Mechanism Study Of Emodin And 3-dehydroandrographolide On Attenuating Lipopolysaccharide-induced Acute Lung Injury Based On Traditional Chinese Medicine Screening Via Zebrafish Inflammation Models

Background and object:The zebrafish model has played an important role in the study of human diseases in recent years due to its advantages such as simple operation,economy,high homology with the human genome and the genes related to human diseases.Acute lung injury(ALI)can induce systemic uncontrolled inflammatory response with high incidence and mortality,and there is still no effective treatment.In this study,we evaluated the anti-:inflammatory activities of emodin and 3-dehydroandrographolide(3-DA)via zebrafish inflammation models and explored underlying anti-ALI mechanisms.Methods and results:1.Emodin and 3-DA improved the survival ability of LPS-stimulated zebrafish,and inhibited the neutrophil migration in inflammation zebrafish caused by copper sulphate,tail transection or LPS microinjection.2.The non-toxic dosages of emodin in mouse macrophage cell line(RAW264.7)were evaluated by cytotoxicity assay.ELISA results showed that emodin decreased the levels of tumor necrosis factor-a(TNF-a)and interleukin-6(IL-6)in a concentration-dependent manner.Western blotting data showed that emodin inhibited the phosphorylation of JNK(Thrl83/Tyrl85),but not the phosphorylation of p38(Thr180/Tyrl82)and ERK(Thr202/Tyr204).Moreover,emodin up-regulated the expression of Nur77,decreased the phosphorylation of Nur77(Ser351).Emodin also inhibited the expression and the phosphorylation of c-Jun(Ser73).SP600125(a specific inhibitor of JNK)enhanced the effects of emodin on the Nur77/c-Jun signaling pathway,while anisocymin(a specific agonists of JNK)counteracted the effects of emodin on the Nur77/c-Jun signaling pathway.Dual-luciferase reporter assay results showed that the emodin increased the activity of Nur77 promoter by inhibiting JNK.siRNA transfection and EMSA results showed Nur77 siRNA treatment blocked the inhibitory effects of emodin on the expression,the phosphorylation levels and DNA-binding activity of c-Jun,respectively.The ALI mice model was established by intratracheal instillation of LPS.H&E staining results showed emodin alleviated the lung histopathological changes in ALI mice.W/D index indicated that emodin decreased the lung water content.The BCA data showed emodin decreased the total protein in bronchoalveolar lavage fluid(BALF).The ELISA results demonstrated emodin decreased the production of TNF-? and IL-6 in BALF.Western blotting results showed emodin regulated the Nur77/c-Jun signaling pathway in ALI.These protective effects of emodin on ALI were counteracted by the treatment of anisomycin.Surface plasmon resonance(SPR)and HPLC-MS/MS results found 106 potential target proteins were captured by emodin in RAW264.7 cells.Western blotting results showed emodin decreased the expression of ROCK1 and up-regulated the phosphorylation of PAK2 in LPS-stimulated RAW264.7 cell and ALI mice.3.The non-toxic dosages of 3-DA on RAW264.7 cells were evaluated by cytotoxicity assay.The ELISA data showed that 3-DA decreased the expression of TNF-a and IL-6 in a concentration-dependent manner.Western blotting results found 3-DA inhibited the degradation and phosphorylation of I?B?(Ser32).Confocal microscope observation found 3-DA blocked the nuclear.import of NF-?B p65.Western blotting results showed 3-DA inhibited the phosphorylation of Akt(Ser473),and up-regulated the expression of a7nAchR.SPR results revealed the combination of 3-DA and ?7nAchR.Western blotting results showed the inhibitory effects of 3-DA on the NF-?B/Akt signaling pathway were counteracted in the presence of a7nAchR siRNA or methyllycaconitine(MLA,a a7nAchR specific inhibitor).Moreover,3-DA significantly inhibited inflammation in LPS-induced ALI mice,which was related to the alleviated lung histopathological changes,decreased lung water content and the production of inflammatory cytokines TNF-? and IL-6 as well as the suppression of the NF-KB/Akt signaling pathway.However,these effects of 3-DA were attenuated by the treatment with of MLA.Couclusion:Our research frist demonstrates that emodin exerts anti-inflammatory effects through regulation of the JNK/Nur77/c-Jun signaling pathway and 3-DA inhibits inflammation through up-regulation of the cholinergic anti-inflammatory pathway.