ABSTRACT
Objective To evaluate the effect of ulinastatin on γ-aminobutyric acid (GABA) signal pathway in mice with ventilator-induced lung injury (VILI).Methods Thirty-six male Wister mice were randomly divided into 3 groups using a random number table:control group (group C), ventilator-induced lung injury group (group VILI),and ventilator-induced lung injury+ ulinastatin group (group UTI),n =12 in each group.VILI was induced by 4 h mechanical ventilation with tidal volume 40 ml/kg in groups VILI and UTI.Ulinastatin 1×10 5 U/kg was injected intraperitoneally 1 h before ventilation in group UTI,while the equal volume of normal saline was given in groups C and VILI.The mice were then sacrificed,the left lung was lavaged,and broncho-alveolar lavage fluid (BALF)was collected for determination of concentrations of protein,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and intercellular adhesion molecule-1 (ICAM-1).The lung tissues were re-moved for determination of the wet to dry lung weight (W/D)ratio,the mRNA expression level of IL-1β,TNF-αand ICAM-1.The pathological changes of the lungs were determined under light micro-scope and the lung injury scores were also determined.Immunohistochemistry and Western blot were used to detected the protein expression level of GAD and GABAA R.Results The W/D ratio (6.7 ± 2.4 vs.8.5±2.3)and lung scores [(6.9±2.3)scores vs.(1 1.8±2.7)scores]were significantly de-creased in group UTI than those in group VILI.The concentrations of IL-1β[(56±1 1)ng/L vs.(77 ±1 5)ng/L],TNF-α[(105±29)ng/L vs.(1 58±37)ng/L]and ICAM-1 [(205±46)ng/L vs.(293 ±61)ng/L]in BALF in group UTI were significantly decreased than those in group VILI.The mRNA ex-pression levels of IL-1β(1.81±0.26 vs.2.58±0.34),TNF-α(1.61±0.15 vs.2.94±0.27)and ICAM-1 (1.74±0.27 vs.2.79±0.31)were significantly decreased in group UTI than those in group VILI.The protein expression levels of GAD (0.44±0.08 vs.0.18 ±0.04)and GABAA R (0.30 ±0.09 vs.0.15 ± 0.04)were significantly increased in group UTI than those in group VILI.Conclusion Ulinastatin can at-tenuate VILI probably through activating GABA signaling pathway.
ABSTRACT
Objective To evaluate the effect of mechanical stretch preconditioning on pathological stretch-induced activation of γ-aminobutyric acid (GABA) signaling pathway in human type Ⅱ alveolar epithelial cells (AEC Ⅱ).Methods AEC Ⅱ cell line (A549 cells) culturedin vitro were divided into control group (group C), pathological stretch group (group P1) and mechanical stretch preconditioning group (group P2). In group C, A549 cells were cultured routinely. In group P1, A549 cells were exposed to 20% cyclic stretch for 6 hours. In group P2, A549 cells were exposed to 5% cyclic stretch for 60 minutes, and then exposed to 20% cyclic stretch for 6 hours. The cells were harvested for determination of the cell viability by methyl thiazolyl tetrazolium assay, lactate dehydrogeuase (LDH) release was determined by colorimetric method, the levels of interleukin (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA), the mRNA expressions of IL-1β, IL-6 and TNF-α were determined by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expressions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid A receptor (GABAAR) were determined by Western Blot.Results Compared with group C, the cell viability of group P1 was significantlydecreased (A value: 0.196± 0.071 vs. 0.886±0.107), the release rate of LDH was significantly increased [(12.3±2.4)% vs. (1.9±0.5)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly increased [IL-1β (ng/L): 138.6±19.7 vs. 32.7±7.4, IL-6 (ng/L): 196.5±31.7 vs. 55.4±13.8, TNF-α (ng/L): 111.3±21.8 vs. 20.8±7.6; IL-1β mRNA (2-ΔΔCT): 2.79±0.44 vs. 0.83±0.12, IL-6 mRNA (2-ΔΔCT): 1.99±0.25 vs. 0.56±0.11, TNF-α mRNA (2-ΔΔCT): 2.54±0.37 vs. 0.72±0.09]; the protein expressions of GAD and GABAAR were significantly decreased [GAD (gray value): 0.38±0.12 vs. 1.75±0.45, GABAAR (gray value): 0.29±0.09 vs. 1.68±0.39; allP < 0.05]. Compared with group P1, the cell viability of group P2 was significantly increased (A value: 0.523±0.132 vs. 0.196±0.071),the release rate of LDH was significantly decreased [(6.9±1.7)% vs. (12.3±2.4)%]; the contents and mRNA expressions of IL-1β, IL-6 and TNF-α in cell culture medium were significantly decreased [IL-1β (ng/L): 79.2±11.6 vs. 138.6±19.7, IL-6 (ng/L): 89.6±15.6 vs. 196.5±31.7, TNF-α (ng/L): 55.9±11.4 vs. 111.3±21.8; IL-1β mRNA (2-ΔΔCT): 1.92±0.36 vs. 2.79±0.44, IL-6 mRNA (2-ΔΔCT): 1.09±0.18 vs. 1.99±0.25, TNF-α mRNA (2-ΔΔCT): 1.77±0.25 vs. 2.54±0.37]; the protein expressions of GAD and GABAAR were significantly increased [GAD (gray value): 1.26±0.33 vs. 0.38±0.12, GABAAR (gray value): 1.04±0.15 vs. 0.29±0.09; allP < 0.05]. Conclusion The mechanism by which mechanical stretch preconditioning attenuates pathological stretch-induced injury in human AECⅡ is related to the activation of GABA signaling pathway.