| Objective Apoptosis is a key factor in the occurrence of acute lung injury(ALI).The major aim of this study is to explore the protective effect of small molecule drug ITE on ALI,and to further explore its influence on ALI through PI3K/Akt signaling pathway.Methods(1)A total of 36 male SD rats were randomly divided into saline group(Control group),lipopolysaccharide group(LPS group)and small molecule drug ITE group(LPS+ITE group).Constructing rat ALI model by instilling LPS in airway.The rats in the 3groups were instilled LPS(5mg/kg)or the same amount of normal saline into the tracheal tube for 2 hours,and then the small molecule drug ITE(2mg/kg)was injected intraperitoneally in the ITE group.In the Control group and LPS group,the same amount of saline was injected,and the administration was repeated 24 hours and 48 hours after the drug injection.(2)The rats were sacrificed 72 hours after the first intraperitoneal administration,and the lung tissues were taken out for further testing.Observe and record the living condition of the rat and the general condition of the lung tissue of the rat.Use hematoxylin-eosin staining(HE)staining method to observe the pathological morphology of rat lung tissue.The apoptosis of lung tissues determined by terminal-deoxynucleoitidy1 transferase mediated nick end labeling(TUNEL)staining.The expression level of PI3K,Akt,Bcl-2,Bax,cleaved Caspase-3 and cleaved Caspase-9 protein and the phosphorylation level of p-Akt were evaluated by Western blot.Results(1)Successfully established a rat ALI model.The results of HE staining showed that a large number of inflammatory cells infiltrated the lung tissues of the LPS group,and the alveolar structure was destroyed.Compared with the LPS group,the lung tissue structure of the ITE group tended to be normal,and ITE could significantly alleviate the lung tissue damage caused by LPS stimulation.(2)The results of lung tissue apoptosis detected by TUNEL staining showed that compared with the Control group,the number of apoptosis in the LPS group was significantly increased(p<0.05);compared with the LPS group,the number of apoptosis in the ITE group was significantly reduced(p<0.05).(3)Western blot detection of related apoptotic proteins in lung tissue showed that the expression of Bax,cleaved Caspase-9 and cleaved Caspase-3 proteins in the LPS group was significantly increased compared with the Control group(p<0.05);compared with the LPS group,ITE The protein expression of Bax,cleaved caspase-9 and cleaved Caspase-3 decreased in the group(p<0.05).Compared with the Control group,the Bcl-2 protein expression in the LPS group was significantly decreased(p<0.05);compared with the LPS group,the Bcl-2 protein expression in the ITE group was increased(p<0.05).(4)Immunohistochemical results showed that the expression of CYP2J2 in the lung tissue of the Control group was less;compared with the Control group,the expression of CYP2J2 in the lung tissue of the LPS group was significantly reduced;compared with the LPS group,the expression of CYP2J2 in the lung tissue of the ITE group A significant increase.(5)The effect of ITE on PI3K/Akt signaling pathway: Compared with Control group,PI3K protein expression and p-Akt(Ser473)phosphorylation level in LPS group were significantly decreased(p<0.05);Compared with LPS group,PI3K protein in ITE group The expression and phosphorylation level of p-Akt(Ser473)increased significantly(p<0.05).Conclusion As the endogenous ligand of Ah R,the small molecule drug ITE can upregulate the expression of CYP2J2 protein and generate a large number of EETs,which reducing the apoptotic response,improving LPS-induced ALI,and reducing lung tissue apoptosis.The possible mechanism is related to the PI3K/Akt signaling pathway. |
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