the signaling pathway and the key signal molecules of protein kinase (RIPK)3 in SH-SY5Y cells. Methods SH-SY5Y cells were transfected with RIPK3 expression plasmid vector to upregulate intracellular RIPK3, while the SH-SY5Y cells were transfected with empty vector plasmid, which was considered as control group. Western blot assay was used to check the expression of exogenous RIPK3 in cells. The proliferation rate of SH-SY5Y cells was determined by MTT assay at designated time to detect exogenous RIPK3 activity. Whole transcriptome sequencing (RNAseq) was used to detect the transcription of genes. Whole-transcriptomic gene transcription was measured by following Ingenuity Pathway Analysis (IPA) to obtain downstream signaling pathways and the key molecule, which were partly confirmed by following droplet digital PCR (ddPCR). Results Exogenous RIPK3 showed biological activity in SH-SY5Y, which inhibited the proliferation of cells. IPA showed that znic finger protein 36 (ZFP36) was significantly up-regulated as compared with that of the control group. The tran?scription levels of ZFP36 downstream genes such as tumor necrosis factor (TNF), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF) and mRNA-decapping enzyme 2 (DCP2) were affected at the same time. Conclusion Within the limitations of this study, it seems that RIPK3 is notable for the development, inflammation and tumorigenesis of the nervous system as an independent regulator of ZFP36 gene and downstream effectors.