Expression Of Receptor-interacting Protein 1 Research Articles

The Role of Sgt1 in Methamphetamine/Hyperthermia-induced Necroptosis.

Methamphetamine (METH) is a synthetic drug widely abused globally and can result in hyperthermia (HT) and psychiatric symptoms. Our previous studies showed that heat shock protein 90 alpha (HSP90α) plays a vital role in METH/HT-elicited neuronal necroptosis; however, the detailed mechanism of HSP90α regulation remained obscure. Herein, we demonstrated a function of the suppressor of G-two allele of SKP1 (Sgt1) in METH/HT-induced necroptosis. Sgt1 was mainly expressed in neurons, co-located with HSP90α, and increased in rat striatum after METH treatment. METH/HT injury triggered necroptosis and increased Sgt1 expression in PC-12 cells. Data from computer simulations indicated that Sgt1 might interact with HSP90α. Geldanamycin (GA), the specific inhibitor of HSP90α, attenuated the interaction between Sgt1 and HSP90α. Knockdown of Sgt1 expression did not affect the expression level of HSP90α. Still, it inhibited the expression of receptor-interacting protein 3 (RIP3), mixed lineage kinase domain-like protein (MLKL), p-RIP3, and p-MLKL, as well as necroptosis induced by METH/HT injury. In conclusion, Sgt1 may regulate the expression of RIP3, p-RIP3, MLKL, and p-MLKL by assisting HSP90α in affecting the METH/HT-induced necroptotic cell death.

Receptor-interacting protein 3-phosphorylated Ca2+ /calmodulin-dependent protein kinase II and mixed lineage kinase domain-like protein mediate intracerebral hemorrhage-induced neuronal necroptosis.

Necroptosis-mediated cell death is an important mechanism in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Our previous study has demonstrated that receptor-interacting protein 1 (RIP1) mediated necroptosis in SBI after ICH. However, further mechanisms, such as the roles of receptor-interacting protein 3 (RIP3), mixed lineage kinase domain-like protein (MLKL), and Ca2+ /calmodulin-dependent protein kinase II (CaMK II), remain unclear. We hypothesized that RIP3, MLKL, and CaMK II might participate in necroptosis after ICH, including their phosphorylation. The ICH model was induced by autologous blood injection. First, we found the activation of necroptosis after ICH in brain tissues surrounding the hematoma (propidium iodide staining). Meanwhile, the phosphorylation and expression of RIP3, MLKL, and CaMK II were differently up-regulated (western blotting and immunofluorescent staining). The specific inhibitors could suppress RIP3, MLKL, and CaMK II (GSK'872 for RIP3, necrosulfonamide for MLKL, and KN-93 for CaMK II). We found the necroptosis surrounding the hematoma and the concrete interactions in RIP3-MLKL/RIP3-CaMK II also both decreased after the specific intervention (co-immunoprecipitation). Then we conducted the short-/long-term neurobehavioral tests, and the rats with specific inhibition mostly had better performance. We also found less blood-brain barrier (BBB) injury, and less neuron loss (Nissl staining) in intervention groups, which supported the neurobehavioral tests. Besides, oxidative stress and inflammation were also alleviated with intervention, which had significant less reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, lactate dehydrogenase (LDH), Iba1, and GFAP surrounding the hematoma. These results confirmed that RIP3-phosphorylated MLKL and CaMK II participate in ICH-induced necroptosis and could provide potential targets for the treatment of ICH patients.

Retinoic acid-inducible gene 1 (RIG-1) and IFN-β promoter stimulator-1 (IPS-1) significantly down-regulated in the severe coronavirus disease 2019 (COVID-19).

Retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA5) are the well-known cytoplasmic sensors that recognize microbial DNA or RNA and active down-stream molecules, including IFN-β promoter stimulator-1 (IPS-1) and receptor interacting protein 1 (RIP1). The roles played by the networked molecules on the infection with SARS-CoV-2 needs more investigations. In this project MDA5, RIG-1, IPS-1 and RIP1 mRNA levels were evaluated in 45 hospitalized patients suffering from coronavirus disease of 2019 (COVID-19) and 45 healthy subjects using Real Time-qPCR technique. The results showed significant decreased RIG-1 and IPS-1 in the SARS-CoV-2 infected patients when compared to healthy cases. MDA5 and RIP1 did not change when compared two groups. Male patients had similar expression of MDA5, RIG-1, IPS-1 and RIP1 when compared to female patients. Based on the results, it seems that RIG-1 and its signaling molecule, IPS-1, play key roles in the peripheral blood immune cells against SARS-CoV-2 and, their down-regulation may be induced by the virus to escape from immune responses.

Pramipexole alleviates traumatic brain injury in rats through inhibiting necroptosis

Traumatic brain injury (TBI) has high morbidity, mortality and disability. Cell death runs through its occurrence and development. Necroptosis is a recently discovered mode of cell death. Its mechanism still has not been fully resolved. Studies which researcher published before showed that: pramipexole could play a neuroprotective role by inducing hypothermia; receptor interacting protein 1 (RIP1) could play a neuroprotective role by regulating necroptosis. On this basis, we carried out the experiments and it was observed that we could establish a hypothermia model of SD rats safely and effectively via pramipexole. Meanwhile, necroptosis and expression of RIP1 and its related proteins did change. As a result, the prognosis of TBI rats did improve. In brief, we found that pramipexole could play a protective role after TBI by inhibiting necroptosis. We hope our investigation would provide new theoretical basis to improve the outcome of clinical TBI patients.

Role of spinal RIP3 in inflammatory pain and electroacupuncture-mediated analgesic effect in mice

AimsElectroacupuncture (EA) is a potentially useful treatment for inflammatory pain. Receptor-interacting protein 3 (RIP3) triggers the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome; activation independent of necroptosis has been reported. However, the role of RIP3 in inflammatory pain and its EA-induced analgesic effects remains unclear. Main methodsMice were treated with EA (2 Hz, 2 mA) after complete Freund's adjuvant (CFA) pain models were established. Inhibition or activation of spinal RIP3 was achieved by intrathecal administration of GSK-843 (a specific RIP3 inhibitor) or microinjection of lentivirus-RIP3, respectively. Mechanical analgesiometry and thermal analgesiometry were used to assess paw withdrawal threshold and paw withdrawal latency in mice. Quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to evaluate the expression of RIP3 and NLPR3 in spinal dorsal horn (SDH) of mice. Key findingsThe expression of spinal RIP3 and NLPR3 increased significantly after CFA injection. Both intrathecal administration of GSK-843 and EA alleviated mechanical and thermal pain behaviors induced by CFA and inhibited the expression of RIP3 and NLRP3 in the SDH of CFA mice. Over-expression of RIP3 induces pain-like symptoms in mice and inhibits the regulatory effects of EA on inflammatory pain. SignificanceOur results indicate that the EA analgesia effect may be related to suppression of RIP3 and NLRP3 expression in the SDH. This study could provide potential insights into the underlying spinal mechanisms involved in the analgesic effect of EA.

Contribution of Necroptosis to Myofiber Death in Idiopathic Inflammatory Myopathies.

Myofiber necrosis is a significant pathologic characteristic of idiopathic inflammatory myopathies (IIMs), and its molecular mechanism is largely unknown. Necroptosis is a recently identified form of regulated necrotic cell death, and its activation might have crucial biologic consequences. The aim of the present study was to investigate the role of necroptosis in IIM muscle damage. Western blot and immunohistochemistry analyses were performed to examine the expression of receptor-interacting protein 3 (RIP-3) and mixed-lineage kinase domain-like (MLKL) proteins in 26 IIM patients and 4 healthy controls, as well as necroptosis-related damage-associated molecular pattern molecules. Tumor necrosis factor (TNF) was used to stimulate cultured C2C12 myoblasts, and the involvement of necroptosis in cell death of C2C12 cells was studied in vitro. The expression of RIP-3 and MLKL proteins and their phosphorylated forms was significantly increased in the muscle tissue of IIM patients compared to that of healthy controls. The expression levels of RIP-3 and MLKL proteins were associated with the severity of muscle damage in patients with IIM. Significant colocalization of MLKL with high mobility group box chromosomal protein 1 in necrotizing myofibers was observed in muscle biopsy tissue from patients with IIM. Stimulation of C2C12 myoblasts with TNF and a pan-caspase inhibitor, Z-VAD, resulted in the overactivation of necroptosis and significantly increased necrotic cell death. Strategies involving either inhibition of necroptosis with necrostatin-1 or knockdown of MLKL expression successfully prevented necroptosis-induced cell death of C2C12 cells. These findings demonstrate that overactivated necroptosis contributes to muscle damage in IIMs and suggest that necroptosis inhibitors could represent a new therapeutic target in the treatment of IIMs.

RIP3 Contributes to Cardiac Hypertrophy by Influencing MLKL-Mediated Calcium Influx.

Receptor-interacting protein 3(RIP3), a RIP family member, has been reported as a critical regulator of necroptosis and involves in the pathogenesis of various heart diseases. However, its role in the development of myocardial hypertrophy after pressure overload is unclear. We aimed to investigate the roles of RIP3 in pathological cardiac hypertrophy. A rat model of myocardial hypertrophy induced by the aortic banding method was used in this study. Neonatal rat cardiomyocytes (NRCMs) were stimulated with angiotensin II (Ang-II) or phenylephrine (PE) to induce neurohumoral stress. Our results showed that RIP3 level was significantly elevated in the hypertrophic myocardium tissues from patients, rats subjected to AB surgery, and NRCMs treated with Ang-II or PE. After downregulation of RIP3 expression in NRCMs, the phenotypes of myocardial hypertrophy were obviously alleviated. In mechanism, we demonstrated that RIP3 interacts with mixed lineage kinase domain-like protein (MLKL) and promotes its cell membrane localization to increase the influx of calcium within cells, thereby mediating the development of myocardial hypertrophy. More interestingly, we found the blockage of calcium influx by 2-aminoethoxydiphenyl borate, and lanthanum chloride efficiently reverses RIP3-induced cardiac remodeling in NRCMs. Taken together, our findings indicate a key role of the RIP3-MLKL signaling pathway in myocardial hypertrophy, which may be a novel promising treatment strategy for myocardial hypertrophy.

Augmenter of liver regeneration protects the kidney against ischemia-reperfusion injury by inhibiting necroptosis

ABSTRACT Necroptosis plays an important role in the pathogenesis of acute kidney injury (AKI), and necroptosis-related interventions may therefore be an important measure for the treatment of AKI. Our previous study has shown that augmenter of liver regeneration (ALR) inhibits renal tubular epithelial cell apoptosis and regulates autophagy; however, the influence of ALR on necroptosis remains unclear. In this study, we investigated the effect of ALR on necroptosis caused by ischemia-reperfusion and the underlying mechanism. In vivo experiments indicated that kidney-specific knockout of ALR aggravated the renal dysfunction and pathological damage induced by ischemia-reperfusion. Simultaneously, the expression of renal necroptosis-associated protein receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and mixed-lineage kinase domain-like protein (MLKL) significantly increased. In vitro experiments indicated that overexpression of ALR decreased the expression of hypoxia-reoxygenation-induced kidney injury molecules, the inflammation-associated factor tumor necrosis factor-alpha (TNF-α), and monocyte chemotactic protein. Additionally, the expression of RIP1, RIP3, and MLKL, which are elevated after hypoxia and reoxygenation, was also inhibited by ALR overexpression. Both in vivo and in vitro results indicated that ALR has a protective effect against acute kidney injury caused by ischemia-reperfusion, and the RIP1/RIP3/MLKL pathway should be further verified as a probable necroptosis regulating mechanism.

RIP1 Regulates Mitochondrial Fission during Skeletal Muscle Ischemia Reperfusion Injury

Background Dynamin related protein-1 (Drp1)-mediated mitochondrial fission relates to ischemia reperfusion (IR) injury, and its association with necroptosis is implied. We hypothesized that receptor-interacting protein 1 (RIP1), a key kinase in necroptosis, acted as an upstream of Drp1-mediated mitochondrial fission during skeletal muscle IR. Methods Thirty rats were randomized into the SM, IR, NI, MI, and DI group (n = 6). The rats in the SM group were shamly operated, and those in the IR group were subjected to 4-hour ischemia of the right hindlimb that was followed by 4-hour reperfusion. Intraperitoneal administration of Nec-1 1 mg/kg, Mdivi-1 1.2 mg/kg and same volume of DMSO were given before ischemia in the NI, MI and DI groups, respectively. Upon reperfusion, the soleus muscles were harvested to determine morphological changes and the expression of RIP1, total Drp1 and p-Drp1 (Ser616). Moreover, the muscular oxidative stress indicators and plasma muscle damage biomarkers were detected. Results IR led to impaired histopathological structures and mitochondrial fragmentation in the soleus muscle tissue, accompanied with increased muscular oxidative stress and muscle injury biomarkers, which could be similarly alleviated by Mdivi-1 and Nec-1 (p < 0.05). RIP1 and p-Drp1 (Ser616) protein levels were significantly upregulated in the soleus muscle subjected to IR injury, this upregulation was attenuated in the NI group, and Mdivi-1 downregulated the protein expression of p-Drp1 (Ser616) but not of RIP1 (p < 0.05). Conclusion RIP1 functions as an upstream of Drp1-mediated mitochondrial fission in the execution of necroptosis during skeletal muscle IR.

Role and mechanism of necrostin-1 in promoting oxidative stress response of macrophages in high glucose condition.

To investigate the role and molecular mechanism of necrostatin-1 (Nec-1), a specific programmed cell necrosis inhibitor, in promoting the oxidative stress response of macrophages under high glucose (HG) environment. Macrophages were cultured in control (5.5 mmol·L-1 glucose) or HG (25 mmol·L-1 glucose) medium for 72 h. The HG+Nec-1 group was given HG and 5 μmol·L-1 Nec-1. Reactive oxygen species (ROS) level, malondialdehyde (MDA) activity, and superoxide dismutase (SOD) activity were measured by 2'-7'dichlorofluorescin diacetate, MDA, and SOD enzyme linked immunosorbent assay kits, respectively. Moreover, receptor interacting protein 1 (RIP1) expression was assessed through real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot (WB). Finally, after the expression of RIP1 in macrophages was silenced, the effect of HG environment on oxidative stress response was evaluated in the gene-deficient cells. The HG group had increased ROS level and MDA activity (P<0.000 1) and decreased SOD activity (P<0.000 1) compared with the control group. The HG+Nec-1 group had higher ROS level and MDA activity (P<0.000 1) and lower SOD activity (P<0.01) than the HG group. The qRT-PCR and WB results showed that RIP1 mRNA level (P<0.001) and protein expression level (P<0.000 1) in the HG group were significantly higher than those in the control group, and RIP1 mRNA and protein expression levels in the HG+Nec-1 group were significantly lower than those in the HG group (P<0.000 1). After RIP1 was silenced effectively (P<0.001) with si-RNA, the ROS level and MDA activity of the HG+si-RIP1 group decreased compared with those of the HG+si-negative control (si-NC) group (P<0.001), and SOD activity in the HG+si-RIP1 group increased than that in the HG+si-NC group (P<0.000 1). HG promotes oxidative stress on macrophages by upregulating RIP1 expression.

Downregulation of RIP3 Improves the Protective Effect of ATF6 in an Acute Liver Injury Model.

Background Activating transcription factor 6 (ATF6) and receptor-interacting protein 3 (RIP3) are important signaling proteins in endoplasmic reticulum (ER) stress and necroptosis, respectively. However, their regulatory relationship and clinical significance are unknown. We investigate the impact of ATF6 on RIP3 expression, and its role in hepatocyte necroptosis in an acute liver injury model. Methods In vivo and in vitro experiments were carried out. LO2 cells were treated with thapsigargin (TG). In vivo, male BALB/c mice were treated with carbon tetrachloride (CCl4, 1 mL/kg) or tunicamycin (TM, 2 mg/kg). Then, the impact of ATF6 or RIP3 silencing on liver injury, hepatocyte necroptosis, and ER stress-related protein expression was examined. Results TG induced ER stress and necroptosis and ATF6 and RIP3 expression in LO2 cells. The knockdown of ATF6 significantly decreased RIP3 expression (p < 0.05) and increased ER stress and necroptosis. The downregulation of RIP3 significantly reduced necroptosis and ER stress (p < 0.05). Similar results were observed in CCl4 or the TM-induced mouse model. The knockdown of ATF6 significantly decreased CCl4-induced RIP3 expression and increased liver injury, necroptosis, and ER stress in mice livers (p < 0.05). In contrast, the downregulation of RIP3 significantly reduced liver injury, hepatocyte necroptosis, and ER stress. Conclusions Hepatocyte ATF6 has multiple roles in acute liver injury. It reduces hepatocyte necroptosis via negative feedback regulation of ER stress. In addition, ATF6 can upregulate the expression of RIP3, which is not helpful to the recovery process. However, downregulating RIP3 reduces hepatocyte necroptosis by promoting the alleviation of ER stress. The findings suggest that RIP3 could be a plausible target for the treatment of liver injury.

Upregulation of RIP3 promotes necroptosis via a ROS‑dependent NF‑κB pathway to induce chronic inflammation in HK‑2 cells

Tubular atrophy/interstitial fibrosis (TA/IF) is a major cause of late allograft loss, and inflammation within areas of TA/IF is associated with adverse outcomes in kidney transplantation. However, there is currently no satisfactory method to suppress this inflammation to improve TA/IF. The present study aimed to determine the proinflammatory role of receptor-interacting protein 3 (RIP3) in TA/IF to discover a novel therapeutic target. Reverse transcription-quantitative PCR and western blotting were performed to detect the expression of RIP3 and inflammation-associated factors. Lactate dehydrogenase release assay was used to determine necroptosis. Fluorescent 2,7-dichlorodihydrofluorescein diacetate was used to detect the levels of reactive oxygen species (ROS). The results demonstrated that patients with chronic TA/IF exhibited upregulated receptor-interacting protein 3 (RIP3) expression compared with the patients who had a favorable recovery after renal transplant. Therefore, the current study used normal renal tubular epithelial cells HK-2 to establish a cellular model with a high expression level of RIP3 in order to investigate the effect of RIP3 on renal epithelial cells after transplantation. The western blotting results demonstrated that overexpression of RIP3 could significantly increase the phosphorylation level of the necroptosis executive molecule mixed lineage kinase domain-like protein. Lactate dehydrogenase release, a key feature of necroptosis, was also markedly improved by RIP3 overexpression. Moreover, a higher inflammatory response was detected in HK-2 cells with RIP3 overexpression, and this elevated inflammation could be restored by the necroptosis inhibitor necrosulfonamide. Of note, it was found that overexpression of RIP3 activated the NF-κB signaling pathway via the excessive accumulation of ROS to induce necroptosis, which ultimately led to inflammation. Collectively, these findings indicated that overexpression of RIP3 promoted necroptosis via a ROS-dependent NF-κB pathway to induce chronic inflammation, suggesting that RIP3 may have the potential to be a therapeutic target against inflammation in TA/IF.

MyD88 determines the protective effects of fish oil and perilla oil against metabolic disorders and inflammation in adipose tissue from mice fed a high-fat diet

BackgroundThe beneficial effects of ω−3 polyunsaturated fatty acids (PUFA) vary between different sources. However, there is a paucity of comparative studies regarding the effects and mechanisms of marine and plant ω−3 PUFA on obesity.ObjectiveThe aim of this study was to evaluate the effects of fish oil (FO) and perilla oil (PO) on glucolipid metabolism, inflammation, and adipokine in mice fed a high-fat (HF) diet in association with the contribution of toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88) pathway.MethodsC57BL/6J mice and MyD88−/− mice were randomly divided into 4 groups: normal chow diet, HF diet, HF diet accompanied by daily gavage with either FO or PO. After 4 weeks, blood biochemistries, adipocyte histology, mRNA, and protein expression of MyD88-dependent and -independent pathways of TLR4 signaling in epididymal adipose tissue were measured.ResultsIn C57BL/6J mice, there were no statistical differences between FO and PO in decreasing body weight, glucose, insulin, triglyceride, total cholesterol, interleukin-6, and increasing adipocyte counts. FO and PO decreased mRNA and protein expression of TLR4, MyD88, tumor necrosis factor receptor-associated factor 6, inhibitor of nuclear factor kappa B kinase beta and nuclear factor-kappa B p65. In MyD88−/− mice, the beneficial effects of FO and PO on HF diet-induced metabolism abnormalities and inflammation were abolished. FO and PO had no impacts on mRNA and protein expression of receptor-interacting protein-1, interferon regulate factor 3, and nuclear factor-kappa B p65.ConclusionFO and PO exhibit similar protective effects on metabolic disorders and inflammation through inhibiting TLR4 signaling in a manner dependent on MyD88. These findings highlight plant ω−3 PUFA as an attractive alternative source of marine ω−3 PUFA and reveal a mechanistic insight for preventive benefits of ω−3 PUFA in obesity and related metabolic diseases.

Chlorpromazine and promethazine reduces Brain injury through RIP1-RIP3 regulated activation of NLRP3 inflammasome following ischemic stroke

ABSTRACT Objectives: Stroke is an important cause of death and disability. Recent evidence suggests that post-stroke inflammation is an important factor in stroke pathology and a root cause of its lasting consequences. Phenothiazine drugs, like chlorpromazine and promethazine (C + P), induce hypothermia and have been shown to play a major role in neuroprotection. In the present study, we investigated this neuroprotective mechanism by assessing the anti-inflammatory effect of these drugs. Methods: Adult Sprague-Dawley rats underwent 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion, with or without C + P (8 mg/kg). Infarct volumes, neurological deficits, along with mRNA and protein quantities of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), NLRPyrin domain containing 3 (NLRP3), and interleukin-1β (IL-1β) were assessed, as well as the infiltration of neutrophils and macrophages. Results: C + P induced hypothermia that significantly reduced RIP1, RIP3, NLRP3 and IL-1β expression, infarction, and immune cell infiltration, while C + P treatment with temperature control at 37°C induced lesser effect. Conclusion: These findings suggest that the anti-inflammatory effect of C + P may be dependent on drug-induced hypothermia and regulation of the NLRP3 inflammasome via the RIP1/RIP3 complex. Future investigations are needed regarding C + P as potential treatment of ischemic stroke.

Relationship between the expression of TNFR1-RIP1/RIP3 in peripheral blood and cognitive function in occupational Al-exposed workers: A mediation effect study

Aluminium (Al), not essential for biological activities, accumulates in the tissues. It exerts toxic effects on the nervous system, inducing in humans’ irreversible cognitive impairment. In this study, a cluster sampling method was used to observe the cognitive function of long-term occupational Al-exposed workers in a large Al factory, and determine the expression of peripheral blood tumour necrosis factor receptor 1 (TNFR1), receptor-interacting protein 1 (RIP1), and RIP3. TNF-alpha, expressed in blood macrophages and microglia, with its receptors TNFR1, TR1 and TR3, enhances the necroptosis of neurons. Additionally, the relationship between the expression of TNFR1, RIP1, and RIP3 in the peripheral blood of long-term occupational Al-exposed workers and changes in their cognitive function was explored. The differences in the distributions of clock drawing test (CDT) scores among the three groups were statistically significant (P < 0.05). The results of correlation analysis showed that RIP1 and RIP3 protein contents were negatively correlated with mini-mental state examination (MMSE) and CDT scores (P < 0.05). Plasma Al content was positively correlated with other biological indicators (P < 0.05), and negatively correlated with MMSE and CDT scores (P < 0.05). Results showed that RIP3 protein had an incomplete mediation effect between plasma Al content and cognitive function. This suggests that Al may affect cognitive function by influencing the expression of TNFR1, RIP1, and RIP3 in the nervous system.

Exogenous sodium hydrosulfide protects against high glucose‑induced injury and inflammation inhuman umbilical vein endothelial cells by inhibiting necroptosis via the p38 MAPK signaling pathway.

In recent years hydrogen sulfide (H2S) has demonstrated vasculoprotective effects against cell death, which suggests its promising therapeutic potential for numerous types of disease. Additionally, a protective effect of exogenous H2S in HG-induced injuries in HUVECs was demonstrated, suggesting a potential protective effect for diabetic vascular complications. The present study aimed to investigate the mechanism accounting for the cytoprotective role of exogenous H2S against high glucose [HG (40 mM glucose)]-induced injury and inflammation in human umbilical vein endothelial cells (HUVECs). HUVECs were exposed to HG for 24 h to establish an in vitro model of HG-induced cytotoxicity. The cells were pretreated with sodium hydrosulfide (NaHS), a donor of H2S, or inhibitors of necroptosis and p38 MAPK prior to the exposure to HG. Cell viability, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), IL-1β, IL-6, IL-8, TNF-α, phosphorylated-(p)38 and receptor-interacting protein 3 (RIP3) expression levels were detected using the indicated methods, including Cell Counting Kit 8, fluorescence detection, western blotting, immunofluorescence assay and ELISAs. The results demonstrated that necroptosis and the p38 MAPK signaling pathway mediated HG-induced injury and inflammation. Notably, NaHS was discovered to significantly ameliorate p38 MAPK/necroptosis-mediated injury and inflammation in response to HG, as evidenced by an increase in cell viability, a decrease in ROS generation and loss of MMP, as well as the reduction in the secretion of proinflammatory cytokines. In addition, the upregulated expression of RIP3 induced by HG was repressed by treatment with SB203580, while the HG-induced upregulation of p-p38 expression levels were significantly downregulated following the treatment of Nec-1 and RIP3-siRNA. In conclusion, the findings of the present study indicated that NaHS may protect HUVECs against HG-induced injury and inflammation by inhibiting necroptosis via the p38 MAPK signaling pathway, which may represent a promising drug for the therapy of diabetic vascular complications.

Dihydrotanshinone I Attenuates Plaque Vulnerability in Apolipoprotein E-Deficient Mice: Role of Receptor-Interacting Protein 3.

Aims: Vulnerable plaque disruption in advanced atherosclerosis leads to acute thrombus and subsequent myocardial infarction and severely threatens human health. Necroptosis of macrophage involved in the necrotic core is one key factor for plaque vulnerability. Dihydrotanshinone I (DHT) is a natural diterpenoid isolated from Danshen demonstrating effective anti-inflammatory property. It is accepted that inflammation plays a crucial role in the process of atherogenesis. However, whether DHT prevents atherosclerosis is poorly understood. Here, we investigated the effect of DHT on vulnerable plaque in an apolipoprotein E-deficient (ApoE-/-) mice model of atherosclerosis and the underlying protective mechanisms. Results: In the in vitro experiment, first LPS/ZVAD (LPS, lipopolysaccharide; ZVAD, ZVAD-FMK, a cell-permeable pan-caspase inhibitor) stimulated necroptosis of macrophage in a receptor-interacting protein 3 (RIP3)-dependent pathway, which was regulated by Toll-like receptor 4 (TLR4) dimerization. Further study illustrated that activated RIP3 evoked endoplasmic reticulum stress as well as reactive oxygen species generation. Both DHT and RIP3 silence reversed the above phenomena. In the in vivo experiment, aorta and serum samples were collected to determine features of plaque stability, including plaque size, necrotic core area, as well as collagen content in fibrous cap and the expression of related protein molecules. Both DHT and RIP3 inhibitor GSK872 significantly enhanced plaque stability in ApoE-/- mice by reducing oxidative stress, shrinking necrotic core area, increasing collagen content, and decreasing RIP3 expression. Innovation and Conclusion: Our study showed that DHT may stabilize vulnerable plaque by suppressing RIP3-mediated necroptosis of macrophage, which indicates its potential application as a lead compound for cardiovascular treatments, especially for advanced atherosclerosis. Antioxid. Redox Signal. 34, 351-363.

RSK3 mediates necroptosis by regulating phosphorylation of RIP3 in rat retinal ganglion cells.

Receptor-interacting protein 3 (RIP3) plays an important role in the necroptosis signaling pathway. Our previous studies have shown that the RIP3/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis occurs in retinal ganglion cell line 5 (RGC-5) following oxygen-glucose deprivation (OGD). However, upstream regulatory pathways of RIP3 are yet to be uncovered. The purpose of the present study was to investigate the role of p90 ribosomal protein S6 kinase 3 (RSK3) in the phosphorylation of RIP3 in RGC-5 cell necroptosis following OGD. Our results showed that expression of RSK3, RIP3, and MLKL was upregulated in necroptosis of RGC-5 after OGD. A computer simulation based on our preliminary results indicated that RSK3 might interact with RIP3, which was subsequently confirmed by co-immunoprecipitation. Further, we found that the application of a specific RSK inhibitor, LJH685, or rsk3 small interfering RNA (siRNA), downregulated the phosphorylation of RIP3. However, the overexpression of rip3 did not affect the expression of RSK3, thereby indicating that RSK3 could be a possible upstream regulator of RIP3 phosphorylation in OGD-induced necroptosis of RGC-5 cells. Moreover, our in vivo results showed that pretreatment with LJH685 before acute high intraocular pressure episodes could reduce the necroptosis of retinal neurons and improve recovery of impaired visual function. Taken together, our findings suggested that RSK3 might work as an upstream regulator of RIP3 phosphorylation during RGC-5 necroptosis.

RIP3/MLKL-mediated neuronal necroptosis induced by methamphetamine at 39°C.

Methamphetamine is one of the most prevalent drugs abused in the world. Methamphetamine abusers usually present with hyperpyrexia (39°C), hallucination and other psychiatric symptoms. However, the detailed mechanism underlying its neurotoxic action remains elusive. This study investigated the effects of methamphetamine + 39°C on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats. Primary cortex neurons were exposed to 1 mM methamphetamine + 39°C. Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39°C triggered obvious necrosis-like death in cultured primary cortical neurons, which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially. Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39°C for 3 hours. After pre-treatment with RIP3 inhibitor GSK'872, propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased; RIP3 and MLKL protein expression significantly decreased. Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse. Taken together, the above results suggest that methamphetamine + 39°C can induce RIP3/MLKL regulated necroptosis, thereby resulting in neurotoxicity. The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University, China (approval numbers: 2017-S026 and 2017-S033) on March 7, 2017.

Expression of receptor interacting protein 3 and mixed lineage kinase domain-like protein-key proteins in necroptosis is upregulated in ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory colonic disease strongly associated with intestinal epithelial cell (IEC) death. Necroptosis is characterized by a newly programmed cell death through a caspase-independent pathway. Receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) are very important in the pathway of necroptosis. However, their expression in UC remains unelucidated. This study aimed to investigate the expression of RIP3 and MLKL in patients with UC, along with its correlation with disease activity. Colonic tissue samples were taken from 22 UC patients and 19 healthy controls. RIP3 and MLKL expression levels were evaluated by immunohistochemical staining and western blotting. RIP3 and MLKL were upregulated in inflamed tissues of UC (P<0.01). No variations were observed in healthy control subjects and non-inflamed colons (P>0.05). RIP3 and MLKL were positively correlated with UC disease activity (P<0.01). Our results suggest that necroptosis is strongly associated with intestinal inflammation in patients with UC. Further studies of necroptosis may be helpful in future treatments of UC.