清燥救肺汤对Lewis肺癌荷瘤小鼠肺癌组织氧化磷酸化能量代谢的影响

Effect of Qingzao Jiufei Decoction(清燥救肺汤)on Energy Metabolism of Oxidative Phosphorylation in Lung Carcinoma Tissue of Lewis Tumor-Bearing Mice

目的探讨清燥救肺汤治疗肺癌的可能作用机制。方法50只C57BL/6J小鼠随机分为模型组,环磷酰胺(CTX)组,清燥救肺汤高、中、低剂量组,每组10只。各组小鼠分别接种Lewis肺癌细胞建立肺癌荷瘤小鼠模型。24 h后,模型组每日给予0.2 ml生理盐水灌胃,每日2次;CTX组隔日1次以50 mg/kg CTX腹腔注射;清燥救肺汤高、中、低剂量组分别以11、5.5、2.75 g/(kg·d)清燥救肺汤灌胃,每日2次。清燥救肺汤各剂量组在接种前后14天均灌胃,其余各组在接种细胞24 h后继续给药14天。Real-time PCR法检测肺癌组织中三磷酸腺苷合酶β亚基(ATP 5B)mRNA表达,蛋白免疫印迹法检测细胞色素C氧化酶亚单位Ⅳ(COXⅣ)蛋白表达,高效液相色谱法检测一磷酸腺苷(AMP)、二磷酸腺苷(ADP)、三磷酸腺苷(ATP)的含量;JC-1法检测线粒体膜电位(MMP)变化。结果与模型组比较,CTX组及清燥救肺汤高、中剂量组ATP 5B mRNA,COXⅣ蛋白表达、ATP含量和线粒体膜电位显著降低(P<0.05或P<0.01);与CTX组比较,清燥救肺汤高、中、低剂量组ATP 5B mRNA、COXⅣ蛋白表达升高,清燥救肺汤中、低剂量组ATP含量、MMP水平,清燥救肺汤高剂量组AMP/ATP显著升高,清燥救肺汤中、低剂量组AMP/ATP及清燥救肺汤低剂量组ADP/ATP显著降低(P<0.05或P<0.01);与清燥救肺汤高剂量组比较,清燥救肺汤中、低剂量组ATP 5B mRNA、COXⅣ蛋白表达、ATP含量升高,AMP/ATP值、ADP/ATP值显著降低,清燥救肺汤低剂量组MMP水平明显升高(P<0.05或P<0.01)。与清燥救肺汤中剂量组比较,清燥救肺汤低剂量组ATP 5B mRNA、COXⅣ蛋白表达、ATP含量上调(P<0.01)。结论清燥救肺汤能抑制肺癌细胞增殖,其机制可能与抑制氧化磷酸化能量代谢途径关键限速酶COXⅣ蛋白表达,降低线粒体膜电位,抑制ATP 5B活性,减少ATP生成及升高AMP/ATP值有关。

Objective To explore the underlying mechanism of Qingzao Jiufei Decoction(清燥救肺汤,QZJFD)in the treatment of lung carcinoma.Methods Fifty C57BL/6J male mice were randomly divided into five groups,with 10 mice in each group.The groups included a model group,a cyclophosphamide(CTX)group,and QZJFD high,medium and low dose group.Lewis lung carcinoma cells were used to establish the mice tumor-bearing models of lung carcinoma.Twenty-four hours later,the model group was administered with 0.2 ml normal saline by gavage,twice daily;CTX group was injected intraperitoneally with 50 mg/kg cyclophosphamide,once every other day;the high,medium and low dose QZJFD group received 11,5.5 and 2.75 g/(kg·d)Qingzao Jiufei Decoction by gavage respectively,twice daily.QZFJD groups were given intragastric administration 14 days before and after the modelling,while other groups only received treatment 24 hours after the modelling for 14 days.The mRNA expression of adenosine triphosphate synthaseβsubunit(ATP 5B)in lung carcinoma tissues was detected by real-time PCR;the protein expression of cytochrome c oxidase subunitⅣ(COXⅣ)was detected by western blotting;the adenosine monophosphate(AMP),adenosine diphosphate(ADP),and adenosine triphosphate(ATP)were detected by high performance liquid chromatography.JC-1 method was used to detect the changes of mitochondrial membrane potential(MMP).Results The expression of ATP 5B mRNA,COXⅣprotein,ATP,and MMP significantly reduced in CTX group and QZJFD high and medium dose group compared to those in model group(P<0.05 or P<0.01).Compared to those in CTX group,the expression of ATP 5B mRNA and COX IV protein in all QZJFD groups,ATP and MMP levels in QZJFD medium and low dose group,as well as AMP/ATP in QZJFD high dose group significantly increased,while AMP/ATP in QZJFD medium and low dose group,and ADP/ATP in QZJFD low dose group significantly decreased(P<0.05 or P<0.01).Compared to QZJFD high dose group,both the medium and low dose group had higher expression of ATP 5B mRNA and COXⅣprotein,higher ATP level,and lower levels of AMP/ATP and ADP/ATP,and the low dose group also had significantly higher MMP level(P<0.05 or P<0.01).A statistically significant increase was seen in QZJFD low dose group when compared to the medium dose group in terms of ATP 5B mRNA and COXⅣprotein expression,and the ATP level(P<0.01).Conclusion QZJFD may inhibit the proliferation of lung carcinoma cells,by inhibiting the protein expression of COXⅣ,the key rate-limiting enzyme of oxidative phosphorylation energy metabolism,reducing mitochondrial membrane potential,inhibiting ATP 5B activity,reducing ATP production,and increasing the ratio of AMP/ATP.