Effect Of Qingzao Jiufei Decoction On Apoptosis And Oxidative Phosphorylation Energy Metabolism Of Lung Cancer Cells

Objective:To explore the effect of Qingzao Jiufei Decoction（QZJFD）on apoptosis and oxidative phosphorylation energy metabolism of lung cancer cells.Method:50 C57BL/6J male mice were randomly divided into five groups with 10 mice in each group.The groups included a model group,a chemotherapy（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.Two weeks before modeling,the high,medium and low dose QZJFD group received 11,5.5,2.75g·kg^-1·d^-1Qingzao Jiufei Decoction by gavage respectively,twice daily;the model group was administered with 0.2 m L normal saline by gavage,twice daily.After 24hours of inoculation,the model group and each dose group of QZJFD continued to be administered according to the designed dose;CTX group was injected intraperitoneally with 50 mg·kg^-1·（2d）^-1cyclophosphamide,once every other day.After 2 weeks,the mice in each group were killed and the tumor tissues were taken.The apoptosis of lung carcinoma cells was observed by transmission electron microscopy（TEM）;the Janus protein tyrosine kinase 2（JAK2）protein phosphorylation level was detected by immunohistochemistry（IHC）;the protein expression of signal transducer and activator of transcription 3（STAT3）,BCL2-Associated X（Bax）,Cyclin D1and cytochrome c oxidase subunitⅣ（COXⅣ）were detected by western blotting;The m RNA expression of adenosine triphosphate synthaseβsubunit（ATP5B）in lung carcinoma was detected by real-time PCR;JC-1 method was used to detect the changes of mitochondrial membrane potential（MMP）;the adenosine monophos-phate（AMP）,adenosine diphosphate（ADP）,and adenosine triphosphate（ATP）were detected by high performance liquid chromatography.Result:1.Effect of QZJFD on tumor weight of Lewis-bearing miceCompared to model group,the tumor weight of CTX group,high,medium and low dose group of QZJFD was decreased（P<0.01）.Compared to CTX group,the tumor weight of high,medium and low dose group was increased（P<0.01）.Compared to high dose group,the tumor weight of medium and low dose group was increased（P<0.01）.Compared to medium dose group,the tumor weight of low dose group was increased（P<0.05）.From this we can know that positive drugs and QZJFD all have higher anti-tumor effect,among which positive drugs have the best effect,and there is a dose effect relationship between the doses of QZJFD.2.Effect of QZJFD on apoptosis in lung cancer cells（1）Observation on apoptosis of lung cancer cells by transmission electron microscopeIn the model group,the nuclei were swollen or shriveled,some mitochondria were swollen,the cristae of mitochondria was shortened,and a small amount of vacuoles were found in the cytoplasm.In the CTX group,mitochondria were swollen,mitochondrial cristae shortened,reduced or even disappeared,cell morphology changed,nuclear chromatin constricted,marginalized,nuclear membrane wrinkled,cell nucleus was broken into fragments,and large area of apoptosis was observed.In the QZJFD low dose group,the cell structure was disordered,nuclear chromatin was pyknotic,marginalized,and a few vacuoles in the cytoplasm.In the QZJFD medium dose group,the cell structure was disordered,organelles disappeared,mitochondria were swollen,the mitochondrial cristae was shortened,reduced or even disappeared,and the nuclear chromatin was constricted,marginalized,nuclear membrane folds,vacuoles in cytoplasm,and overall apoptosis was obvious.In the QZJFD dose group,the cell structure was disordered,organelles disappeared,mitochondria were swollen,mitochondrial cristae shortened,and reduced or even disappeared,the nuclear chromatin constricted,marginalized,nuclear membrane wrinkled,there are cell nucleus cleavage into fragments,and a large number of apoptosis.（2）Effect of QZJFD on p-JAK2 protein expression in lung cancer tissue of Lewis-bearing miceCompared to model group,the expression of p-JAK2 protein in CTX group and high,medium dose QZJFD group was decreased（P<0.05,P<0.01）.Compared to CTX group,the expression of p-JAK2 protein in low dose QZJFD group was increased（P<0.05）.Compared to high dose QZJFD group,the expression of p-JAK2protein in low dose group was decreased（P<0.05）.（3）Effect of QZJFD on p-STAT3 protein expression in lung cancer tissue of Lewis-bearing miceCompared to model group,the expression of p-STAT3 protein in CTX group and high,medium dose QZJFD group was decreased（P<0.05,P<0.01）.Compared to CTX group,the expression of p-STAT3 protein in medium and low dose QZJFD group was significantly increased（P<0.01）.Compared to high dose QZJFD group,the expression of p-STAT3 protein in low dose group was significantly increased（P<0.01）.（4）Effect of QZJFD on Bax protein expression in lung cancer tissue of Lewis-bearing miceCompared to model group,the expression of Bax protein in high and medium dose QZJFD group was increased（P<0.05,P<0.01）.Compared to CTX group,the expression of Bax protein in high dose QZJFD group was increased（P<0.05）.Compared to high dose group,the expression of Bax protein in low dose QZJFD group was significantly decreased（P<0.01）.Compared to medium dose group,the expression of Bax protein in low dose QZJFD group was decreased（P<0.05）.（5）Effect of QZJFD on Cyclin D1 protein expression in lung cancer tissue of Lewis-bearing miceCompared to model group,the expression of Cyclin D1 protein in CTX group and high,medium dose QZJFD group was decreased（P<0.05,P<0.01）.Compared to CTX group,the expression of Cyclin D1 protein in low dose QZJFD group was increased（P<0.05）.3.Effect of QZJFD on oxidative phosphorylation energy metabolism of lung cancer（1）Effect of QZJFD on COXⅣprotein expression in lung cancer tissue of Lewis-bearing miceCompared to model group,the expression of COXⅣprotein in CTX group and high,medium and low dose QZJFD group was down regulated（P<0.01）.Compared to CTX group,the expression of COXⅣprotein in high,medium and low dose QZJFD group was up regulated（P<0.01）.Compared to high dose group,the expression of COXⅣprotein in medium and low dose QZJFD group was up regulated（P<0.01）.Compared to medium dose group,the expression of COXⅣprotein in low dose QZJFD group was up regulated（P<0.01）.（2）Effect of QZJFD on ATP5B m RNA expression in lung cancer tissue of Lewis-bearing miceCompared to model group,ATP5B m RNA expression in CTX group,high and medium dose QZJFD group was decreased（P<0.05,P<0.01）.Compared to CTX group,the ATP5B m RNA expression in high,medium and low dose QZJFD group was increased（P<0.05,P<0.01）.Compared to high dose group,the ATP5B m RNA expression in medium and low dose QZJFD group was increased（P<0.05,P<0.01）.（3）Effect of QZJFD on MMP（polymer/monomer）in lung cancer cells of Lewis-bearing miceThere were statistical differences in the overall distribution of polymer/monomer ratio among the five groups（H=47.06,P=0.000）.Compared to model group,MMP in CTX group,high and medium dose QZJFD group was significantly decreased（P<0.05,P<0.01）.Compared to CTX group,MMP in middle and low dose QZJFD group was increased（P<0.05,P<0.01）.Compared to high dose group,MMP in low dose QZJFD group was significantly increased（P<0.05）.（4）Effect of QZJFD on ATP,ADP/ATP,AMP/ATP contents in Lewis-bearing miceCompared to model group,the content of ATP in CTX group,high,medium and low dose QZJFD group was significantly decreased;ADP/ATP and AMP/ATP in CTX group,high and medium dose QZJFD group was significantly increased（P<0.05,P<0.01）.Compared to CTX group,ATP content in middle and low dose group,AMP/ATP in high dose group was significantly increased;AMP/ATP in middle and low dose group and ADP/ATP in low dose QZJFD group was significantly decreased（P<0.05,P<0.01）.Compared to high dose group,ATP content in middle and low dose group was significantly increased,AMP/ATP and ADP/ATP significantly decreased（P<0.05,P<0.01）.Compared to middle dose group,ATP content in low dose QZJFD group was significantly increased（P<0.05）.Conclusion1.QZJFD can inhibit the proliferation of lung cancer cells in Lewis-bearing mice.2.QZJFD can inhibit the proliferation of lung cancer cells,and its mechanism may be related to the following two aspects.（1）Its mechanism may be related to the inhibition of the phosphorylation of JAK2 and STAT3 protein,the promotion of its downstream Bax protein expression and the inhibition of its downstream Cyclin D1 protein expression.（2）Its mechanism may be related 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.