中药药理与临床

目的:检测自噬体形成(FOXK-FOXO-SKP2)与溶酶体合成(ACSS2-TFEB-CARM1)通路蛋白表达，结合透射电镜观察自噬溶酶体，探究清燥救肺汤抑制荷Lewis小鼠肺癌增殖机制。方法:以雄性C57BL/6J小鼠右腋皮下注射Lewis细胞建立动物模型，随机分为模型对照组、环磷酰胺50 mg/kg组(腹腔注射，隔日1次)、清燥救肺汤2.7、5.5、11 g/kg组。各组造模前2 w给药，接种后连续给药2 w。试验结束后处死小鼠并取荷瘤组织，称瘤质量、瘤体积、计算体质量变化及瘤质比；透射电镜观察肺癌细胞自噬溶酶体的形成；qRT-PCR法检测肺癌组织Foxk1、Foxk2、Foxo3、Acss2、Skp2、Carm1 mRNA的表达；Western blot法检测肺癌组织p-TFEB-1蛋白表达；荧光显微镜下观察肺癌组织GFP-LC3蛋白表达。结果:与模型对照组比较，环磷酰胺组与清燥救肺汤2.7、5.5、11 g/kg组瘤体积及瘤质比显著降低、p-TFEB-1蛋白表达显著下调(P<0.01);环磷酰胺组和清燥救肺汤5.5、11 g/kg组GFP-LC3蛋白、Acss2 mRNA表达明显上调，Foxk2 mRNA表达明显下调(P<0.05或P<0.01);环磷酰胺组和清燥救肺汤11 g/kg组瘤质量显著降低(P<0.01),Foxk1、Foxk2、Skp2 mRNA表达下调，Foxo3、Carm1 mRNA表达明显上调(P<0.05或P<0.01);电镜下清燥救肺汤5.5、11 g/kg组可见自噬溶酶体。结论:清燥救肺汤可诱导肺癌细胞自噬，抑制肺癌增殖，减小瘤体积及瘤质比，其机制可能为通过上调GFP-LC3蛋白及Foxo3 mRNA的表达，下调Foxk1、Foxk2和Skp2的mRNA表达，促进自噬体的形成；下调p-TFEB-1蛋白表达、上调Acss2、Carm1 mRNA的表达促进溶酶体的形成，最终促进自噬融酶体的形成实现。

Objective:To detect the autophagosome formation(FOXK-FOXO-SKP2) and lysosome synthesis(ACSS2-TFEB-CARM1) pathway's protein expression, combined with transmission electron microscopy observation of autophagic 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 cyclophosphamide(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 expressions 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 ratio by inducing autophagy in lung cancer cells. The underlying mechanism may be related to up-regulating GFP-LC3 protein 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 decreasing p-TFEB-1 protein levels, elevating Acss2 mRNA,and Carm1 mRNA expression.