The Study Of The Role Of Angiogenin-1 And Angiogenin-2 In Phosgene Induced Acute Lung Injury Of Rats

Part 1Study of the the effect of Angiogenin-1 in phosgene induced ALI of ratsObjective:To measure the level of angiogenin-1 in serum and assess the severity of acute lung injury to explore the effect of angiogenin-1 in phosgene induced ALI of rats to provide theoretical basis of phosgene induced ALI.Method:①12 clean healthy male SD rats were randomly divided into 2 groups:The phosgene group (rats inhaled with phosgene 100% 8.33mg/L 5 minutes) and the control group (rats inhaled with fresh air for 5 minutes),6 rats in each group(n=6).②After inhaled phsogene or fresh air, the rats were fed with food and water in normal way.③The Angiogenin-1 levels in the serum were measured by enzyme-linked immunosorbent assay(ELISA), histopathological examinations (hematoxylin and eosin stain) of the lung of the rats were performed and lung wet/dry weight ratio of the rats was performed 6 hours after inhaled the air or phosgene. The counter of the white cells in the bronchia alveolus lavage fluid (BALF) and the measurement of the angiogenin-1 concentration in bronchoalveolar lavage fluid of rats were performed 6 hours after the rats inhaled the air or phosgene. The data do the statistical analysis using SPSS 16.0, t-test, P<0.05.Result:The lung in the phosgene inhaled rats were more seriously dameged comparing with that in control group. The level of the angiogenin-1 in the serum in the phogene inhaled rats were lower than that in the control group, P<0.05.Conclusion:The concentration of the angiogenin-1 in the serum in phogene inhaled rats were lower than that in the control group, p<0.05, which indicates that the decreased expression of angiopoietin-1 may be associated with acute lung injury induced by phosgene. So it is worthwhile to do the further research in angiogenin-1 in rats phosgene induced ALI. Party 2The study of the change of the ratio of angiopoietin-2 to angiopoietin-1 in phosgene induced ALI ratsObjective:This study observe the relationgship of phosgene-induced acute lung injury rats serum ratio of angiopoietin-2 to angiopoietin-1 and lung damage index (lung wet to dry ratio, bronchoalveolar lavage fluid cells (BALF) protein concentration, BALF leukocyte count) to discuss the mechanism of phosgene inhaled induced acute lung injury, which provide theoretical foudation of prevention and treatment of it.Methods:①Selected healthy adult male SD rats were randomly divided into 13 experimental groups (phosgene group):13 group after rats inhaled with phosgene for 5 mintues Groups and control group (air group), n=6.②Rats were given different gas treatment:control group (air group) rats stayed in exposure cabinet (only fresh air without phosgene) for 5 minutes; the experimental group (phosgene group) stayed in the exposure cabinet for 5 minutes (100% purity of 8.33mg/L phosgene), the rats were fed with clean water and food after inhaled with air or phosgene.③ake lungs of rats and calculated wet/dry weight ratio, measure bronchoalveolar lavage fluid cells (BALF) white blood cell count, protein concentration; Draw blood, do centrifugation, gather supernatant fluid and measure the level of angiopoietin-1 and angiopoietin-2 with the method of ELISA and calculate ratio of angiopoietin-2 to angiopoietin-1.④data were analyzed using SPSS 16.0, ANOVA test and LSD-t test P<0.05.Results:①Rats inhaled with Phosgene within 48 hours, serum angiopoietin-2, lung wet/dry weight ratio, bronchoalveolar lavage fluid cells (BALF) white blood cell count, protein concentration tended to increase with time elapsed; Rats serum angiopoietin-1 tended to decrease with time elapsed.②orrelation coefficient of angiopoietin-2 and lung wet/dry ratio was 0.441, P<0.01, that of angiopoietin-2 and lavage fluid cells (BALF) protein concentration was 0.283, P<0.01, that of angiopoietin-2 and BALF WBC count was 0.439, P<0.01. Correlation coefficient of angiopoietin-1 and lung wet/dry ratio was -0.286 P<0.01, that of angiopoietin-1 and lavage fluid cells (BALF) protein concentration was-0.198, P= 0.071, that of angiopoietin-1 and BALF WBC count was-0.362 P<0.05= 0.001. Correlation coefficient of Ratio of angiopoietin-2 to angiopoietin-1 and lung wet/dry ratio was 0.683, P <0.01, that of ratio of angiopoietin-2 to angiopoietin-1 and lavage fluid cells (BALF) protein concentration was 0.534, P<0.01, that of ratio of angiopoietin-2 to angiopoietin-1 and BALF leukocyte count was 0.710, P <0.01.Conclusion:Rats inhaled with this time*concentration of phosgene, ratio of angiopoietin-2 to angiopoietin-1 and lung damage index (lung wet to dry ratio, bronchoalveolar lavage fluid(BALF) cell counts、protein concentration were correlative, ratio of angiopoietin-2 to angiopoietin-1 may be an early indicator of the severity of phosgene-induced acute lung injury.Party 3The protective mechanism of angiogenin-1 in phosgene induced acute lung injury model in ratsObjective:Observe the level of angiogenin-1, NF-κB and aseess the severity phosgene induced acute lung injury in rats and discuss the mechanism of the protective effect of angiogenin-1 in phosgene induced acute lung injury, which provide theoretical foudation of the study of phosgene induced acute lung injury.Methods:①18 clean healthy male SD rats were randomly divided into 3 groups:the experimental group (phosgene group), PDTC-treated group and the control group 1(air group),6 rats in each group (n=6).②ats inhale with 100% 8.33mg/L fresh air or phosgene for 5 minutes, rats treated with PDTC via intraperitoneal injection with the dose of 100 mg/kg 30 minutes after the rats inhaled with phosgene in PDTC-treated group, rats treated with normal saline via intraperitoneal injection with the dose of 100 mg /kg 30 minutes after the rats inhaled with phosgene in phosgene group and air group, after finished that, all rats were raised with clean water and food for 12 hours.③Do the arterial blood gas analysis after the rats raised for 12 hours, assess the lung damage index (lung wet/dry weight ratio, cell count of bronchoalveolar lavage fluid (BALF), protein concentration in BALF, lung injury score of HE stained tissure); Measure the angiogenin-1 level in serum using ELISA; Measure angiopoietin-1 and NF-κB level in lung tissue using westblot and immunohistochemical. Data were analyzed using SPSS 16.0 statistical software, ANOVA and LSD t test, P<0.05.Results:①Wet-to-dry ratio of lung tissue white Blood Cell Count in BALF, lung injury score, partial pressure of carbon dioxide in arterial blood, protein concentration in BALF were significantly elevated in phosgene group [6.56±1.36、10.28±2.06,5.83±1.16,55.6±2.5,70.67±29.29], comparing with that in the air group [3.64±0.72,2±0.82, 0.33±0.051,42.67±4.40,37.00±13.44] (P<0.05); Serum Concentration of angiopoietin-1, partial pressure of oxygen in arterial blood in arterial were significantly lower in phosgene group[1.70±0.14,6.71±0.97] comparing with that in the air group 1.93±0.12,13.13±1.53 (P<0.05); (2)Wet-to-dry ratio of lung tissue, phosgene group, white Blood Cell Count in BALF, lung injury score, partial pressure of carbon dioxide in arterial blood, protein concentration in BALF were significantly elevated in phosgene group [6.56±1.36,10.28±2.06,5.83±1.16,55.6±2.5,70.67±29.29], comparing with that in the PDTC-treated group [5.15±1.17, 7.14±1.35,3.61±0.58,46.370±3.62,57.33±27.5] (P<0.05); Serum Concentration of angiopoietin-1, partial pressure of coxygen in arterial blood in arterial were significantly lower in phosgene group[1.70±0.14, 6.71±0.97] comparing with that in PDTC-treated group[55.6+2.5, 46.370±3.62] (P>0.05). Angiopoietin-1 level of immunohistochemistry of lung tissure in experimental group was lower than that in air group; while angiopoietin-1 level of immunohistochemistry of lung tissure in the PDTC-treated group was higher than that in phosgene group, which was lower than that in the air group. The result of West-blot of lung tissure indicated the same thing as that of immunohistochemistry of Angiopoietin-1 level. NF-κB level of immunohistochemistry of lung tissure in experimental group was higher than that in air group; while NF-κB level of immunohistochemistry of lung tissure in the PDTC-treated group was lowerer than that in phosgene group, which was lower than that in the air group. The result NF-κB of West-blot of lung tissure indicated the same thing as that of immunohistochemistry of NF-κB level.Conclusion:Angiopoietin-1 may have an effect on NF-κB signaling pathway to inhabit the elavated of inflammatory cytokines to reduce inflammation, lung endothelial permeability, which lessen the severity of acute lung injury.