Protective Effects And Mechanisms Of Isorhapontigenin Against Lipopolysaccharide-induced Acute Lung Injury Via Nrf2 Signaling Pathway

OBJECTIVE:To establish an animal model of acute lung injury and a macrophage inflammation model expose to LPS,and to explore whether ISO can promote autophagy by activating the expression of SIRT1 in macrophages,leading to the P62-dependent autophagic degradation of Keap1,which in turn promotes translocation of Nrf2 into the nucleus,exerts anti-oxidative and anti-inflammatory effects and protects ALI mice.METHODS:The first part is to explore the protective effect of ISO on LPS-induced acute lung injury via Nrf2 pathway.In Vitro study: Firstly,we chose RAW264.7 cell line to explore whether ISO could alleviate LPS-induced inflammatory response and oxidative stress;secondly,to explore whether ISO could alleviate oxidative stress and inflammation by activating the Nrf2 pathway.Lastly,we used Nrf2 inhibitor to rescue our experiments.In Vivo study: C57BL/6 mice were selected and Nrf2 inhibitor was used to explore whether ISO had a protective effect on LPS-induced ALI mice via Nrf2 pathway.The second part is to explore the protective effect of ISO on LPS-induced acute lung injury via SIRT1/Nrf2 pathway.In Vitro study: Firstly,we determined whether ISO could activate the SIRT1/Nrf2 pathways,and explored the regulatory relationship between the SIRT1 and Nrf2 pathways;secondly,to explore whether ISO could alleviate oxidative stress and inflammation by activating the Nrf2 pathway.In Vivo study: C57BL/6 mice were selected and SIRT1 inhibitor was used to explore whether ISO had a protective effect on LPS-induced ALI mice via SIRT1/Nrf2 pathway.The third part is to explore the protective effect of ISO on LPS-induced acute lung injury via SIRT1/autophagy/Nrf2 pathway.In Vitro study: firstly we determined whether ISO could activate SIRT1,induce autophagy and the regulatory relationship between SIRT1 and autophagy;secondly,we explored the regulatory relationship between autophagy and Nrf2 pathways;Lastly,to explore whether ISO could alleviate oxidative stress and inflammation by activating the SIRT1/autophagy/Nrf2 signaling pathway,and we used autophagy inhibitor to rescue our experiments.In Vivo study:C57BL/6 mice were selected and autophagy inhibitor was used to explore whether ISO had a protective effect on LPS-induced ALI mice by promoting autophagy.RESULTS:The first part confirmed that ISO could reduce the inflammatory response and oxidative stress in macrophages through antioxidant and anti-inflammatory effects,and alleviate the severity of ALI mice exposed to LPS.In the mechanism study,we confirmed that ISO could promote translocation of Nrf2 into the nucleus,promote HO-1,thereby exerting an anti-oxidative and anti-inflammatory effect.By inhibiting the Nrf2/HO-1 pathway,the anti-inflammatory and antioxidant effects of ISO were reversed.In the second part,we confirmed that ISO could enhance the translocation of Nrf2 into the nucleus by activating SIRT1,and then increased the expression of its downstream target proteins HO-1 and GCLM,thereby exerting anti-oxidative and antiinflammatory effects.By inhibiting the expression of SIRT1,the Nrf2 pathway was also inhibited,and the anti-inflammatory and antioxidant effects of ISO were reversed.In the third part,we confirmed that ISO could promote autophagy by activating SIRT1,which in turn induced the P62-mediated autophagic degradation of Keap1,and finally activated the Nrf2 pathway to exert its anti-oxidative and anti-inflammatory effects.By inhibiting the expression of SIRT1,the autophagy pathway and the P62/Keap1/Nrf2 pathway were inhibited;meanwhile inhibiting autophagy,the antiinflammatory and antioxidative effects of ISO were reversed.CONCLUSION:Our study confirmed that ISO could inhibit oxidative stress and inflammation,exert a protective effect on LPS-induced macrophage inflammation models and ALI mice.The mechanism of this protective effect might be by activating the expression of SIRT1,promoting autophagy,which leads to the autophagic degradation of P62-dependent Keap1,which in turn promoted translocation of Nrf2 into the nucleus.