ABSTRACT
Programmed cell death (PCD) is a genetically determined, active and orderly cell death in the organism, and it affects the evolution of the organism, maintenance of its homeostasis, and development of several tissues and organs. The abnormal regulation of this process is closely related to various human diseases, including cancer. The identified pathways of PCD include apoptosis, autophagy, necroptosis, pyroptosis, and ferroptosis, which can be activated when cells are stimulated by various internal and external environmental factors. These pathways can induce cell death or maintain cell survival in kidney cancer cells under the regulation of various signaling molecules, thus affecting tumor progression or therapeutic efficacy. In this paper, the role of these PCD pathways in the development of kidney cancer was reviewed in light of recent research advances to provide new directions for the in-depth study of the pathogenesis of kidney cancer and the development of targeted antitumor drugs.
ABSTRACT
Necroptosis is one of the regulated cell death, which involves receptor interacting protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain like protein (MLKL) signaling pathway. Among them, MLKL is the final execution of necroptosis. The formation of RIPK1/RIPK3/MLKL necrosome induces the phosphorylated MLKL, and the activated MLKL penetrates into the membrane bilayer to form membrane pores, which damages the integrity of the membrane and leads to cell death. In addition to participating in necroptosis, MLKL is also closely related to other cell death, such as NETosis, pyroptosis, and autophagy. Therefore, MLKL is involved in the pathological processes of various diseases related to abnormal cell death pathways (such as cardiovascular diseases, neurodegenerative diseases and cancer), and may be a therapeutic target of multiple diseases. Understanding the role of MLKL in different cell death can lay a foundation for seeking various MLKL-related disease targets, and also guide the development and application of MLKL inhibitors.
Subject(s)
Protein Kinases/metabolism , Necroptosis/physiology , Receptor-Interacting Protein Serine-Threonine Kinases , Signal Transduction , Pyroptosis , ApoptosisABSTRACT
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.
Subject(s)
Animals , Mice , Necroptosis , Resveratrol/therapeutic use , Vibrio vulnificus , Sepsis/drug therapy , Blotting, WesternABSTRACT
Previous studies focused on the unique regulatory mechanisms of different cell death pathways. However， recent studies highlight crosstalk and co-ordination between these pathways and initiate a new cell death process called PANoptosis （pyroptosis， apoptosis， necroptosis）. PANoptosis is an inflammatory programmed cell death pathway regulated by the PANoptosome complex with critical features of pyroptosis， and/or necroptosis but cannot be characterized by any of the death modes of pyroptosis， apoptosis or necroptosis alone. By activating the PANoptosis pathway， some triggers like bacterial， viral， and fungal infections can cause death of the host. This review explains the PANoptosis-related routes， regulators and their potential effects on blinding eye diseases.
ABSTRACT
Aluminum is a light metal which is rich in the earth's crust and widely used. Recently, the adverse health effects of environmental and occupational aluminum exposure on human have attracted more and more attention. Aluminum exposure has toxic effects on the central nervous system and is believed to be closely related to the development and progression of Alzheimer's disease. The neurotoxic mechanism of aluminum is complex, especially the role of regulated cell death (RCD) in aluminum-induced neuronal death remains to be further studied. RCD refers to all modes of cell death regulated by multiple intracellular signal transduction pathways under physiological and pathological conditions, including apoptosis, necroptosis, autophagy, pyroptosis, and ferroptosis. This review summarized the morphological characteristics and mechanisms of each RCD mode in the process of aluminum-induced neuronal death, and discussed the relationship and transformation between different RCD modes, providing a new scientific basis for future studies on the treatment and intervention of neurotoxicity induced by aluminum exposure.
ABSTRACT
Objective To investigate the protective effect of human umbilical cord mesenchymal stem cell-derived exosome (hucMSC-Exo) on renal ischemia-reperfusion injury (IRI), and to clarify the critical role and regulating mechanism of transient receptor potential canonical (TRPC) 6/poly adenosine-diphosphate-ribose polymerase (PARP) 1 signaling pathway during this process. Methods The hucMSC-Exo was extracted by ultracentrifugation, and identified by transmission electron microscope (TEM), nanoparticle tracing analysis and Western blot. SD rats were randomly divided into the sham operation group (group S), sham operation+TRPC6 inhibitor SKF96365 group (group SS), renal IRI group (group IRI), exosome treatment group (group EXO) and exosome +TRPC6 inhibitor SKF96365 group (group ES), with 6 rats in each group. Serum creatinine and blood urea nitrogen levels were detected. Pathological changes of renal tissues were observed by hematoxylin-eosin (HE) staining and Paller score was calculated. The expression levels of key molecules of necroptosis in rat renal tissues, including receptor-interacting protein kinase (RIPK)1, RIPK3 and mixed-lineage kinase domain-like protein (MLKL), TRPC6 and PARP1, were detected by Western blot. Results Typical saucer-like structure was observed under TEM. Nanoparticle tracing analysis showed that the average diameter of the extracted substance was 125.9 nm. Western blot revealed that the surface markers of CD9, CD63 and CD81 were positively expressed, confirmed that the extracted substance was exosome. Compared with group S, the serum creatinine and blood urea nitrogen levels were up-regulated, the pathological damage of renal tissues was worsened, Paller score was elevated, the relative expression levels of TRPC6 and PARP1 proteins were down-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were up-regulated in group IRI (all P < 0.05). Compared with group IRI, the serum creatinine and blood urea nitrogen levels were down-regulated, the pathological damage of renal tissues was mitigated, Paller score was decreased, the relative expression levels of TRPC6 and PARP1 proteins were up-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were down-regulated in group EXO (all P < 0.05). Compared with group EXO, the serum creatinine and blood urea nitrogen levels were up-regulated, the pathological damage of renal tissues was aggravated, Paller score was increased, the relative expression levels of TRPC6 and PARP1 proteins were down-regulated, and the relative expression levels of RIPK1, RIPK3 and MLKL proteins were up-regulated in group ES (all P < 0.05). Conclusions hucMSC-Exo may alleviate the necroptosis induced by renal IRI in rat models, which is related to the activation of TRPC6/PARP1 signaling pathway.
ABSTRACT
Objective:To study the effects of arsenic exposure on necroptosis pathway and inflammatory response of mouse myocardial cells.Methods:Sixty male C57BL/6J mice were randomly divided into control group (group C) and low, medium, and high dose arsenic exposure groups (groups L, M, H) based on body weight using a random number table method. Each group had 15 mice, and they drank 0.00, 0.15, 1.50, and 15.00 mg/L arsenic trioxide (As 2O 3) solution prepared with deionized water. The exposure period was 12 weeks. Hematoxylin-eosin (HE) staining and Masson trichrome staining of paraffin-embedded heart tissues were used to observe the histopathology changes of the heart. Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes of myocardial cells. The quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of inflammatory genes [tumor necrosis factor (TNF)-α and interleukin(IL)-6] and the genes involved in necroptosis pathway [receptor-interacting protein (RIP) 1, RIP3 and mixed-lineage kinase domain-like protein (MLKL)]. Protein expressions of RIP1 and RIP3 in the heart were assessed by western blotting. Results:Histopathological examination results showed there were myocardial necrosis, inflammatory cells infiltration and fibroblasts hyperplasia and other changes in groups M and H. TEM analysis revealed marked ultrastructural changes in groups M and H, including fractured myofibril, fractured Z lines of sarcomere, and swollen mitochondria with fractured cristae. Compared with group C (1.00 ± 0.00), the mRNA expression of RIP1 in group H was significantly up-regulated (1.41 ± 0.06, P < 0.05); the mRNA expressions of RIP3 (1.29 ± 0.14, 1.56 ± 0.08), MLKL (1.23 ± 0.05, 1.36 ± 0.07), TNF-α (2.20 ± 0.10, 2.23 ± 0.18) and IL-6 (1.87 ± 0.16, 1.63 ± 0.15) were significantly up-regulated in groups M and H ( P < 0.05). The protein expressions of RIP1 (0.43 ± 0.04, 0.50 ± 0.04) and RIP3 (0.68 ± 0.02, 0.84 ± 0.05) in groups M and H were higher than those in group C (0.25 ± 0.01, 0.45 ± 0.04, P < 0.05). Conclusion:Subchronic arsenic exposure induces histopathological changes such as myocardial necrosis and fibrosis in mice, inducing necroptosis and inflammatory reactions in myocardial cells.
ABSTRACT
ABSTRACT Background: Polysaccharides from edible mushrooms possess immunomodulatory, anti-inflammatory, and anti-tumor activities. Recent studies indicated that necroptosis plays a role in the pathogenesis of inflammatory diseases and mediates increased expression of inflammatory cytokines. Objective: Therefore, it is imperative to determine the impact of polysaccharide extract from Lentinula edodes (L. edodes) on inflammatory cytokines in experimental model of colitis in mice. Methods: Female C57BL/6 mice divided into three or four mice per group were used for this study. Polysaccharide sample was orally administered to mice prior to (7 days) and during colitis induction with 2.5% dextran sodium sulfate (7 days), followed by additional 3 days of administration. Changes in body weight and colon length were used as markers for colitis, and pro-inflammatory cytokines and tumor necrosis factor receptor 1 (TNFR1) expressions, as well as necroptosis were analyzed in the colon of colitis mice. Data obtained were analysed by Tukey-Kramer and two-tailed standard t tests. Results: The results indicated that the polysaccharide sample suppressed colitis in mice using effects on the body weight and colon length as markers. Also, it was demonstrated that necrostatin-1, a specific inhibitor of necroptosis, suppressed the expression of interleukin (IL)-8, a pro-inflammatory chemokine, in Caco-2 cells induced necroptosis induced by zVAD and TNF-α, an indication that necroptosis may be involved in the expression of pro-inflammatory cytokines. Moreover, the polysaccharide sample suppressed the expression of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-6, IL-1β, and interferon (IFN)-γ in the colon of mice. Conclusion: These results suggested that the suppressive effects of the polysaccharide sample on inflammatory cytokines expression may contribute to its anti-colitis effect, and so may serve as a potent therapeutic agent against inflammatory bowel disease.
RESUMO Contexto: Polissacarídeos de cogumelos comestíveis possuem atividades imunomodulatórias, anti-inflamatórias e anti-tumorais. Estudos recentes indicaram que a necroptose desempenha um papel na patogênese de doenças inflamatórias e regula o aumento da expressão de citocinas inflamatórias. Objetivo: Torna-se imprescindível determinar o impacto do extrato de polissacarídeo de Lentinula edodes (L. edodes) em citocinas inflamatórias em modelo experimental de colite em camundongos. Métodos: Foram utilizados para este estudo os camundongos C57BL/6 femininos divididos em três ou quatro camundongos por grupo. A amostra de polissacarídeo foi administrada oralmente em camundongos antes (7 dias) e durante a indução de colite com sulfato de dextran sulfato de sódio (7 dias), seguido por 3 dias adicionais de administração. Alterações no peso corporal e comprimento do cólon foram utilizadas como marcadores para colite, e citocinas pró-inflamatórias e tumores receptor fator 1 (TNFR1), bem como necroptose foram analisadas no cólon de camundongos colite. Os dados obtidos foram analisados por testes Tukey-Kramer e testes padrão t de duas caudas. Resultados: Os resultados indicaram que a amostra de polissacarídeo suprimiu colite em camundongos usando efeitos sobre o peso corporal e o comprimento do cólon como marcadores. Além disso, foi demonstrado que a necrostatina-1, inibidora específica da necroptose, suprimiu a expres são de interleucina (IL)-8, uma quimiocina pró-inflamatória, em células caco-2 induzidas necroptose induzidas por zVAD e TNF-α, uma indicação de que a necroptose pode estar envolvida na expressão de citocinas pro-inflamatórias. Além disso, a amostra de polissacarídeo suprimiu a expressão de citocinas pró-inflamatórias, como o fator de necrose tumoral (TNF)-α, IL-6, IL-1β e interferon (IFN)-γ no cólon dos camundongos. Conclusão: Esses resultados sugeriram que os efeitos supressivos da amostra de polissacarídeo na expressão de citocinas inflamatórias podem contribuir para o seu efeito anti-colite, podendo, portanto, servir como um potente agente terapêutico contra doença inflamatória intestinal.
ABSTRACT
Mitophagy is the selective degradation of damaged mitochondria, and it is of great significance to maintain the normal quantity and quality of mitochondria to ensure cell homeostasis and survival. Necroptosis is a type of programmed cell necrosis that can be induced by excessive mitophagy. Reactive oxygen species (ROS) are produced mainly by mitochondria and can damage mitochondria. Hyperoxic acute lung injury (HALI) is a serious complication of clinical oxygen therapy, and its pathogenesis is not clear. Existing studies have shown that mitophagy and necroptosis are involved in the occurrence of HALI. There are many mechanisms regulating mitophagy and necroptosis, including tumor necrosis factor-α (TNF-α), E3 ubiquitin protein ligase (PINK1/Parkin) protein pathway encoded by PTEN-induced kinase 1/PARK2 gene, phosphoglycerate mutase 5 (PGAM5), etc. PGAM5 has been proved to be a key factor linking mitophagy and necroptosis. Previous studies of our team found that the mechanism of microRNA-21-5p (miR-21-5p) alleviating HALI was related to its pGAM5-mediated inhibition of mitophagy, but the mechanism of PGAM5-mediated mitophagy and necroptosis remains unclear. Therefore, this paper reviews the targets of PGAM5-mediated mitophagy and necroptosis, in order to find clues of lung protection of pGAM5-mediated mitophagy and necroptosis in HALI, and provide theoretical basis for subsequent basic research.
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
Objective: To investigate the effect of berberine on programmed necrosis of hepatocytes induced by metabolic-associated fatty liver disease (MAFLD) in mice and its related molecular mechanism. Methods: Twenty male C57BL/6N mice were randomly divided into four groups (n=5 in each group): control group (S), fatty liver group (H), berberine group(B), nuclear factor erythroid 2-related factor 2 inhibitor group (Nrf2), and all-trans-retinoic acid (ATRA) group (A). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TC), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) concentrations were detected at the end of week 12 to calculate fatty liver index (liver mass/body mass ratio). Liver tissue was stained with HE, Masson and Oil Red O, and SAF score was used to evaluate the degree of liver injury. The expression levels of hepatic programmed necrosis-related proteins, namely receptor-interacting protein kinase 3 (RIPK3), phosphorylated mixed series protease-like domain (p-MLKL) and Nrf2 were detected by Western blot method. One-way ANOVA was used for intragroup comparisons and LSD-t tests were used for intergroup comparisons. Results: Compared with S group, H group serum ALT, AST, LDH, TG, TC, TNF-α, IL-1β levels and fatty liver index were significantly increased. The liver tissue was filled with vacuolar-like changes and inflammatory cell infiltration. Numerous red lipid droplets were observed with oil red O staining. Collagen fiber hyperplasia was evident with Masson staining. SAF scores (6.60 ± 0.55 and 0.80 ± 0.45) were significantly increased. The expressions of RIPK3 and p-MLKL were up-regulated. Nrf2 level was relatively increased, and the differences were statistically significant (P < 0.05). Compared with H group, berberine intervention group liver biochemical indexes, lipid levels, pro-inflammatory mediator expression, fatty liver index, and SAF score were significantly reduced, and the expression of RIPK3 and p-MLKL were down-regulated, while Nrf2 levels were further increased, and the differences were statistically significant (P<0.05). Compared with B group, treatment with Nrf2 inhibitor had antagonized the protective effect of berberine on fatty liver. Serum ALT, AST, LDH, TG, TC and TNF-α, IL-1β levels, fatty liver index, and SAF scores were significantly increased and the expressions of RIPK3 and p-MLKL were relatively increased, and the differences were statistically significant (P < 0.05). Conclusion: Berberine can significantly improve the metabolic-associated fatty liver disease injury in mice, and its mechanism is related to activation of Nrf2 and inhibition of programmed necrosis of hepatocytes.
Subject(s)
Animals , Male , Mice , Berberine/therapeutic use , Fatty Liver , Mice, Inbred C57BL , NF-E2-Related Factor 2/metabolism , NecrosisABSTRACT
Objective:To evaluate the effects of berberine on necroptosis of non-alcoholic fatty liver disease in mice and its relationship with adenosine monophosphate-activated protein kinase(AMPK)/ signal transducer and activator of transcription 6(STAT6) pathway.Methods:Twenty-five 8-week-old male C57BL/6N mice were divided into control group, steatotic liver group, berberine treatment group(200 mg·kg -1·d -1), AMPK inhibitor Compound C treatment group(0.2 mg·kg -1·d -1), and STAT6 inhibitor AS1517499 treatment group(10 mg·kg -1·d -1). After 12 weeks of intervention, the mice and liver tissue were weighed, and serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), triglyceride, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) as well as liver malondialdehyde and superoxide dismutase were measured; liver tissue HE, Masson, and oil red O staining were performed. Western blotting was used to detect the expressions of necroptosis related proteins[receptor interaction protein kinase 3(RIPK3), phosphorylated(p-) mixed lineage kinase domain-like(MLKL)], AMPK, p-AMPK, and p-STAT6. Results:Compared with control group, the steatotic liver group had higher quality of liver and liver index, and higher levels of serum AST, ALT, triglyceride, TNF-α, IL-1β, and oxidative stress( P<0.05); Liver tissue was full of cavity changes and inflammatory cell infiltration, widely distributed red lipid droplets and obvious blue fiber dyeing; The expressions of RIPK3 and p-MLKL were up-regulated ( P<0.05), but the levels of p-AMPK and p-STAT6 were relatively reduced ( P<0.05). Compared with the steatotic liver group, berberine intervention decreased liver quality and liver index, improved liver function, reduced blood lipid levels, pro-inflammatory factor expression and oxidative stress level, and significantly alleviated the degree of liver steatosis and fibrosis, the levels of RIPK3 and p-MLKL ( P<0.05), while the expressions of p-AMPK and p-STAT6 were increased significantly ( P<0.05). As compared with the berberine treatment, AMPK and STAT6 inhibitor treatment could offset the protective effect of berberine on steatotic liver, moreover, the expressions of RIPK3 and p-MLKL were increased ( P<0.05). There was no statistical difference in AMPK total protein content among the five groups ( P>0.05). Conclusion:Berberine can activate AMPK/STAT6 pathway to inhibit the necroptosis of hepatocyte, thus plays a protective role on non-alcoholic fatty liver disease in mice.
ABSTRACT
Acute pancreatitis (AP) is a common and potentially threatening disease of the pancreas, and some patients eventually develop to severe acute pancreatitis (SAP). Symptomatic support therapies such as rehydration therapy and anti-infection are still the main treatments. Lacking specific therapies is the main reason for the high mortality of AP patients, especially those with SAP. Premature trypsinogen activation is the most important pathologic cellular event in the pathogenesis of AP. The release of trypsin can cause self-digestion inside and outside of acinar cells, especially the release of cathepsin B can also cause a caspase-unrelated regulatory cell death (RCD) known as necroptosis, which is closely related to the development and prognosis of AP. Therefore, it is necessary to further study the mechanism of necroptosis in the occurrence and development of AP. This article reviews the mechanism of necroptosis and the research progress related to AP, in an attempt to provide a new understanding of the pathogenesis and treatment of AP, and promote the better target drug development.
ABSTRACT
Objective:To investigate whether the necroptosis pathway receptor interacting protein 1-receptor interacting protein 3-mixed lineage kinase domain-like protein (RIP1-RIP3-MLKL) is involved in fluoride-induced osteoblastic death.Methods:Human osteosarcoma cell line (Saos-2 cells) were cultured in vitro and divided into NC group, sodium fluoride (NaF) groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF), necroptosis inhibitor Necrostatin-1 (Nec-1) group (50.0 μmol/L Nec-1) and NaF + Nec-1 groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF + 50.0 μmol/L Nec-1). After cultured for 24 and 48 h, respectively, cell proliferation was determined via the CCK-8 method, and the activity of lactate dehydrogenase (LDH) was determined by chemical colorimetry. To further analyze the influence of NaF on RIP1-RIP3-MLKL pathway, Saos-2 cells were divided into NCⅡgroup and NaFⅡgroups (2.5, 5.0, 10.0, 20.0 and 40.0 mg/L NaF). After cultured for 24 and 48 h, respectively, the protein expression levels of RIP1, RIP3 and MLKL were determined by Western blotting. Results:The cell proliferation rates (%: 100.00 ± 0.59, 104.90 ± 0.44, 104.16 ± 0.41, 82.45 ± 1.91, 64.59 ± 1.83, 103.56 ± 0.41, 107.18 ± 0.87, 105.35 ± 1.28, 89.63 ± 1.20, 77.51 ± 1.30; 100.00 ± 0.33, 107.92 ± 0.44, 101.78 ± 1.06, 75.45 ± 0.39, 57.94 ± 1.17, 106.74 ± 0.21, 111.85 ± 0.21, 107.82 ± 0.68, 82.34 ± 0.56, 70.19 ± 0.99) among all groups were significantly different at both 24 and 48 h ( F = 77.13, 2 313.43, P < 0.05). Except the cell proliferation rate of the 10.0 mg/L NaF + Nec-1 group that was not significantly different with that of the 10.0 mg/L NaF group at 24 h ( P > 0.05), the cell proliferation rates of other NaF + Nec-1 groups were significantly higher than those of corresponding NaF groups at both 24 and 48 h ( P < 0.05). The proliferation rate was negatively correlated with fluoride concentration ( r24 h = - 0.976, r48 h = - 0.969, P < 0.001). The LDH activity in all concentrations of NaF groups was significantly higher than that in NC group and corresponding NaF + Nec-1 groups at both 24 and 48 h ( P < 0.05). The LDH activity was positively correlated with fluoride concentration ( r24 h = 0.985, r48 h = 0.988, P < 0.001). The protein expression levels of RIP1, RIP3 and MLKL of 5.0 mg/L NaFⅡ group at 24 h, RIP3 of 5.0 mg/L NaFⅡ group at 48 h, and RIP1, RIP3 and MLKL of 10.0, 20.0 and 40.0 mg/L NaFⅡ groups at both 24 and 48 h were higher than that in NCⅡ group ( P < 0.05). The protein expression levels of RIP1, RIP3 and MLKL were positively correlated with fluoride concentration ( r24 h-RIP1 = 0.881, r48 h-RIP1 = 0.952, r24 h-RIP3 = 0.867, r48 h-RIP3 = 0.938, r24 h-MLKL = 0.758, r48 h-MLKL = 0.907, P < 0.001). Conclusion:Fluoride can directly cause necroptosis of osteoblasts through the RIP1-RIP3-MLKL pathway, and the severity of cell damage is closely related to fluoride concentration, Nec-1 has partially reversed the effects of fluoride.
ABSTRACT
Objective:To investigate the inhibitory effect of specific inhibitor of necroptosis necrostatin-1 (Nec-1) on necroptosis of retinal ganglion cells (RGCs) in rats with acute ocular hypertension.Methods:Twenty-four adult male Sprague Dawley rats were randomly divided into normal control group, model control group, Nec-1 treatment group and negative control group by random number table method, with 6 rats in each group.High intraocular pressure (IOP)-induced ischemia and reperfusion model was established through anterior chamber irrigation of 0.9% sodium chloride solution in left eyes of the rats, raising the IOP to 110 mmHg (1 mmHg=0.133 kPa) for 60 minutes.Nec-1 (4 mmol/L, 2 μl) or dimethyl sulfoxide (2 μl) was intravitreally injected immediately in Nec-1 treatment group and negative control group following modeling, respectively, according to grouping.No intervention was administered to the normal control group.Paraffin sections of rat retinas of the left eyes in different groups were prepared seven days after modeling.The retinal structure was observed by hematoxylin-eosin staining, and the expression levels of thymocyte antigen-1 (Thy-1) and glial fibrillary acidic protein (GFAP) were detected via immunohistochemical staining.All animal experiments were approved by an Ethics Committee of Tianjin Union Medical Center (No.2017 Quick audit C01).Results:Seven days after modeling, compared with normal control group, the retinal nerve fiber layer was thinner in model control group and negative control group, and the RGCs were arranged loosely, and cells in the inner nuclear layer were reduced and arranged disorderly, and cells in the outer nuclear layer were normal or enlarged.Compared with model control group and negative control group, the nerve fiber layer was thickened and the number of RGCs was significantly increased in Nec-1 treatment group.The number of Thy-1-positive RGCs was decreased in model control group, negative control group and Nec-1 treatment group than normal control group, and there were more Thy-1-positive RGCs in Nec-1 treatment group than model control group and negative control group.The integrated absorbance ( A) value of GFAP protein in normal control group, model control group, negative control group and Nec-1 treatment group was 47.209±15.311, 116.220±18.194, 116.382±19.020, 92.818±10.236, respectively, showing statistically significant differences among them ( F=24.675, P<0.001). The integrated A value of GFAP protein was significantly increased in model control group, negative control group and Nec-1 treatment group than normal control group, and the integrated A value of GFAP protein in Nec-1 treatment group was lower than that in model control group and negative control group, with statistically significant differences (all at P<0.05). Conclusions:Nec-1 can promote RGCs survival by inhibiting the necroptosis of RGCs in rats with acute intraocular hypertension.
ABSTRACT
Objective:To explore the regulatory effect of cellular FLICE-like inhibitory protein (cFLIP) on myocardial ischemia-reperfusion injury based on the RIPK1/RIPK3/MLKL-mediated necroptosis pathway.Methods:The cardiomyocyte hypoxia/reoxygenation (H/R) model was constructed by hypoxia for 4 h/reoxygenation for 12 h, and the rat ischemia reperfusion (I/R) model was constructed by ligating the left anterior descending artery for 30 min and reperfusion for 3 h. CCK-8 method was used to detect the viability of cardiomyocytes in each group. DAPI/PI double staining was used to observe changes in necrosis rate of myocardial cell. STRING database was used to predict the protein interaction network of cFLIP. TTC staining was used to detect the area of myocardial infarction in each group of rats, and the protein expression of cFLIPL, cFLIPS, p-RIPK1, p-RIPK3 and p-MLKL were detected by Western blot.Results:In cardiomyocyte H/R injury and myocardial tissue I/R injury, the protein expressions of cFLIPL and cFLIPS were significantly down-regulated, while the levels of p-RIPK1, p-RIPK3 and p-MLKL were increased significantly. Up-regulating the protein expression of cFLIPL and cFLIPS could significantly reduce the damage of cardiomyocytes and the rate of cell necrosis induced by H/R, and decrease the area of myocardial infarction caused by I/R. STRING database results showed that cFLIP had direct protein interactions with RIPK1 and RIPK3. Overexpression of cFLIP in cardiomyocyte and myocardial tissue significantly inhibited H/R or I/R induced the phosphorylation levels of RIPK1, RIPK3 and MLKL.Conclusions:Overexpression of cFLIP can significantly inhibit the RIPK1/RIPK3/MLKL-mediated necroptosis, thereby reducing myocardial cell damage and decreasing the area of myocardial infarction.
ABSTRACT
Liver transplantation is an effective treatment for the end-stage liver disease. However, hepatic ischemia-reperfusion injury (HIRI) will inevitably occur during liver transplantation, which might lead to early graft dysfunction or aggravate rejection. The underlying protective mechanism remains to be further elucidated. Programmed cell death is an important mechanism of HIRI, and multiple novel types of programmed cell death participate in the pathological process of HIRI. In-depth study of programmed cell death is expected to further improve the therapeutic effect of liver transplantation. In this article, research progresses on apoptosis, autophagy and autophagy-dependent cell death, ferroptosis, necroptosis, pyroptosis, pathanatos and other common programmed cell death patterns in HIRI were reviewed, aiming to provide reference for enhancing the success rate of liver transplantation and improving clinical prognosis of the recipients.
ABSTRACT
ObjectiveTo observe the mechanism of modified Shengjiangsan in necroptosis and renal fibrosis of rats with diabetic nephropathy based on receptor-interacting protein （RIP）1/RIP3/mixed lineage kinase domain-like protein （MLKL） signaling pathway. MethodSeventy-five SD rats were randomly divided into a model group， a normal group， three high， medium， and low-dose modified Shengjiangsan groups （4.365， 8.73， 17.46 g·kg-1）， and an irbesartan group （0.013 5 g·kg-1）. After 4 weeks of intragastric administration， the levels of 24 h urine protein （UTP）， tumor necrosis factor-α （TNF-α）， and interleukin-1β （IL-1β） of rats in each group were determined， as well as the changes in degree of renal pathology. Real-time quantitative polymerase chain reaction （Real-time PCR） and immunohistochemistry were used to detect the mRNA and protein expression levels of IL-1β， TNF-α， monocyte chemoattractant protein-1 （MCP-1）， transforming growth factor-β1 （TGF-β1）， and nuclear factor kappa B （NF-κB） in kidney tissues of rats. Western blot assay was used to detect the expression levels of key proteins in the RIP1/RIP3/MLKL signaling pathway. ResultAs compared with the normal group， the renal interstitial fibrosis in the model group was obvious， and the 24 h UTP， IL-1β， TNF-α levels were significantly increased （P<0.05）. In the model group， the mRNA and protein expression levels of IL-1β， TNF-α， MCP -1， TGF-β1， and NF-κB in the kidney tissues were significantly increased （P<0.05）， and protein expression levels of RIP1， RIP3， p-MLKL， and MLKL were significantly increased （P<0.05）. Compared with the model group， all modified Shengjiangsan groups and the irbesartan group improved the levels of renal interstitial fibrosis in rats to varying degrees. As compared with the model group， the 24 h UTP levels in all modified Shengjiangsan groups and the irbesartan group were decreased to varying degrees （P<0.05）， the content of IL-1β and TNF-α in the serum were decreased （P<0.05）， the mRNA and protein expression levels of IL-1β， TNF-α， MCP-1， TGF-β1， and NF-κB in renal tissues was down-regulated （P<0.05）， and the protein expression levels of RIP1， RIP3， p-MLKL， and MLKL were down-regulated （P<0.05）. ConclusionModified Shengjiangsan ameliorates renal injury of rats with diabetic nephropathy， and the mechanism may be related to the down-regulation of the RIP1/RIP3/MLKL signaling pathway， the prevention of renal tissue necroptosis， and the inhibition of renal fibrosis.
ABSTRACT
The increasing incidence of obesity and diabetes has made diabetic kidney disease （DKD） the main cause of chronic kidney disease and end-stage renal disease. Despite current pharmacological interventions for blood glucose control and renin-angiotensin system inhibition， the risk of kidney disease progression and complications remains high. At present， the pathogenesis of DKD has been clarified to be related to chronic inflammatory response， oxidative stress， glucose and lipid metabolism disorders， and hemodynamic abnormalities. According to recent studies， the programmed cell deaths （PCD） of renal intrinsic cells such as pyroptosis and necroptosis play a key role in the occurrence and development of DKD. Pyroptosis and necroptosis， the two newly discovered routes of PCD， can protect the hosts from being invaded by microbial pathogens， but their dysregulation is associated with multiple autoimmunity and autoinflammatory responses. Pyroptosis and necroptosis are closely interlinked and cross-regulated. Different from apoptosis， these two cellular suicide mechanisms cause membrane rupture and release of cell contents through their respective gasdermin D （GSDMD） and mixed lineage kinase domain-like protein （MLKL）， with damage-associated molecular patterns （DAMPs） and inflammatory cytokines like interleukin-1β （IL-1β） involved to trigger inflammation， and chronic inflammatory responses are key factors leading to the progression of DKD. Traditional Chinese medicine （TCM） has long been employed for the prevention and treatment of DKD and the resulting clinical outcomes are remarkable. TCM has been proved to exert a protective effect against DKD by affecting the expression of nucleotide oligomerization domain-like receptor protein 3 （NLRP3） inflammasome， receptor-interacting protein kinase 3 （RIPK3）， and MLKL. This paper reviewed the relationship of pyroptosis and necroptosis with DKD and its intervention with TCM.
ABSTRACT
We investigated the ability of Dracocephalum moldavica (EPDM) flavonoids to protect human brain microvascular endothelial cells (HBMECs) from necroptosis induced by ischemia-reperfusion injury. To mimic the process of cerebral ischemia-reperfusion injury, a necroptosis model was established by treatment with the pan-cysteine aspartic acid protease (caspase) inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation (OGD/R) injury using HBMECs. Cell proliferation and cytotoxicity (cell counting kit-8, CCK-8) was used to measure cell viability. A Hoechst33342/PI fluorescent double-staining method was exploited to determine the rate of cell necroptosis. A commercial kit was used to detect lactate dehydrogenase in the cell culture supernate. DCFH-DA probes, calcein AM and JC-1 probes were used to measure changes in ROS production, mitochondrial membrane permeability transformation pore (MPTP) opening and mitochondrial membrane potential (MMP), respectively. Enzyme-linked immunosorbent assay (ELISA) kits were chosen to detect the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Western blotting was used to detect necroptosis-related proteins. The results show that relative to control group, Z-VAD-FMK combined with OGD/R injury reduced cell viability, increased the necroptosis rate and the levels of LDH and ROS in HBMECs. The MPTP of the model group cells opened and the MMP reduced. TNF-α, IL-1β, and IL-6 levels were significantly elevated. Furthermore, the expression of receptor-interacting protein kinase 3 (RIP3) and mitochondrial phosphoglycerate mutase 5 (PGAM5) was significantly increased, accompanied by an increase of phosphorylated mixed-lineage kinase domain-like protein (p-MLKL)/MLKL. EPDM partially reversed the changes of the above-mentioned factors in HBMECs induced by Z-VAD-FMK plus OGD/R injury. These results indicate that EPDM may protect HBMECs from cerebral ischemia-reperfusion injury by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening to maintain mitochondrial function, thereby providing a scientific basis for the use of EPDM in the treatment of cerebral ischemia-related diseases.