Study On The Effect And Mechanism Of Stellera Chamaejasme L.Flavonoids TT-11 In Alleviating Acute Lung Injury

Objective Acute lung injury(ALI)and its more serious form,acute respiratory distress syndrome(ARDS),have a high incidence rate in the world,with complex etiology and unclear pathogenesis.At present,there is still a lack of specific drugs and treatment methods in the clinic.Therefore,the purpose of this study is to explore the protective effect of TT-11 on ALI and its molecular mechanism by using molecular biology,experimental zoology,and other techniques:1.To investigate the protective effect of TT-11 on LPSinduced ALI in mice;2.To explore the inhibitory effects of TT-11 on LPS-induced inflammation;3.Clarify the protective mechanism of TT-11 on LPS-induced ALI.Methods1.Study on the effect of TT-11 on LPS-induced ALI in mice:the mouse model of acute lung injury was established by dropping LPS into the throat,the lungs were taken,the pathological changes of lung tissue were observed by H&E staining,the wet-dry weight ratio of the lung was measured,and the MPO activity was detected by MPO assay kit,alveolar lavage fluid(B ALF)was taken for cell count and total protein concentration.2.Effect of TT-11 on LPS-induced inflammatory response in vivo and in vitro:LPS-induced ALI in vivo:BALF was taken,the secretion of inflammatory factors was detected by ELISA assay,and the release of NO was detected by Griess method;The homogenate of lung tissue was taken,and the expression levels of inflammation-related proteins were detected by Western Blotting.LPS induced Raw 264.7 cells in vitro:MTT assay was used to detect the effect of TT11 on cell viability.The concentration without obvious cytotoxic effect was selected for in vitro anti-inflammatory experiments:the supernatant was taken,the secretion of inflammatory factors was detected by ELISA assay kit,the release of NO was detected by Griess method,and the expression levels of inflammation-related proteins were detected by Western Blotting.3.Study on the mechanism of TT-11 alleviating LPS-induced ALI via Nrf2-ROSNLRP3 signaling axis:LPS-induced ALI in vivo:BALF was taken,ROS level was detected by flow cytometry,and MDA level was detected by MDA assay kit;the lung was taken,and the SOD activity was detected by SOD assay kit,and the proteins were detected by Western Blotting which were related with Nrf2-ROS-NLRP3 signaling axis.LPS-induced Raw 264.7 cells in vitro:ROS level was detected by flow cytometry,MDA level was detected by MDA assay kit,and SOD activity was detected by SOD assay kit,NLRP3 inflammasome,and Nrf2 related proteins were detected by Western Blotting assay,Nrf2 nuclear translocation was detected by nucleocytoplasmic isolation and immunofluorescence,ROS inhibitor,Nrf2 activator,and Nrf2 inhibitor were used to explore the role of Nrf2-ROS-NLRP3 signaling axis in TT-11 alleviating LPS-induced ALI.Results1.TT-11 alleviated ALI induced by LPS,inhibited pathological changes of lung tissue,pulmonary edema,and permeability changes,and inhibited MPO activity induced by LPS.2.TT-11 inhibited the expression and secretion of proinflammatory cytokines IL-6,IL1/3,and TNF-α,and reduced the release of NO by inhibiting the expression of iNOS,and inhibited the phosphorylation of MAPKs induced by LPS in vivo and in vitro.3.TT-11 activated Nrf2 nuclear translocation by promoting Keapl degradation,activated downstream antioxidant proteins GCLM and HO-1,inhibited LPS-induced ROS and MDA levels,and restoreed SOD activity,moreover,TT-11 inhibited LPS-induced inflammatory response and oxidative stress via Nrf2-ROS-NLRP3 signaling axis.Conclusion TT-11 ameliorates LPS-induced ALI,and its mechanism is related to the activation of Nrf2 and the inhibition of NLRP3 inflammasome activation mediated by ROS.