补中益气汤联合顺铂调控PDK1/Akt信号通路抑制糖酵解影响肺腺癌细胞增殖OA

Objective:To investigate the impact of Buzhong Yiqitang combined with cisplatin on the proliferation of human lung adenocarcinoma(A549)cells through regulation of the pyruvate dehydrogenase kinase 1(PDK1)/protein kinase B(Akt)signaling pathway and influence on glycolysis.Methods:Transcriptome sequencing(RNA-seq)was employed to compare the expression of glycolysis-related genes between A549 cells and cisplatin-resistant human lung adenocarcinoma cells(A549/DDP).Small interfering RNA(siRNA)was employed to knock down PDK1,and the knockdown efficiency was verified by Western blot and Read-time PCR.The cell counting kit-8(CCK-8)assay was used to assess the survival and viability of A549 cells under the following conditions:siRNA negative control+cisplatin(128,64,32,16,8,4,0 μmol·L-1),siPDK1+cisplatin(128,64,32,16,8,4,0 μmol·L-1),and siPDK1+cisplatin(128,64,32,16,8,4,0 μmol·L-1)+Buzhong Yiqitang(10%)-containing serum.The 20%inhibitory concentration(IC20)of the siRNA negative control+cisplatin group(7.832 μmol·L-1)was calculated and used as the subsequent cisplatin concentration.Colony formation assay was performed to evaluate the proliferation of A549 cells.Lactate and adenosine triphosphate(ATP)assay kits were used to measure lactate and ATP production.The mitochondrial membrane potential was detected with the fluorescent probe JC-1.Western blotting was conducted to examine the expression levels of PDK1,phosphorylated(p)-Akt,Akt,pyruvate kinase M2(PKM2),glucose transporter 1(GLUT1),pyruvate dehydrogenase(PDH),and lactate dehydrogenase A(LDHA).Confocal immunofluorescence was employed to detect PDK1 and p-Akt.Results:RNA-seq results identified PDK1 as a highly expressed differential gene in glycolysis metabolism between A549 cells and A549/DDP cells,and it was highly expressed in tumor cells.Gene Set Enrichment Analysis(GSEA)revealed upregulated and downregulated genes in glycolysis and gluconeogenesis pathways.Western blot and RT-qPCR confirmed that PDK1-si-2 had the highest transfection efficiency,with a PDK1 knockdown rate exceeding 60%.CCK-8 assay determined the half-maximal inhibitory concentration(IC50)values for each group as(30.698±5.348),(16.372±3.562),(13.237±1.573)μmol·L-1,while the IC20 of cisplatin in siRNA negative control-transfected A549 cells was(7.832±0.672)μmol·L-1.Compared with the siRNA negative control group,the siRNA negative control+cisplatin group showed decreased colony formation rate,reduced lactate production,lowered mitochondrial membrane potential,downregulated protein levels of p-Akt,GLUT1,PDK1,PDH,and LDHA,and reduced PDK1 fluorescence intensity(P<0.05).The siPDK1 group exhibited decreased colony formation rate,reduced lactate production,increased ATP production,lowered mitochondrial membrane potential,downregulated protein levels of p-Akt,PKM2,GLUT1,PDK1,PDH,and LDHA,and reduced PDK1 and p-Akt fluorescence intensity(P<0.05).Compared with the siPDK1 group,the siPDK1+Buzhong Yiqitang group showed decreased colony formation rate,reduced lactate production,downregulated protein levels of PKM2,GLUT1,and PDK1,and reduced PDK1 fluorescence intensity(P<0.05).The siPDK1+cisplatin group exhibited decreased colony formation rate,reduced lactate production,increased ATP production,lowered mitochondrial membrane potential,downregulated protein levels of PKM2,GLUT1,PDK1,and PDH,and reduced PDK1 and p-Akt fluorescence intensity(P<0.05).The siPDK1+cisplatin+Buzhong Yiqitang group demonstrated decreased colony formation rate,reduced lactate production,increased ATP production,lowered mitochondrial membrane potential,downregulated protein levels of p-Akt,PKM2,GLUT1,PDK1,PDH,and LDHA,and reduced PDK1 and p-Akt fluorescence intensity(P<0.05).Compared with the siPDK1+Buzhong Yiqitang group,the siPDK1+cisplatin group showed decreased colony formation rate,downregulated protein levels of p-Akt and PDH,and reduced PDK1 and p-Akt fluorescence intensity(P<0.05).The siPDK1+cisplatin+Buzhong Yiqitang group exhibited decreased colony formation rate,reduced lactate production,increased ATP production,lowered mitochondrial membrane potential,downregulated protein levels of p-Akt,GLUT1,PDH,and LDHA,and reduced PDK1 and p-Akt fluorescence intensity(P<0.05).Compared with the siPDK1+cisplatin group,the siPDK1+cisplatin+Buzhong Yiqitang group showed decreased colony formation rate,increased ATP production,and downregulated protein levels of p-Akt,PKM2,and LDHA(P<0.05).Conclusion:Buzhong Yiqitang combined with cisplatin can suppress lung adenocarcinoma cell proliferation by modulating glycolysis through the PDK1/Akt signaling pathway.