Effect And Mechanism Of Tetramethylpyrazine On Mice With Acute Lung Injury Through MiR-194-5p/Rac1/LIMK1 Pathway

Objective:The improving effect of tetramethylpyrazine（TMP）on Lipopolysaccharide（LPS）induced acute lung injury in mice and the role and mechanism of miR-194-5p.Methods:Acute lung injury mouse models were established using a"second hit",and healthy C57/BL6 mice were randomly grouped.ALI model was induced by intraperitoneal injection of LPS for the first time and intratracheal injection of LPS for the second time16h later.The mice in treatment groups had TMP or dexamethasone administered intraperitoneally 30 min prior to the 1st hit and 30 min past the 2nd hit.The knockout and overexpressing miR-194-5p group mice were transfected with adenovirus with knockout and overexpressing miR-194-5p by tracheal drip one week before modeling.Peripheral blood oxygen saturation（Sp O₂）was measured after 24h of endotracheal infusion of LPS;The ratio of wet weight to dry weight（W/D）was calculated from lung tissue.Interleukin1β（IL-1β）,interleukin6（IL-6）and tumor necrosis factorα（TNF-α）in serum of mice were determined by ELISA kit.The expression of IL-1β,IL-6 and TNF-αwas detected by q PCR;HE staining was used to observe the pathological changes of lung tissue.Immunohistochemical staining（IHC）assessed MPO,p-Rac1and p-LIMK1 expression.Western blot detected the expression of Rac1 and LIMK1 in lung tissue,and tight junction related proteins ZO-1 and occludin.The total number of white blood cells and protein concentration in bronchoalveolar lavage solution were detected.Evans Blue detects leakage of lung capillaries;TUNEL was used to detect cell apoptosis in mouse lung tissue.Results:The study was divided into two parts（1）TMP inhibits the activation of Rac1/LIMK1 signaling pathway to improve the hyperpermeability and pulmonary inflammation of lung capillary endothelial cells in LPS-induced ALI mice.C57/BL6 mice were treated with LPS,and it was found that LPS induced lung inflammation and pulmonary edema in mice,and p-Rac1 and p-LIMK1 proteins were increased in lung tissue,Rac1/LIMK1 signaling pathway was activated,and TMP treatment could reverse the above changes.Through ELISA,q PCR,HE staining,Evans Blue,lung tissue wet/dry ratio,immunohistochemical staining,Western blot and other experiments,it was confirmed that TMP could inhibit the activation of Rac1/LIMK1signaling pathway to reduce the permeability of lung capillary endothelial cells and lung inflammation.（2）Effects of miR-194-5p on tetramethylpyrazine in mice with acute lung injuryDual luciferin assay reports confirmed that Rac1 is the target gene of miR-194-5p.In order to further study whether TMP can inhibit Rac1/LIMK1 signaling pathway through miR-194-5p,ALI mice were transfected with miR-194-5p knockout and adenovirus overexpressing miR-194-5p one week before TMP intervention.By Western blot,bronchoalveolar lavage fluid test,Evans Blue test and other tests,it was found that over-expression of miR-194-5p（miR-194-5p）inhibited the Rac1/LIMK1/ZO-1/occludin pathway,thereby reducing the permeability of pulmonary capillary endothelial cells in ALI mice.ALI mice treated with TMP and miR-194-5p（spong-miR-194-5p）could partially block the protective effect of TMP on LPS-induced acute lung injury compared with mice treated with TMP only.These results suggested that TMP mediated by miR-194-5p improved the permeability and inflammation of lung capillary endothelial cells in LPS-induced ALI mice.To sum up,the following conclusions were obtained through in vivo experiments in this study:（1）Activation of Rac1/LIMK1/ZO-1/occludin pathway can lead to pulmonary capillary endothelial cytoskeletal remodeling and increased permeability,and TMP can relieve LPS induced endothelial cytoskeletal remodeling and increased permeability by inhibiting Rac1/LIMK1/ZO-1/occludin pathway.（2）Overexpression of miR-194-5p improves LPS-induced acute lung injury in mice by inhibiting Rac1/LIMK1/ZO-1/occludin pathway activity by targeting Rac1.（3）TMP inhibits LPS-induced ALI in C57/BL6 mice,possibly through the miR-194-5p/Rac1/LIMK1 pathway;miR-194-5p may be an important molecular target for TMP to inhibit ALI mice.