| ObjectiveIn this study,we investigated the protective effects of keratinocyte growth factor-2(KGF-2)on lung tissue induced by oleic acid induced acute lung injury(ALI)in rats by observing changes in pulmonary microvascular endothelial barrier and related pathways.Methods1.Group: 45 male Sprague-Dawley rats were randomly divided into control group,ALI group and ALI+KGF-2 group.2.Treatment: The control group was injected with normal saline(NS)in the tail vein,and NS was injected into the lung 72 hours before the tail vein injection;oleic acid was injected into the tail vein of the ALI group,and the lung was instilled NS 72 hours before modeling;ALI+KGF-2 The group was injected with oleic acid in the tail vein,and KGF-2was instilled into the lungs 72 hours before modeling.The rat ALI model was replicated by tail vein injection of oleic acid.3.After different treatment according to the group,the abdominal aorta of the rats was sacrificed at 8 h,and lung tissue samples and bronchoalveolar lavage fluid(BALF)were obtained.4.The changes of lung tissue injury were observed: the lung tissue changes were observed in general;the lung histopathological examination and lung injury score were used to evaluate the degree of lung injury in HE staining;the ultrastructural changes of rat lung tissue were observed under electron microscope.5.To observe the changes of inflammation and apoptosis in lung tissue: TUNEL was used to detect the apoptosis of lung tissue epithelial cells and endothelial cells;ELISA was used to detect the levels of TNF-a and IL-10,and the degree of inflammation in the lung was evaluated;Wright-Gemsa staining was used to observe cell changes in BALF.6.To observe the changes of alveolar-capillary barrier permeability: Calculate the wet weight to dry weight ratio of rat lung tissue,and evaluate the edema of lung tissue in rats;Calculate the permeability index of rat lung tissue and evaluate the protein of rat lung tissue Leakage;Evans blue test to assess pulmonary vascular permeability in rat lung tissue.7.The expressions of tight junction proteins Claudin-5,Zo-1 and VE-cadherin in lung microvascular endothelial cells were observed.Immunohistochemistry was used to determine the localization and expression of Claudin-5,Zo-1 and VE-cadherin in rat lung tissues.The expressions of Claudin-5,Zo-1 and VE-cadherin in rat lung tissue were detected by Western blot.The expression levels of Claudin-5,Zo-1 and VE-cadherin mRNA were detected by RT-PCR.8.The expressions of Wnt/?-catenin signaling pathway proteins Wnt5 a and ?-catenin were observed: Immunohistochemistry was used to determine the localization and expression of Wnt5 a,?-catenin in rat lung tissue;Western blot was used to determine the expression of Wnt5 a,?-catenin in rat lung tissue.Results1.Changes in lung tissue damage:1.1 Gross observation: There was no obvious abnormal change in the lung tissue of the control group;the lung tissue of the ALI group was severely damaged;the lung injury of the ALI+KGF-2 group was alleviated.1.2.1 Observation under light microscope: no obvious abnormalities were found in the lung tissue of the control group.In the ALI group,the alveolar space of the lung tissue wasthickened,and a large number of inflammatory cells accumulated in the alveolar space.In the ALI+KGF-2 group,lung tissue was relieved.1.2.2 ALI lung injury score(LIS): LIS value of ALI group was higher than that of control group(P<0.01).Compared with ALI group,LIS of ALI+KGF-2 group was lower(P<0.01),but compared with control group LIS high.1.3 Ultrastructural changes of rat lung tissue were observed under electron microscope:the microvascular structure of the lung tissue of the control group was intact.In the ALI group,the lung tissue cells were severely damaged.The ALI+KGF-2 group was less damaged.2.Inflammatory changes and apoptosis in lung tissue:2.1 Apoptosis was observed: scattered apoptotic cells were scattered between the lung tissues of the control group.The number of apoptotic cells in the ALI group was significantly increased(P<0.01).After KGF-2 intervention,the number of apoptotic cells decreased,but it was still higher than the control group(P<0.01).2.2 The expression levels of TNF-a and IL-10 were observed.Compared with the control group,the expression of TNF-a in lung tissue of ALI group was significantly increased(P<0.01),and the expression level of IL-10 was decreased(P<0.01).Compared with ALI group,ALI+KGF-2 group showed a decrease in TNF-a expression level(P < 0.01)and IL-10 expression level(P<0.01).2.3 The changes of BALF cells were observed: compared with the control group,the white blood cells in the BALF of the ALI group were significantly increased,and a large number of red blood cells were present.Compared with the ALI group,the white blood cells in the BALF of the ALI+KGF-2 group were decreased.The number of red blood cells decreased.Compared with the control group,the white blood cells in the BALF of the ALI+KGF-2 group increased,and the number of red blood cells increased significantly.3.Alveolar-capillary barrier permeability change:Lung tissue wet-to-dry ratio(W/D),lung permeability index(LPI),and Evans blue(EB)experiments in each group: lung W/D ratio,LPI value,EB in ALI group compared with control group The exudation content increased significantly(P < 0.01).After KGF-2pretreatment,the lung W/D ratio and LPI value were significantly lower than the ALI model group(both P<0.01)and the EB exudation content decreased(P<0.05).Compared with the control group,the ALI+KGF-2 group had an increase in lung W/D ratio,LPI value,and EB exudation content(P<0.01).4.Changes in expression of tight junction proteins Claudin-5,Zo-1 and VE-cadherin between pulmonary microvascular endothelial cells:Oleic acid-induced ALI can decrease the expression of Claudin-5,Zo-1 and VE-cadherin in pulmonary microvascular endothelial cells(all P<0.01);Pretreatment with ALI+KGF-2 group increased the expression of Claudin-5,Zo-1 and VE-cadherin(all P<0.05).5.Expression of Wnt/?-catenin signaling pathway proteins Wnt5 a and ?-catenin:The expression of Wnt5 a and ?-catenin protein was increased by oleic acid-induced ALI in rats(all P<0.01).After pretreatment with KGF-2,the expression of ?-catenin protein was decreased(P < 0.01),However,Wnt5 a protein expression was not significantly reduced(p>0.05).Conclusion1.KGF-2 has protective effect on oleic acid-induced ALI rats by restoring pulmonary microvascular endothelial barrier.2.The protective effect of KGF-2 on pulmonary microvascular endothelial barrier induced by oleic acid may be related to the regulation of Wnt/?-catenin signaling pathway. |
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