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OBJECTIVES@#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.@*METHODS@#Macrophages were cultured in control (5.5 mmol·L@*RESULTS@#The HG group had increased ROS level and MDA activity (@*CONCLUSIONS@#HG promotes oxidative stress on macrophages by upregulating RIP1 expression.
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Humans , Glucose , Macrophages , Necrosis , Oxidative Stress , Reactive Oxygen SpeciesABSTRACT
Objective To investigate the lethal effect of melittin on human hepatocellular carcinoma cell line SMMC-7721 and its possible mechanism. Methods MTT assay was used to investigate the killing effect of different concentrations melittin on human hepatocellular carcinoma cells SMMC-7721. Meanwhile, the inhibitory effect of specific programmed necrosis inhibitors necrostain-1(Nec-1) on melittin killing SMMC-7721 cells was detected. Programmed cell necrosis observed by Hoechst 33342 and PI double staining. The necrotosis rate was detected by flow cytometry. Ultrastructural changes of cell was detected by transmission electron microscopy. The expression of receptor interacting protein 1(RIP1) was detected by Western blotting. Results Compared with the control group, the proliferation activity of SMMC-7721 cells was significantly decreased after treatment with different concentrations of melittin for 24 hours (P < 0. 05) . Cells stained in dark blue and red. Cell membrane integrity was destroyed, organelle swelling, organelle membrane was destroy, that demonstrates cell was necrosis. Westen blotting result showed an increased proportion of RIP1 expression in SMMC-7721 cells. Compared with the melittin group, cell proliferation activity was significantly increased, cell necrotosis rate was decreased, and intracellular RIP1 expression was down-regulated in the Nec-1 pretreatment group. Conclusion Melittin induces cell death in SMMC-7721 cells through the RIP1-mediated programmed necrosis pathway.
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Objective To evaluate the effect of dexmedetomidine on receptor-interacting protein 1 (RIP1) signaling pathway during brain injury after cardiac arrest and resuscitation in pigs.Methods Twenty-one healthy domestic male white pigs,weighing 33-41 kg,were divided into 3 groups (n =7 each) using a random number table method:sham operation group (group S),cardiac arrest-resuscitation group (group CA-R) and dexmedetomidine group (group D).Ventricular fibrillation was electrically induced and untreated for 8 min followed by 5 min of cardiopulmonary resuscitation to establish the model of brain injury after cardiac arrest and resuscitation in anesthetized domestic white pigs.Dexmedetomidine was infused via the femoral vein in a loading dose of 0.5 μg/kg at 5 min after successful resuscitation,followed by an infusion of 0.5 μg · kg-1 · h-1 for 6 h in group D.The equal volume of normal saline was given instead in S and CA-R groups.The concentrations of neuron-specific endase (NSE) and S-100β protein in serum were measured at 1,3,6 and 24 h after resuscitation (T1-4).Neurologic deficit score (NDS) was evaluated at T4.The animals were sacrificed at T4,brains were removed and cerebral cortex tissues were obtained for determination of the expression of RIP1,RIP3 and mixed lineage kinase domain-like protein (MLKL) by Western blot.Results Compared with group S,the serum concentrations of NSE and S-100β protein were significantly increased at T1-4,the NDS was increased at T4,and the expression of RIP1,R1P3 and MLKL in cerebral cortex tissues was up-regulated in CA-R and D groups (P＜0.05).Compared with group CA-R,the serum concentrations of NSE and S-100β protein were significantly decreased at T3,4,the NDS was decreased at T4,and the expression of RIP1,RIP3 and MLKL in cerebral cortex tissues was down-regulated in group D (P＜0.05).Conclusion The mechanism by which dexmedetomidine reduces brain injury after cardiac arrest and resuscitation may be related to inhibiting the activation of RIP 1 signaling pathway in pigs.
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Objective To study the relationship between receptor interacting protein (RIP)1 and hepatocyte necropto?sis in isoniazid (INH) induced mouse model. Methods Kunming male mice were randomly divided into three groups. Con?trol group (C) received 0.3 mL of normal saline one time per day. INH group (INH) was injected intraperitoneally INH 100 mg/kg body weight, one time per day. Nec-1+INH group was injected intraperitoneally Nec-1 in 0.1%DMSO and 1 mg/kg body weight one time/12 hours, and INH was injected intraperitoneally at the same dose with that of INH group. All animals were treated for 7 days. Pathological changes of liver tissues were studied by HE staining. RIP1 expression was detected by immunohistochemical, Western blot and real-time PCR analysis. Levels of malondialdehyde (MDA), reactive oxygen species (ROS), glutathione (GSH) and superoxide dismutase (SOD) in liver homogenate were determined by colorimetric method. Re?sults Hepatocytes were arranged orderly in C group. The degeneration and necrosis of hepatocytes were found in Nec-1+INH group, and severe degeneration and necrosis of hepatocytes were found in INH group. Compared with C group, the ex?pression levels of RIP1, ROS and MDA were increased significantly, and the expression levels of GSH and SOD were de?creased significantly in INH group (P<0.05). INH-induced acute liver necroptosis was significantly alleviated after treat?ment with Nec-1. Compared with INH group, the expression levels of RIP1, MDA and ROS were significantly decreased, and the expression levels of GSH and SOD were significantly increased in Nec-1+INH group (P<0.05). Conclusion These re?sults suggest that RIP1 is involved in INH-induced hepatocyte necroptosis in mice. The inhibition of RIP1 expression might be a treatment strategy for prohibition of INH-induced acute liver necroptosis.
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Objective To investigate the location of receptor interacting protein 3( RIP3) in Necroptosis and its function in this signal passage, and explore the relationship between receptor interacting protein 1 ( RIP1 ) and RIP3 in nuclear translocation. Methods Primary cerebrocortical neurons were cultured for 12 days,then pre-treated with zVAD-fmk(20μ,mol/L) for half an hour to block apoptosis. ①Extracting nuclear and cytoplasmic protein after neurons were exposed to TNF for different time ,then protein levels of RIP3 were analyzed by western blot and immunofluorescence for qualitative observation;②In the following research,the neurons were treated with Nec-1 and shRlPl ,then the protein level of RIP1 and RIP3 with western blot were analyzed, cell viability were determined by measuring LDH levels. Results ①In signaling pathways of necroptosis, the protein level of RIP3 in cytoplasmic decreased gradually with prolonged TNF exposure, to the corresponding it rolled up in nucleus and a-chieved the peak in 12 hours of TNF treatment ( Cytoplasmic 0. 45 ± 0. 03 ,0. 41 ± 0. 02,0. 73 ± 0. 03 ,0. 90 ± 0.01,1.15 ±0.04,1.30 ±0.02,0.99 ±0.03,0.63 ±0. 03;Nucleus 0. 07 ±0.02,0. 26 ±0.02,0. 57 ±0. 02,0. 68 ± 0.02,0. 80 ± 0.01,0.92 ± 0.02,1.28 ± 0.03,0. 87 ± 0.02) (P < 0.01). ②Blocking the relationship between RIP1 and RIP3 with necrostatin-1 and shRIPl , nuclear translocation of RIP3 decreased and caused a great increase in cell viability( 1.00 ±0.05,0.39 ±0.03,0.50 ±0. 03) (P<0. 01). Conclusion RIP3 mainly locates in cy-tolymph of normal cells,it translocates into nucelus as necroptosis takes place. RIP1 function with RIP3 in nuclear translocation. Block nuclear translocation of RIP3 is a potential way to protect cells.
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Objective To prepare anti-activin receptor-interacting protein 1 (ARIP1) polyclonal antibody.Methods The GST-ARIP1 fusion protein was expressed in E.coli BL21.A polyclonal antibody against ARIP1 was obtained by immunizing a rabbit with the purified GST-ARIP1 and the localization of mature ARIP1 protein in mouse brain tissues was detected by immunohistochemistry using the prepared anti-ARIP1 antibody.Results Anti-ARIP1 polyclonal antibody could bind specifically with recombinant ARIP1,but not recombinant ARIP2.Immunohistochemical staining showed that ARIP1 mainly expressed in hypothalamus and hippocampus using the prepared anti-ARIP1 antibody.Conclusion The polyclonal antibody against ARIP1 has been successfully prepared and can be used to do immunohistochemical analysis for ARIP1 protein expression.