| Background:Septic shock refers to shock caused by pathogenic microbial infection.It is a common type of shock in clinical practice.The main symptoms of septic shock are dropped blood pressure,insufficient perfusion of tissues and organs,which lead to ischemic necrosis,dangerous condition and high mortality rate(about 60%).Although various positive anti-shock measures or methods of antagonizing cytokines are currently used,there is no remarkable effect in clinically.Septic shock is still a serious problem need to be solved.The most common type of septic shock is that caused by Gram-negative bacilli(G-)infection.Lipopolysaccharide(LPS)plays a major role in septic shock caused by G-bacteria.Thus,the similar performance occurred in experimental animals caused by LPS injection was called endotoxin shock,which is widely used to study septic shock.Endotoxin shock is initiated by the dysregulation of cytokine production which due to the LPS stimulation of macrophages and neutrophils.These cytokines cause vasodilation,increasing the vascular bed volume,decreasing the effective circulating blood volume,and causing symptoms associated with endotoxic shock.Thus,macrophages play a crucial role in the development of endotoxic shock.And,regulation of macrophages activation and quantity is expected to alleviate and cure endotoxin shock.Ojective:We investigated the mechanism of zVAD on macrophages activation and necroptosis as well as the effects of it on the pathogenesis of endotoxic shock,which could provide a novel basis for the clinical treatment of endotoxic shock.Method:1.C57BL/6 mice were stimulated with LPS(10 ?g/g)for 0?3?6 or 12 h.(1)qRT-PCR was used to detect the expression levels of RIP1,RIP3 and MLKL in peripheral blood mononuclear cells(PBMCs)and spleen tissues.(2)Immunofluorescence staining was used to detect RIP1 and RIP3 protein expression in liver and lung tissue sections.2.Groups of C57BL/6 mice were pretreated with various doses of zVAD(5,10 or 20 ?g/g body weight)or vehicle(saline)for 2 h followed by LPS challenge(40 ?g/g body weight),and mortality was observed.Groups of C57BL/6 mice were pretreated with zVAD(20 ?g/g body weight)or vehicle(saline)followed by LPS challenge(25,37.5,or 50 ?g/g body weight),and mortality was observed.3.C57BL/6 mice were pretreated with different doses of zVAD(5,10 or 20 ?g/g body weight)or vehicle(saline)prior to LPS challenge(10?g/g body weight).(1)The degree of pathological damage in liver and lung tissues was observed by H&E staining.(2)Apoptotic cells in liver and lung tissues were detected by TUNEL.(3)Levels of TNF-?,IL-12 and IL-6 in serum were measured by ELISA.(4)The neutrophil infiltration in liver and lung was measured by MPO activity detection kit.(5)The percentage of MDSCs,G-MDSCs and M-MDSCs in spleens were determined using flow cytometry.4.The mice were challenged with LPS(10 ?g/g body weight)for 1 hour followed by zVAD(20?g/g body weight)treatment.(1)After 12 hours,the expression levels of CD86 on F4/80+ cells and CD11c+cells in spleens were measured by flow cytometry.(2)After 6 hours,levels of TNF-? and IL-6 in serum were measured by ELISA.5.C57BL/6 mice were injected with zVAD(20 ?g/g body weight)by intraperitoneal or intravenous injection followed by LPS(10 ?g/g body weight)challenge.(1)The degree of pathological damage in liver and lung tissues was observed by H&E staining.(2)Apoptotic cells in liver and lung tissues were detected by TUNEL.(3)Levels of TNF-?,IL-12 and IL-6 in serum were measured by ELISA.(4)The percentage of F4/80+cells and PI uptake of F4/80+cells at 6 hours and 12 hours were measured by flow cytometry.6.Pretreated with zVAD(20 ?g/g body weight)for 2 hours(i.p.or i.v.),C57BL/6 mice were challenged with LPS(25 ?g/g body weight,i.p.).The mortality of mice was recorded and analysed.7.C57BL/6 mice bone marrow-derived macrophages(BMDMs)were induced with GM-CSF,and BMDMs were treated with different concentrations of zVAD.The cell viability was measured by CCK8,which determined the appropriate concentration.8.2 ml 3%thioglycollate medium was injected into the abdominal cavity of mice.After 3 days,the peritoneal cells were rinsed out with PBS and were planted on the medium subsequently.The suspended cells were discarded after 3 h and the medium was replaced by fresh DMEM.(1)The adherent peritoneal macrophages were pretreated with different concentrations of zVAD for 30 min.The cell viability was measured by CCK8,which determined the appropriate concentration.(2)The peritoneal macrophages were pretreated with zVAD(45 ?M)or PBS followed by LPS stimulation(100 ng/ml).Proteins extracted from the peritoneal macrophages were used for immunoblotting analysis.9.Bone marrow-derived macrophages(BMDMs)or peritoneal macrophages induced by thioglycolate medium were pretreated with zVAD(5,15,45 ?M)or with vehicle(PBS)for 30 min followed by LPS administration(100 ng/ml)for 24 or 48 h.Levels of secreted TNF-?,IL-12 and IL-6 in culture supernatants of BMDMs or peritoneal macrophages were analyzed by ELISA.10.C57BL/6 and iNOS-/-mice were injected with zVAD(20 ?g/g body weight)or vehicle through abdominal cavity prior to LPS challenge for 12 hours.(1)The degree of pathological damage in liver and lung tissues was observed by H&E staining.(2)Levels of TNF-?,IL-12 and IL-6 in serum were measured by ELISA.11.BMDMs generated from C57BL/6 and iNOS-/-mice.(1)BMDMs were pretreated with or without zVAD(20,40,or 80?M)for 30 min followed by LPS stimulation(100 ng/ml).PI uptake by BMDMs was assessed by flow cytometry at 24 h.(2)BMDMs were pretreated with zVAD(20?M)followed by LPS stimulation(100 ng/ml)for different time intervals respectively(0,10,20 or 40 min).The expression of p-RIP3 and t-RIP3 were determined by Western blot.12.After intraperitoneal injection of zVAD(20 ?g/g body weight)or vehicle(saline)for 2 h,mice were challenged with lipopolysaccharide(LPS,10 ?g/g body weight)or not for 12 h.The expression levels of CD86 and TNF-? in F4/80+cells in spleens and livers were measured by flow cytometry.13.BMDMs generated from C57BL/6 mice were pretreated with zVAD(80 ?M)for 30 min or not.Cells were then stimulated with LPS for 24 h.The cells were collected to detect the concentration of intracellular IL-6 and TNF-?.14.After being pretreated with zVAD(80 ?M)or PBS for 30 min,BMDMs were stimulated with LPS for 20 or 40 minutes.Cell lysates were prepared and subjected to immunoblotting with the indicated antibodies.Results:1.(1)The mRNA transcript levels of RIP1,RIP3,and MLKL significantly increased in PBMCs and spleens from mice challenged with LPS.(2)The protein levels of RIP1 and RIP3 were both significantly increased in livers and lungs.2.Treatment with zVAD could significantly extend mice survival by hours and increased the survival rate.3.(1)The H&E staining showed that zVAD could alleviate LPS induced liver and lung pathology damage.(2)TUNEL staining revealed that the percentage of apoptotic cells in lung and liver tissues from LPS and zVAD treated mice was significantly reduced compared to that from LPS-treated mice.(3)Treatment with LPS and zVAD markedly reduce the concentrations of TNF-?,IL-12 and IL-6 in endotoxin shock mice.(4)The MPO activity indicated that the infiltration of neutrophils in liver and lung were decreased after treated with zVAD.(5)zVAD markedly increased the proportion of MDSCs and G-MDSCs in a concentration-dependent manner while decreased the proportion of M-MDSCs.4.(1)Post-treatment of zVAD had no significant effect on the CD86 expression of macrophages and dendritic cells(DCs).(2)Post-treatment of zVAD had no significant effect on the levels of TNF-? and seemly increased the levels of IL-6 in LPS-challenged mice.5.(1)The H&E staining showed that intraperitoneal injection of zVAD could alleviate liver and lung tissue damage,while intravenous injection of zVAD showed no significant effect.(2)TUNEL staining revealed that intraperitoneal injection of zVAD could decreased the percentage of apoptotic cells in the liver and lung,whereas intravenous injection of zVAD showed no significant effect.(3)The same trend occurred for levels of TNF-?,IL-12 and IL-6.(4)Compared with LPS-treated mice,intraperitoneal injection of zVAD significantly reduced the percentage of F4/80+macrophages and increased the PI uptake of F4/80+macrophages in the abdominal at 6 h and 12 h,whereas intravenous injection of zVAD showed no significant effect.6.Intraperitoneal injection of zVAD significantly decreased the mortality of endotoxin shock mice,whereas intravenous injection of zVAD showed no significant effect.7.The 80 ?M concentration of zVAD would not decrease the cell viability of BMDMs.8.(1)The 45 ?M concentration of zVAD would not decrease the cell viability of peritoneal macrophages.(2)zVAD could increase the expression of p-RIP1 in LPS stimulated peritoneal macrophages.9.zVAD reduced the secretion of TNF-?,IL-12 and IL-6 in LPS challenged BMDMs and peritoneal macrophages.10.(1)The H&E staining showed that zVAD alleviated the liver and lung tissues damage of WT mice,but no significant effect on iNOS-/-mice.(2)zVAD reduced the levels of TNF-?,IL-12 and IL-6 in WT mice,but no effect on iNOS-/-mice.11.(1)The PI uptake of BMDMs generated from iNOS-/-mice is lower than that from WT mice.(2)The p-RIP3/RIP3 ratio of BMDMs generated from iNOS-/-mice is lower than that from WT mice.12.Treatment with zVAD(i.p.)could significantly block the secretion of TNF-? and inhibit LPS-induced CD86 expression on macrophages in spleens and livers.13.zVAD could not affect LPS-induced production of IL-6 and TNF-? in BMDMs.14.zVAD could not affect LPS-induced activation of MAPKs and NF-?B signaling pathways in BMDMs.Conclusion:1.Necroptosis-related molecules increase in endotoxic shock.2.zVAD can alleviate LPS-induced endotoxic shock.3.zVAD can alleviate LPS-induced endotoxic shock by inducing the necroptosis of macrophages.4.zVAD can alleviate LPS-induced endotoxic shock by promoting MDSCs-mediated inhibition of macrophage activation. |
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