The Role Of LRRC8A On Lipopolysaccharides-induced Acute Lung Injury In Rat

Part 1 The construction of LPS induced ALI model and changes of LRRC8A expression.Objective: Construct the acute lung injury rat model by tracheal injecting LPS,observe the expression of the LRRC8 A with or without intervene LPS.Methods: Thirty healthy adult Sprague-Dawley male rats were randomly divided into negative control group,ALI group.Negative control group treat with saline.ALI group treat with LPS(5mg/kg).The rats were sacrificed after intervention of saline or LPS at 6,12,24 hours three time points.The pathological changes in lung tissue were analyzed,alveolar fluid clearance(AFC)and the ratio of wet/dry were observed,the level of IL-1? and TNF-? were assessed in bronchoalveolar lavage fluid(BALF),the expression of LRRC8 A were measured by western blot and real-time PCR.Results:1.An acute lung injury rat model was constructed.HE stain indicate that LPS could induced acute lung pathological injury and inflammatory responses.With time passing by,the damage of lung tissue increase significantly,especially at 24 h.Comparing with negative control group,ALI group demonstrate the decrease of AFC(P<0.01),the increases of W/D,the level of TNF-? and IL-1? in BALF raised obviously.2.The LRRC8 A protein measured by western blot analysis and expression of LRRC8 A mRNA were detected by realtime-PCR analysis.Compared with negative control group,ALI group showed significantly decreased in both expression of LRR8 A protein and mRNA,particularly at24h(P<0.05).Conclusions:1.After the inducement of LPS,the lung tissue damage increased withtime elapsing,the peak of injury was at 24 h.2.The changes of these indexes including TNF-?,IL-1?,W/D and AFC,which indicate that the model of ALI was successfully created.3.Under the situation of ALI,the expression of LRRC8 A protein and mRNA both decreased,hypothesizing that LRRC8 A maybe take a part in ALI progress.Part 2 Effects of Ad-sftpc-LRRC8 A on AFC in LPS induced lung injury rats.Objective: To investigate the role of LRRC8 A in acute lung injury.Construct the model of overexpression LRRC8 A in the lung by nasal injection of Ad-sftpc-LRRC8 A and evaluate the AFC levels.Methods: Eighteen SD rats were randomly divided into three groups:group A(Ad-sftpc-EGFP),group B(Ad-sftpc-lrrc8a-EGFP),group C(Ad-EGFP).Three groups through nasal administration of different adenovirus,Ad-sftpc-EGFP,Ad-sftpc-lrrc8a-EGFP,Ad-EGFP respectively.Two weeks later,each group sacrifice 3 rats to take out lung,spleen,liver and kidney tissues,use confocal microscopy to detective the distribution of adenovirus vector.The levels of LRRC8 A protein were tested by western blot analysis.The other 3 rats of each group treated with LPS follow part 1protocol,then evaluate the AFC after 24 h later.Results: Pathology of lung tissues indicate no severe damages and inflammation response.Confocal microscopy can observe the green fluorescence under the excitation of 488 nm laser.The green fluorescence of group A and B can be found only in lung,other liver,spleen and kidney tissues could not detect.Group C can detected green fluorescence in all parts of lung,liver,spleen and kidney tissues.Compared with group A and C,western blot revealed that the protein of LRCC8 A increased obviously in group B.It indicates that the gene transfer was totally successful.During acute lung injury,Group B with overexpression of LRRC8 A has an enhanced AFC,comparing with the other two groups(P<0.01).Conclusions: After nasal administration of Ad-sftpc-lrrc8a-EGFP,the expression of LRRC8 A in the lung tissues of rats is obvious upregulated.After administration of adenovirus vector,there are no significant side effects,and no obvious inflammation response were observed.The adenovirus vector of group A and B mainly distribute in lung,the other organs such as liver,kidney and spleen does not spread.The results proved that the LRRC8 A overexpression model was well constructed.Overexpression of LRRC8 A enhance the AFC after ALI.It can conclude that LRRC8 A maybe can alleviate ALI,playing a protective role in ALI process.