Effect of genipin on lipopolysaccharide-mediated vascular hyperpermeability and its underlying mechanism in vitro and in vivo
Received:November 20, 2018  
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DOI:10.11915/j.issn.1671-5403.2019.06.093
Key words:genipin; lipopolysaccharide; vascular permeability; mitochondria
Author NameAffiliationE-mail
CAO Yuan-Yuan Department of Critical Care Medicine, the First People′s Hospital of Chenzhou City in Hunan Province, Chenzhou 423000, China  
DENG Jia-Xiong Department of Critical Care Medicine, the First People′s Hospital of Chenzhou City in Hunan Province, Chenzhou 423000, China  
LI Gui-Cheng Department of Critical Care Medicine, the First People′s Hospital of Chenzhou City in Hunan Province, Chenzhou 423000, China  
AI Chen-Mu Department of Critical Care Medicine, the First People′s Hospital of Chenzhou City in Hunan Province, Chenzhou 423000, China  
LI Yun-Feng Department of Critical Care Medicine, the First People′s Hospital of Chenzhou City in Hunan Province, Chenzhou 423000, China yunfengli_edu@163.com 
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Abstract:
      Objective To investigate the effect of genipin on lipopolysaccharide (LPS)-mediated hyperpermeability and its possible mechanism. Methods In vitro experiment:human umbilical vein endothelial cells (HUVECs) were divided into 4 groups, that is, control group (DMSO, 0.1% DMSO for 24.5h), model group (DMSO+LPS, same concentration of DMSO pretreatment for 0.5h followed by 500ng/ml LPS for 24h), treatment group (genipin+LPS, 50μmol pretreatment with genipin for 0.5h followed by same LPS treatment), and inhibitor group (genipin+EX527+LPS, 0.5h pretreatment of 10μmol EX527 and 50μmol genipin and then same dose of LPS). The Pa value (representing endothelial permeability) was tested by Transwell chamber test, activity of cysteine-containing aspartate-specific proteases 3 (caspase-3) was measured by fluorescence intensity with microplate reader, Sirtuin-1 (SIRT1) level was detected by Western blotting, the change of mitochondrial membrane potential was measured by MitoProbeTM JC-1 assay kit, and apoptosis rate was tested by TUNEL. In vivo experiment:24 female rats were randomly divided into 4 groups. The rats of control, model, treatment and inhibitor groups were respectively injected with 0.5ml DMSO pretreated and 0.5ml normal saline (DMSO+normal saline), 0.5ml DMSO pretreatment followed by 10mg/kg LPS treatment (DMSO+LPS), 5mg/kg of genipin pretreatment and then LPS treatment (genipin+LPS), and pretreatment of 5mg/kg genipin and 5mg/kg EX527 and LPS treatment (genipin+EX527+LPS). The pretreatment time lasted for 30min in all the groups. The vascular permeability ΔI was measured by FITC-albumin fluorescence assay, and expression level of SIRT1 was detected with Western blotting in 60min after LPS or normal saline injection. SPSS statistics 20.0 was used for statistical analysis. According to the data type, single factor analysis of variance, LSD multiple comparison or Tamhane′s T2 test was used for intergroup comparison, and single factor repeated measurement analysis of variance was used for intragroup comparison. Results In vitro experiment:compared with control group [Pa:1.00±0.04; caspase-3 activity:100.0±4.4; JC-1 green/red fluorescence ratio:(100.0±4.8)%; apoptosis rate:(2.3±1.4)%; SIRT1:(100.0±8.9)%], Pa value, caspase-3 activity, JC-1 green/red fluorescence ratio and apoptosis rate were significantly increased in the model group [1.68±0.09, 216.0±23.5, (343.0±28.3)%, (40.8±8.9)%], treatment group [1.35±0.07,5.0±15.0, (220.0±13.3)%, (28.2±6.6)%] and inhibitor group [1.60±0.10,2.0±16.5, (309.0±18.5)%, (38.0±9.5)%], and the expression level of SIRT1 level was decreased[(61.0±8.5)%, (86.7±7.6)%, and (64.3±4.8)% respectively]. Compared with the model group, the former indices in treatment group were reduced obviously, while SIRT1 level was increased (P<0.05). Compared with the treatment group, the Pa, caspase-3, JC-1 green/red ratio and apoptosis rate were elevated significantly in the inhibitor group, while the expression of SIRT1 was decreased significantly (P<0.05). In vivo experiment:the blood pressure of rats in the model, treatment and inhibitor groups were decreased significantly (P<0.05) after LPS injection for 30 and 60min when compared with the values before LPS pretreatment and those of the control group, but there was no significant difference in blood pressure among the 3 groups (P<0.05). Compared with the control group [SIRT1:(100.0±4.0)%, ΔI:(0.12±0.03)], the expression level of SIRT1 was decreased while ΔI was increased in the model group [(49.3±8.3)%, 0.54±0.07], the treatment group [(87.3±4.7)%, 0.32±0.05], and the inhibitor group [(55.3±4.9)%, (0.53±0.06)]. When compared with the model group, the expression of SIRT1 was increased, and ΔI was decreased significantly in the treatment group, but the level was significantly decreased and ΔI was significantly increased in the inhibitor group. Statistical significances were seen in above indicators (P<0.05). Conclusion Genipine inhibits LPS-mediated hyperpermeability by inhibiting the activation of mitochondrial apoptotic signal, and this effect may be related to the up-regulation of SIRT1.
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