清燥救肺汤通过FOXK-FOXO-SKP2/ACSS2-TFEB-CARM1双通路调控肺癌细胞自噬溶酶体形成相关蛋白的表达OA

Objective:To detect the autophagosome formation(FOXK-FOXO-SKP2)and lysosome synthesis(AC-SS2-TFEB-CARM1)pathway's protein expression,combined with transmission electron microscopy observation of auto-phagic lysosomes and explore the mechanism of Qingzao Jiufei Decoction(清燥救肺汤,QJD)to inhibit the proliferation of lung cancer in Dutch Lewis mice.Methods:Male C57BL/6J mice were injected subcutaneously with Lewis cells in the right axilla to establish animal models,and they were randomly divided into model control group,the cyclophospha-mide(CTX)group(daily dose of 50 mg/kg intraperitoneally,once every other day),and QJD groups(daily dose of 11/5.5/2.7 g/kg).Mice in each group were treated 2 weeks before modelling and 2 weeks after inoculation.At the end of the experiment,the mice in each group were euthanized,and the loaded tumor tissue was taken.The tumor mass,tumor volume,body mass changes,and tumor mass ratio of each group were counted.Western Blot was used to detect the effect of p-TFEB-1 protein expression in lung cancer tissue,and fluorescence microscopy was used to observe the expression of GFP-LC3 protein in the cells of lung cancer tissue.Real-time fluorescence quantitative PCR was used to detect gene ex-pressions of Foxk1,Foxk2,Foxo3,Acss2,Skp2,and Carm1 in cells.Transmission electron microscopy was used to observe the formation of autophagic lysosomes in lung cancer cells.Results:Compared with those in the model control group,tumor volume and tumor mass ratio were significantly reduced(P<0.01)in the CTX group and QJD groups(doses of 11,5.5,and 2.7 g/kg).The p-TFEB-1 protein expression was significantly down-regulated(P<0.01).The expression levels of GFP-LC3 protein and Acss2 gene were significantly up-regulated in the CTX group and the QJD groups(doses of 11 and 5.5 g/kg),while Foxk2 gene expression was significantly down-regulated(P<0.01 or P<0.05).Tumor mass was significantly reduced in the CTX group and the QJD group(11 g/kg)(P<0.01).The gene expressions of Foxk1,Foxk2,and Skp2 were down-regulated,while gene expressions of Foxo3 and Carm1 were significantly up-regulated(P<0.01 or P<0.05).Electron microscopy revealed the presence of autolysosomes in the QZD groups(11 g/kg and 5.5 g/kg).Conclusion:QJD can inhibit lung cancer proliferation and reduce tumor volume and tumor-to-body mass ra-tio by inducing autophagy in lung cancer cells.The underlying mechanism may be related to up-regulating GFP-LC3 pro-tein and Foxo3 mRNA expression,down-regulating gene expressions of Foxk1,Foxk2,and Skp2 to promote the formation of autophagosomes.It promotes lysosome formation and ultimately promotes the autophagolysosomes formation by decrea-sing p-TFEB-1 protein levels,elevating Acss2 mRNA,and Carm1 mRNA expression.