Aim To investigate the effect of hypobaric hypoxia on macrophage necroptosis and atherosclerotic plaque instability and explore the underlying mechanisms.Methods Mouse bone marrow-derived macrophages were i-solated and cultured,and divided into control group(21％oxygen concentration)and hypoxia group(3％oxygen concen-tration).After 48 hours,cell necroptosis was detected,and the expression of cell necroptosis related proteins was deter-mined by Western blot.Healthy male ApoE-/-mice were randomly divided into control group and hypobaric hypoxia group.After the intervention for 16 weeks,the plasma lipids and inflammatory cytokines were measured,the areas of ath-erosclerotic plaque and necrotic core were evaluated by HE staining.The content of plaque collagen was detected by Mas-son staining.The number of macrophages in the plaque and the expression of necrotic apoptosis related proteins were de-tected by immunohistochemical staining and Western blot.Results Hypoxia induced increased necrotic apoptosis of macrophages(P＜0.01),while necroptotic inhibitor necrostatin-1(Nec-1)reduced hypoxia induced cell death(P＜0.05);hypoxia leads to a decrease in the expression of adenosine deaminase acting on RNA 1(ADAR1)in macrophages(P＜0.01),and an increase in the expression of Z-DNA binding protein 1(ZBP1),phosphorylated receptor-interacting serine/threonine-protein kinase(p-RIPK3),and phosphorylated mixed lineage kinase domain-like protein(p-MLKL)(all P＜0.01).Compared with the control group,the plasma lipid levels of ApoE-/-mice in the hypobaric hypoxia group did not change significantly(P＞0.05),the plasma inflammatory cytokines(TNF-α,IL-1β,IL-6 and MCP-1)increased(all P＜0.05),the area of atherosclerotic plaque increased(P＜0.05),the area of plaque necrotic core increased,the content of plaque collagen decreased,the number of macrophages increased,the expression of ADAR1 decreased,and the expres-sion of ZBP1 and p-MLKL increased(all P＜0.01).Conclusion Hypobaric hypoxia causes the imbalance of A-DAR1/ZBP1 expression in macrophages,activates RIPK3/MLKL signaling pathway,promotes macrophage necroptosis,in-creases the area of plaque necrosis core,and leads to increase instability of atherosclerotic plaque.

Aim To investigate the effect of hypobaric hypoxia on macrophage necroptosis and atherosclerotic plaque instability and explore the underlying mechanisms.Methods Mouse bone marrow-derived macrophages were i-solated and cultured,and divided into control group(21％oxygen concentration)and hypoxia group(3％oxygen concen-tration).After 48 hours,cell necroptosis was detected,and the expression of cell necroptosis related proteins was deter-mined by Western blot.Healthy male ApoE-/-mice were randomly divided into control group and hypobaric hypoxia group.After the intervention for 16 weeks,the plasma lipids and inflammatory cytokines were measured,the areas of ath-erosclerotic plaque and necrotic core were evaluated by HE staining.The content of plaque collagen was detected by Mas-son staining.The number of macrophages in the plaque and the expression of necrotic apoptosis related proteins were de-tected by immunohistochemical staining and Western blot.Results Hypoxia induced increased necrotic apoptosis of macrophages(P＜0.01),while necroptotic inhibitor necrostatin-1(Nec-1)reduced hypoxia induced cell death(P＜0.05);hypoxia leads to a decrease in the expression of adenosine deaminase acting on RNA 1(ADAR1)in macrophages(P＜0.01),and an increase in the expression of Z-DNA binding protein 1(ZBP1),phosphorylated receptor-interacting serine/threonine-protein kinase(p-RIPK3),and phosphorylated mixed lineage kinase domain-like protein(p-MLKL)(all P＜0.01).Compared with the control group,the plasma lipid levels of ApoE-/-mice in the hypobaric hypoxia group did not change significantly(P＞0.05),the plasma inflammatory cytokines(TNF-α,IL-1β,IL-6 and MCP-1)increased(all P＜0.05),the area of atherosclerotic plaque increased(P＜0.05),the area of plaque necrotic core increased,the content of plaque collagen decreased,the number of macrophages increased,the expression of ADAR1 decreased,and the expres-sion of ZBP1 and p-MLKL increased(all P＜0.01).Conclusion Hypobaric hypoxia causes the imbalance of A-DAR1/ZBP1 expression in macrophages,activates RIPK3/MLKL signaling pathway,promotes macrophage necroptosis,in-creases the area of plaque necrosis core,and leads to increase instability of atherosclerotic plaque.

AIM:To investigate the role of phosphoenolpyruvate carboxykinase 1(PCK1)in the proliferation and migration of mouse vascular smooth muscle cells(VSMCs)and the underlying mechanism.METHODS:The prolif-eration and migration of mouse VSMCs were induced by platelet-derived growth factor(PDGF)-BB.The cells were divided into a vehicle group and a PDGF-BB group.The expression of PCK1 was detected by Western blot and immunofluores-cence staining.The mouse Pck1 siRNA(si Pck1)were transfected into mouse VSMCs to silence PCK1.The cells were di-vided into the vehicle,si Pck1+vehicle,PDGF-BB and si Pck1+PDGF-BB groups.The protein level of PCK1 was detected by Western blot.The proliferation was explored by Ki-67 immunofluorescence staining and the viability was detected by CCK-8 assay.The migration was determined by a scratch test.Mitochondrial dynamics were observed via transmission electron microscopy.A lentivirus carrying dynamin-related protein 1(Drp1)gene(lenti-Drp1)was transfected into VSMCs to induce them to overexpress DRP1.The cells were divided into the PDGF-BB,si Pck1+PDGF-BB,lenti-Drp1+PDGF-BB and lenti-Drp1+si Pck1+PDGF-BB groups.Proliferation,migration and mitochondrial dynamics were measured as described above.RESULTS:PDGF-BB increased the protein expression of PCK1 and DRP1,cell viability,the per-centage of Ki-67-positive cells,the wound healing rate and mitochondrial division in VSMCs.These effects were sup-pressed when PCK1 protein expression was silenced.After DRP1 was overexpressed,the inhibitory effects of PCK1 silenc-ing on cell viability,the percentage of Ki-67-positive cells,the wound healing rate and mitochondrial division were signifi-cantly reversed.CONCLUSION:PCK1 promotes the mitochondrial division,proliferation and migration of VSMCs in mice by upregulating the expression of DRP1.

AIM:To examine the effects of borneol on inflammation and myocardial remodeling after myocar-dial infarction(MI)in mice,and to investigate the underlying mechanisms.METHODS:Eight-week-old wild-type(WT)C57BL/6 mice and transient receptor potential cation channel subfamily M member 8(TRPM8)gene knockout(TRPM8-/-)mice were randomly divided into sham and MI groups,and were subsequently treated with normal saline(control group)or borneol(borneol group)via gavage.Survival curves were plotted for WT and TRPM8-/-mice with MI treated with or with-out borneol.After 28 d,cardiac function of the mice was assessed through echocardiography,and haemodynamic indexes were evaluated using a multi-channel physiological instrument.Infarct size,myocardial hypertrophy and interstitial fibro-sis were assessed via pathological staining.In addition,inflammatory response in the peri-infarct region was detected.RE-SULTS:The TRPM8 expression was up-regulated in the peri-infarct region of the mice with MI(P＜0.05),and borneol had no effect on TRPM8 expression(P＞0.05).Borneol increased the survival rate,reduced the infarct size,inhibited car-diac remodeling and improved cardiac function in WT mice with MI(P＜0.05 or P＜0.01).However,it did not affect the survival rate,infarct size,myocardial hypertrophy,myocardial fibrosis or cardiac function in TRPM8-/-mice(P＞0.05).Furthermore,borneol reduced inflammatory cell infiltration and the expression of inflammatory cytokines,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6 and monocyte chemotactic protein-1(MCP-1),in WT mice(P＜0.05 or P＜0.01)but not in TRPM8-/-mice(P＞0.05).CONCLUSION:Borneol attenuates inflammation,inhibits cardiac re-modeling and improves cardiac function in mice with MI via TRPM8.