BACKGROUND Diabetic retinopathy(DR)is the primary cause of visual problems in patients with diabetes.The Heyingwuzi formulation(HYWZF)is effective against DR.AIM To determine the HYWZF prevention mechanisms,especially...BACKGROUND Diabetic retinopathy(DR)is the primary cause of visual problems in patients with diabetes.The Heyingwuzi formulation(HYWZF)is effective against DR.AIM To determine the HYWZF prevention mechanisms,especially those underlying mitophagy.METHODS Human retinal capillary endothelial cells(HRCECs)were treated with high glucose(hg),HYWZF serum,PX-478,or Mdivi-1 in vitro.Then,cell counting kit-8,transwell,and tube formation assays were used to evaluate HRCEC proliferation,invasion,and tube formation,respectively.Transmission electron microscopy was used to assess mitochondrial morphology,and Western blotting was used to determine the protein levels.Flow cytometry was used to assess cell apoptosis,reactive oxygen species(ROS)production,and mitochondrial membrane potential.Moreover,C57BL/6 mice were established in vivo using streptozotocin and treated with HYWZF for four weeks.Blood glucose levels and body weight were monitored continuously.Changes in retinal characteristics were evaluated using hematoxylin and eosin,tar violet,and periodic acid-Schiff staining.Protein levels in retinal tissues were determined via Western blotting,immunohistochemistry,and immunostaining.RESULTS HYWZF inhibited excessive ROS production,apoptosis,tube formation,and invasion in hg-induced HRCECs via mitochondrial autophagy in vitro.It increased the mRNA expression levels of BCL2-interacting protein 3(BNIP3),FUN14 domain-containing 1,BNIP3-like(BNIP3L,also known as NIX),PARKIN,PTEN-induced kinase 1,and hypoxia-inducible factor(HIF)-1α.Moreover,it downregulated the protein levels of vascular endothelial cell growth factor and increased the light chain 3-II/I ratio.However,PX-478 and Mdivi-1 reversed these effects.Additionally,PX-478 and Mdivi-1 rescued the effects of HYWZF by decreasing oxidative stress and apoptosis and increasing mitophagy.HYWZF intervention improved the symptoms of diabetes,tissue damage,number of acellular capillaries,and oxidative stress in vivo.Furthermore,in vivo experiments confirmed the results of in vitro experiments.CONCLUSION HYWZF alleviated DR and associated damage by promoting mitophagy via the HIF-1α/BNIP3/NIX axis.展开更多
Objective: BNip3 and its homologue Nix are pro-apoptotic factors of the Bcl-2-family and are expressed in malignant tumors. In vitro, this expression was shown to be mediated by hypoxia. Recently, it has been shown th...Objective: BNip3 and its homologue Nix are pro-apoptotic factors of the Bcl-2-family and are expressed in malignant tumors. In vitro, this expression was shown to be mediated by hypoxia. Recently, it has been shown that placental hypoxia as well as apoptosis are pathogenetic factors for pregnancy-induced hypertensive diseases and intrauterine growth retardation (IUGR). The aim of the study was to analyze placental expression of BNip3 and Nix in pregnancies complicated by preeclampsia, hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome and IUGR. Material and methods: Placental tissue was sampled from 10 pregnancies each with preeclampsia, HELLP syndrome, IUGR and gestational age-matched controls. The placental expression of BNip3/Nix has been investigated with immunohistochemistry by the use of specific human BNip3/Nix antibodies. Results: In cytotrophoblastic cells, the BNip3 expression was strong in the control placentas, but only mediate in the placentas from pregnancies with preeclampsia, IUGR or HELLP syndrome. The intensity of the Nix staining showed a similar pattern. In the syncytiotrophoblast, there was a weak BNip3 staining observable in the control as well as IUGR samples, whereas BNip3 was undetectable in preeclamptic placentas or those with HELLP syndrome. For Nix, only in the preeclampsia a weak staining was detectable, whereas all other probes were negative. Conclusions: Our study shows for the first time that the proapoptotic proteins BNip3 and Nix are expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive pregnancy disorders with different clinical manifestations are characterized by a significantly decreased expression of BNip3 and Nix. These results suggest that the hypothesis of generally increased placental apoptosis in pregnancy-induced hypertensive disorders caused by disturbed trophoblast invasion has to be partly reconsidered.展开更多
目的:探讨二氯化钴(cobaltous chloride,Co Cl2)诱导缺氧对肌细胞萎缩的调控机制。方法:本研究采用C2C12小鼠成肌细胞系作为细胞模型,分为正常组、Co Cl2组、正常+3-Methyladenine(3MA)组、Co Cl2+3MA组。正常组不作处理,Co Cl2组加入20...目的:探讨二氯化钴(cobaltous chloride,Co Cl2)诱导缺氧对肌细胞萎缩的调控机制。方法:本研究采用C2C12小鼠成肌细胞系作为细胞模型,分为正常组、Co Cl2组、正常+3-Methyladenine(3MA)组、Co Cl2+3MA组。正常组不作处理,Co Cl2组加入200μM Co Cl2诱导缺氧,正常+3MA组加入5 m M 3MA,Co Cl2+3MA组加入200μM Co Cl2及5 m M 3MA。使用吉姆萨染色观察肌管形态,多功能酶标仪检测活性氧(reactive oxygen species,ROS)表达,电镜观察自噬体形成情况,实时定量聚合酶链反应(quantita-tive real time polymerase chain reaction,QRT-PCR)和免疫印迹技术(western blotting,WB)检测缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、Bcl2/腺病毒E1B 19k Da相关蛋白3(Bcl2/adenovirusE1B 19k Da interacting protein 3,BNIP3)、微管相关蛋白1轻链-3(microtubule associated protein1 lightchain 3,LC3)m RNA及蛋白表达;另外,通过抑制自噬观察肌肉萎缩盒F基因(muscle atrophy F-box,MAFbx)蛋白表达的变化。结果:正常组可见长条状肌管形成,Co Cl2处理后肌管萎缩、断裂。Co Cl2组ROS含量较正常组升高(t=-4.965,P=0.008),电镜可观察到Co Cl2诱导自噬体形成,同时HIF-1α、BNIP3、LC3表达增加(P<0.05)。Co Cl2与3MA共处理可减少MAFbx蛋白的表达(F=18.246,P=0.001)。结论:Co Cl2诱导C2C12骨骼肌细胞萎缩,可能与HIF-1α/BNIP3信号通路促进自噬发生有关,抑制缺氧诱导的自噬可部分减少肌萎缩。展开更多
基金Supported by the National Key Research and Development Project of China,No.2019YFC1711605National Natural Science Foundation of China,No.81904257Medical Innovation Research Project of Science and Technology Commission of Shanghai Municipality,No.21Y11923100.
文摘BACKGROUND Diabetic retinopathy(DR)is the primary cause of visual problems in patients with diabetes.The Heyingwuzi formulation(HYWZF)is effective against DR.AIM To determine the HYWZF prevention mechanisms,especially those underlying mitophagy.METHODS Human retinal capillary endothelial cells(HRCECs)were treated with high glucose(hg),HYWZF serum,PX-478,or Mdivi-1 in vitro.Then,cell counting kit-8,transwell,and tube formation assays were used to evaluate HRCEC proliferation,invasion,and tube formation,respectively.Transmission electron microscopy was used to assess mitochondrial morphology,and Western blotting was used to determine the protein levels.Flow cytometry was used to assess cell apoptosis,reactive oxygen species(ROS)production,and mitochondrial membrane potential.Moreover,C57BL/6 mice were established in vivo using streptozotocin and treated with HYWZF for four weeks.Blood glucose levels and body weight were monitored continuously.Changes in retinal characteristics were evaluated using hematoxylin and eosin,tar violet,and periodic acid-Schiff staining.Protein levels in retinal tissues were determined via Western blotting,immunohistochemistry,and immunostaining.RESULTS HYWZF inhibited excessive ROS production,apoptosis,tube formation,and invasion in hg-induced HRCECs via mitochondrial autophagy in vitro.It increased the mRNA expression levels of BCL2-interacting protein 3(BNIP3),FUN14 domain-containing 1,BNIP3-like(BNIP3L,also known as NIX),PARKIN,PTEN-induced kinase 1,and hypoxia-inducible factor(HIF)-1α.Moreover,it downregulated the protein levels of vascular endothelial cell growth factor and increased the light chain 3-II/I ratio.However,PX-478 and Mdivi-1 reversed these effects.Additionally,PX-478 and Mdivi-1 rescued the effects of HYWZF by decreasing oxidative stress and apoptosis and increasing mitophagy.HYWZF intervention improved the symptoms of diabetes,tissue damage,number of acellular capillaries,and oxidative stress in vivo.Furthermore,in vivo experiments confirmed the results of in vitro experiments.CONCLUSION HYWZF alleviated DR and associated damage by promoting mitophagy via the HIF-1α/BNIP3/NIX axis.
文摘Objective: BNip3 and its homologue Nix are pro-apoptotic factors of the Bcl-2-family and are expressed in malignant tumors. In vitro, this expression was shown to be mediated by hypoxia. Recently, it has been shown that placental hypoxia as well as apoptosis are pathogenetic factors for pregnancy-induced hypertensive diseases and intrauterine growth retardation (IUGR). The aim of the study was to analyze placental expression of BNip3 and Nix in pregnancies complicated by preeclampsia, hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome and IUGR. Material and methods: Placental tissue was sampled from 10 pregnancies each with preeclampsia, HELLP syndrome, IUGR and gestational age-matched controls. The placental expression of BNip3/Nix has been investigated with immunohistochemistry by the use of specific human BNip3/Nix antibodies. Results: In cytotrophoblastic cells, the BNip3 expression was strong in the control placentas, but only mediate in the placentas from pregnancies with preeclampsia, IUGR or HELLP syndrome. The intensity of the Nix staining showed a similar pattern. In the syncytiotrophoblast, there was a weak BNip3 staining observable in the control as well as IUGR samples, whereas BNip3 was undetectable in preeclamptic placentas or those with HELLP syndrome. For Nix, only in the preeclampsia a weak staining was detectable, whereas all other probes were negative. Conclusions: Our study shows for the first time that the proapoptotic proteins BNip3 and Nix are expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive pregnancy disorders with different clinical manifestations are characterized by a significantly decreased expression of BNip3 and Nix. These results suggest that the hypothesis of generally increased placental apoptosis in pregnancy-induced hypertensive disorders caused by disturbed trophoblast invasion has to be partly reconsidered.
文摘目的:探讨二氯化钴(cobaltous chloride,Co Cl2)诱导缺氧对肌细胞萎缩的调控机制。方法:本研究采用C2C12小鼠成肌细胞系作为细胞模型,分为正常组、Co Cl2组、正常+3-Methyladenine(3MA)组、Co Cl2+3MA组。正常组不作处理,Co Cl2组加入200μM Co Cl2诱导缺氧,正常+3MA组加入5 m M 3MA,Co Cl2+3MA组加入200μM Co Cl2及5 m M 3MA。使用吉姆萨染色观察肌管形态,多功能酶标仪检测活性氧(reactive oxygen species,ROS)表达,电镜观察自噬体形成情况,实时定量聚合酶链反应(quantita-tive real time polymerase chain reaction,QRT-PCR)和免疫印迹技术(western blotting,WB)检测缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、Bcl2/腺病毒E1B 19k Da相关蛋白3(Bcl2/adenovirusE1B 19k Da interacting protein 3,BNIP3)、微管相关蛋白1轻链-3(microtubule associated protein1 lightchain 3,LC3)m RNA及蛋白表达;另外,通过抑制自噬观察肌肉萎缩盒F基因(muscle atrophy F-box,MAFbx)蛋白表达的变化。结果:正常组可见长条状肌管形成,Co Cl2处理后肌管萎缩、断裂。Co Cl2组ROS含量较正常组升高(t=-4.965,P=0.008),电镜可观察到Co Cl2诱导自噬体形成,同时HIF-1α、BNIP3、LC3表达增加(P<0.05)。Co Cl2与3MA共处理可减少MAFbx蛋白的表达(F=18.246,P=0.001)。结论:Co Cl2诱导C2C12骨骼肌细胞萎缩,可能与HIF-1α/BNIP3信号通路促进自噬发生有关,抑制缺氧诱导的自噬可部分减少肌萎缩。
