title: [Effects of autophagy on lipopolysaccharide-induced vascular hyper-permeability].
abstract: OBJECTIVE
  To investigate the effects of autophagy on lipopolysaccharide (LPS)-induced vascular hyper-permeability.
  
  
  METHODS
  (1) In vitro: Human umbilical vein endothelial cells (HUVECs) were randomly divided into blank group, LPS group (5 mg/L LPS stimulation), autophagy inhibitor 6-amino-3-methyl purine (3-MA) + LPS group (5 mmol/L 3-MA pretreatment for 30 minutes + 5 mg/L LPS stimulation) and autophagy revulsive Rapamycin (RAP) + LPS group (10 nmol/L RAP pretreatment for 30 minutes + 5 mg/L LPS stimulation). After LPS simulation for 60 minutes in four groups, endothelial permeability was detected by trans-endothelial electrical resistance (TER) determination. The protein expressions of autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3 II/I) and autophagy related gene Beclin-1 were detected by Western Blot. Cell apoptosis was evaluated by using flow cytometry. Caspase-3 activity was detected by fluorometric assay kit. (2) In vivo: 24 Sprague-Dawley (SD) rats were randomly assigned to four groups according to random number table, with 6 rats in each group. The rats in control group received no treatment; rats in model group were tail intravenous injected 10 mg/kg of LPS. The rats in 3-MA pretreatment and RAP pretreatment groups were tail intravenous injected 10 mg/kg of 3-MA or 2 mg/kg of RAP pretreatment for 30 minutes before 10 mg/kg LPS injection. The extravasation of FITC-albumin in mesenteric post-capillary venules was observed by fluorescence microscope. Then the change in fluorescence intensity of FITC-albumin between the intravascular and extravascular space (ΔI) were measured to reflect vascular permeability.
  
  
  RESULTS
  (1) In vitro, compared with blank group, the LC3 II/I protein, Beclin-1 protein, caspase-3 activity and rate of cell apoptosis in LPS group were increased, and the TER was decreased. Compared with LPS group, the LC3 II/I, Beclin-1, caspase-3 activity and rate of cell apoptosis in 3-MA+LPS group were decreased, and the TER was increased [LC3 II/I protein: (288.2±33.3)% vs. (420.5±39.4)%, Beclin-1 protein: (185.3±26.4)% vs. (293.3±36.1)%, caspase-3 activity: (196.6±28.5)% vs. (339.5±25.4)%, rate of cell apoptosis: (9.50±0.99)% vs. (15.40±1.55)%, TER: 0.88±0.09 vs. 0.63±0.05, all P < 0.05]. Compared with LPS group, the LC3 II/I, Beclin-1, caspase-3 activity and rate of cell apoptosis in RAP+LPS group were further increased, and the TER was further decreased [LC3 II/I protein: (519.6±45.2)% vs. (420.5±39.4)%, Beclin-1 protein: (359.0±38.3)% vs. (293.3±36.1)%, caspase-3 activity: (449.1±31.0)% vs. (339.5±25.4)%, rate of cell apoptosis: (19.30±1.72)% vs. (15.40±1.55)%, TER: 0.54±0.05 vs. 0.63±0.05, all P < 0.05]. (2) In vivo, the albumin extravasation and vascular permeability were increased in model group as compared with those of control group (ΔI: 0.54±0.07 vs. 0.13±0.03, P < 0.05). The albumin extravasation and vascular permeability were obviously decreased in 3-MA pretreatment group as compared with those of model group (ΔI: 0.25±0.05 vs. 0.54±0.07, P < 0.05). The albumin extravasation and vascular permeability were obviously increased in RAP pretreatment group as compared with those of model group (ΔI: 0.67±0.07 vs. 0.54±0.07, P < 0.05).
  
  
  CONCLUSIONS
  Inhibition of autophagy can reduce the LPS-induced vascular hyper-permeability, and enhanced autophagy can further increase vascular permeability. The mechanism of autophagy mediate vascular permeability may be related to the endothelial cells apoptosis.
authors.affiliation: Department of Critical Care Medicine, the First People's Hospital of Chenzhou, Chenzhou 423000, Hunan, China (Wang SB, Li YF, Li T); The Graduate School of Guangdong Medical University, Zhanjiang 524000, Guangdong, China (Yin S); Department of Anesthesiology, Shenzhen Hospital, Southern Medical University, Shenzhen 518000, Guangdong, China (Yin S, Li CL, Liu YT). Corresponding author: Liu Youtan, Email: youtanliuhao@163.com.
authors.forename: Shengbiao
authors.initials: S
authors.lastname: Wang
authors.forename: Shuang
authors.initials: S
authors.lastname: Yin
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Li
authors.forename: Cuiling
authors.initials: C
authors.lastname: Li
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Youtan
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis; D001343:Autophagy; D000071186:Beclin-1 / Q000378:metabolism; D002199:Capillary Permeability; D053148:Caspase 3 / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008070:Lipopolysaccharides / Q000009:adverse effects; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010539:Permeability; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000378:metabolism; D020123:Sirolimus / Q000737:chemistry
chemical_list: D000071186:Beclin-1; D053148:Caspase 3; D016650:Fluorescein-5-isothiocyanate; D008070:Lipopolysaccharides; D008869:Microtubule-Associated Proteins; D012709:Serum Albumin; D020123:Sirolimus; C491997:BECN1 protein, human; C512829:Becn1 protein, rat; C518288:FITC-albumin; C526109:Anxa3 protein, rat; C477012:MAP1LC3A protein, human; C505407:CASP3 protein, human; C505410:Casp3 protein, rat