BACKGROUND Liver injury is common in severe acute pancreatitis(SAP).Excessive autophagy often leads to an imbalance of homeostasis in hepatocytes,which induces lipid peroxidation and mitochondrial iron deposition and ...BACKGROUND Liver injury is common in severe acute pancreatitis(SAP).Excessive autophagy often leads to an imbalance of homeostasis in hepatocytes,which induces lipid peroxidation and mitochondrial iron deposition and ultimately leads to ferroptosis.Our previous study found that milk fat globule epidermal growth factor 8(MFG-E8)alleviates acinar cell damage during SAP via binding toαvβ3/5 integrins.MFG-E8 also seems to mitigate pancreatic fibrosis via inhibiting chaperone-mediated autophagy.AIM To speculate whether MFG-E8 could also alleviate SAP induced liver injury by restoring the abnormal autophagy flux.METHODS SAP was induced in mice by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine or 7 hly injections of 50μg/kg cerulein plus lipopolysaccharide.mfge8-knockout mice were used to study the effect of MFG-E8 deficiency on SAPinduced liver injury.Cilengitide,a specificαvβ3/5 integrin inhibitor,was used to investigate the possible mechanism of MFG-E8.RESULTS The results showed that MFG-E8 deficiency aggravated SAP-induced liver injury in mice,enhanced autophagy flux in hepatocyte,and worsened the degree of ferroptosis.Exogenous MFG-E8 reduced SAP-induced liver injury in a dose-dependent manner.Mechanistically,MFG-E8 mitigated excessive autophagy and inhibited ferroptosis in liver cells.Cilengitide abolished MFG-E8’s beneficial effects in SAP-induced liver injury.CONCLUSION MFG-E8 acts as an endogenous protective mediator in SAP-induced liver injury.MFG-E8 alleviates the excessive autophagy and inhibits ferroptosis in hepatocytes by binding to integrinαVβ3/5.展开更多
Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression ...Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.展开更多
目的初步探讨蛋白酶体亚基PSMB8(proteasome subunit beta 8)对人绒毛膜滋养层细胞(HTR-8/Svneo)凋亡及自噬的影响。方法将HTR-8细胞分为转染组和对照组,采用瞬时转染的方法分别将PSMB8 siRNA-1、PSMB8 siRNA-2转入转染组中,将NC siRNA...目的初步探讨蛋白酶体亚基PSMB8(proteasome subunit beta 8)对人绒毛膜滋养层细胞(HTR-8/Svneo)凋亡及自噬的影响。方法将HTR-8细胞分为转染组和对照组,采用瞬时转染的方法分别将PSMB8 siRNA-1、PSMB8 siRNA-2转入转染组中,将NC siRNA转入对照组中。转染48h后用qRT-PCR进行干涉效率验证,CCK-8检测细胞增殖情况,TUNEL和流式细胞术分析细胞凋亡情况,免疫荧光染色检测细胞内caspase-3(cleaved)和LC3蛋白的表达,qRT-PCR检测细胞中自噬相关基因mRNA表达水平。结果qRT-PCR结果显示,PSMB8 siRNA-1组干扰效果较好。与对照组比较,PSMB8 siRNA-1组细胞增殖能力减弱,HTR-8细胞中TUNEL(15.05±2.62 vs 33.40±2.44,P=0.000)和caspase-3(cleaved)(21.70±5.87 vs 53.29±10.29,P<0.01)阳性率增加。流式结果显示,与对照组比较,PSMB8 siRNA-1组存活细胞比例减少(89.10±0.78 vs 77.00±1.68,P=0.000),早期凋亡和晚期凋亡细胞比例增加(9.09±0.65 vs 10.71±0.61,P<0.05),坏死细胞比例也增加(2.49±0.73 vs 12.49±2.02,P<0.01)。与对照组比较,PSMB8 siRNA-1组LC3蛋白阳性细胞率增加(19.02±3.78 vs 31.90±4.87,P<0.05),各自噬相关基因mRNA相对表达水平均上调。结论在HTR-8细胞中沉默PSMB8基因表达可以诱导细胞凋亡并且激活自噬。展开更多
Environmental pollutants,such as bisphenol A(BPA) have recently been implicated in the development of adverse birth outcomes.However,the underlying teratogenic mechanisms remain unclear.We investigated the effects of ...Environmental pollutants,such as bisphenol A(BPA) have recently been implicated in the development of adverse birth outcomes.However,the underlying teratogenic mechanisms remain unclear.We investigated the effects of BPA on the migration and invasion of human primary extravillous trophoblast HTR-8/SVneo cells.Our results indicated that BPA reduced cell migration and invasion.Moreover,it altered the ratio of matrix metalloproteinases(MMPs) and tissue inhibitors of MMPs(TIMPs) by downregulating MMP-2 and MMP-9,and upregulating TIMP-1 and TIMP-2.Furthermore,BPA suppressed integrin β1,integrin α5,and vimentin.Interestingly,BPA-induced invasion was partially restored by G15,a membrane G-protein-coupled estrogen receptor 30 antagonist.We further revealed that 42 proteins were differentially expressed by mass spectrometry analysis,which could be divided into three categories based on gene ontology including biological process,cellular component,and molecular function.These results suggest that BPA reduces HTR-8/SVneo cell migration and invasion by downregulating MMP-2 and MMP-9,up-regulating TIMP-1 and TIMP-2,and suppressing adhesion molecules.展开更多
基金Supported by the National Natural Science Foundation of China,No.82100685the Scientific Research Fund of Xi’an Health Commission,No.2021yb08+1 种基金Scientific Research Fund of Xi’an Central Hospital,No.2022QN07Innovation Capability Support Plan of Xi’an Science and Technology Bureau,No.23YXYJ0097.
文摘BACKGROUND Liver injury is common in severe acute pancreatitis(SAP).Excessive autophagy often leads to an imbalance of homeostasis in hepatocytes,which induces lipid peroxidation and mitochondrial iron deposition and ultimately leads to ferroptosis.Our previous study found that milk fat globule epidermal growth factor 8(MFG-E8)alleviates acinar cell damage during SAP via binding toαvβ3/5 integrins.MFG-E8 also seems to mitigate pancreatic fibrosis via inhibiting chaperone-mediated autophagy.AIM To speculate whether MFG-E8 could also alleviate SAP induced liver injury by restoring the abnormal autophagy flux.METHODS SAP was induced in mice by 2 hly intraperitoneal injections of 4.0 g/kg L-arginine or 7 hly injections of 50μg/kg cerulein plus lipopolysaccharide.mfge8-knockout mice were used to study the effect of MFG-E8 deficiency on SAPinduced liver injury.Cilengitide,a specificαvβ3/5 integrin inhibitor,was used to investigate the possible mechanism of MFG-E8.RESULTS The results showed that MFG-E8 deficiency aggravated SAP-induced liver injury in mice,enhanced autophagy flux in hepatocyte,and worsened the degree of ferroptosis.Exogenous MFG-E8 reduced SAP-induced liver injury in a dose-dependent manner.Mechanistically,MFG-E8 mitigated excessive autophagy and inhibited ferroptosis in liver cells.Cilengitide abolished MFG-E8’s beneficial effects in SAP-induced liver injury.CONCLUSION MFG-E8 acts as an endogenous protective mediator in SAP-induced liver injury.MFG-E8 alleviates the excessive autophagy and inhibits ferroptosis in hepatocytes by binding to integrinαVβ3/5.
文摘Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.
文摘目的初步探讨蛋白酶体亚基PSMB8(proteasome subunit beta 8)对人绒毛膜滋养层细胞(HTR-8/Svneo)凋亡及自噬的影响。方法将HTR-8细胞分为转染组和对照组,采用瞬时转染的方法分别将PSMB8 siRNA-1、PSMB8 siRNA-2转入转染组中,将NC siRNA转入对照组中。转染48h后用qRT-PCR进行干涉效率验证,CCK-8检测细胞增殖情况,TUNEL和流式细胞术分析细胞凋亡情况,免疫荧光染色检测细胞内caspase-3(cleaved)和LC3蛋白的表达,qRT-PCR检测细胞中自噬相关基因mRNA表达水平。结果qRT-PCR结果显示,PSMB8 siRNA-1组干扰效果较好。与对照组比较,PSMB8 siRNA-1组细胞增殖能力减弱,HTR-8细胞中TUNEL(15.05±2.62 vs 33.40±2.44,P=0.000)和caspase-3(cleaved)(21.70±5.87 vs 53.29±10.29,P<0.01)阳性率增加。流式结果显示,与对照组比较,PSMB8 siRNA-1组存活细胞比例减少(89.10±0.78 vs 77.00±1.68,P=0.000),早期凋亡和晚期凋亡细胞比例增加(9.09±0.65 vs 10.71±0.61,P<0.05),坏死细胞比例也增加(2.49±0.73 vs 12.49±2.02,P<0.01)。与对照组比较,PSMB8 siRNA-1组LC3蛋白阳性细胞率增加(19.02±3.78 vs 31.90±4.87,P<0.05),各自噬相关基因mRNA相对表达水平均上调。结论在HTR-8细胞中沉默PSMB8基因表达可以诱导细胞凋亡并且激活自噬。
基金supported by the National Basic Research Program of China(973 Program,No.2014CB943303)。
文摘Environmental pollutants,such as bisphenol A(BPA) have recently been implicated in the development of adverse birth outcomes.However,the underlying teratogenic mechanisms remain unclear.We investigated the effects of BPA on the migration and invasion of human primary extravillous trophoblast HTR-8/SVneo cells.Our results indicated that BPA reduced cell migration and invasion.Moreover,it altered the ratio of matrix metalloproteinases(MMPs) and tissue inhibitors of MMPs(TIMPs) by downregulating MMP-2 and MMP-9,and upregulating TIMP-1 and TIMP-2.Furthermore,BPA suppressed integrin β1,integrin α5,and vimentin.Interestingly,BPA-induced invasion was partially restored by G15,a membrane G-protein-coupled estrogen receptor 30 antagonist.We further revealed that 42 proteins were differentially expressed by mass spectrometry analysis,which could be divided into three categories based on gene ontology including biological process,cellular component,and molecular function.These results suggest that BPA reduces HTR-8/SVneo cell migration and invasion by downregulating MMP-2 and MMP-9,up-regulating TIMP-1 and TIMP-2,and suppressing adhesion molecules.
