ABSTRACT
ObjectiveTo investigate the effect of different oxygen concentration on the proliferation and autophagy of colon cancer cells and to explore the effect of Yangyin Huayu Jiedu Preseription （YHJP） on autophagy and apoptosis of colon cancer cells under hypoxia based on phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway. MethodHCT-116 cells were divided into normoxia group， 1% O2 group， and 5% O2 group. Cell viability was detected by cell proliferation assay （MTS）， and autophagy was observed based on monodansylcadaverine （MDC） staining. HCT-116 cells were treated with YHJP in 5% O2 microenvironment. The cells were divided into normal group， blank serum group， and low-， medium-， high-dose YHJP groups （5%， 15%， 25% serum containing YHJP）. Cell inhibition rate in each group was calculated by MTS， and changes in the rate of autophagy were detected based on MDC staining. Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI） was employed to detect the apoptosis rate of each group. Western blotting was applied to measure the expression of autophagy proteins microtubule-associated protein 1 light chain 3 （LC3Ⅱ/Ⅰ）， yeast Atg6 homolog （Beclin-1）， ubiquitin-binding scaffold protein p62 （p62）， apoptosis-related proteins B-cell lymphoma-2 （Bcl-2）， Bcl-2/adenovirus E1B interacting protein 3 （BNIP-3）， and Bcl-2 associated X protein （Bax）， cleaved cysteine-aspartic acid protease-3 （Caspase-3）， hypoxia-inducible factor-1α （HIF-1α） and pathway proteins PI3K， phosphorylated （p）-PI3K， Akt， and p-Akt. ResultCell survival rates of the 1% O2 and 5% O2 groups were increased compared with that in the normoxia group， particularly the 5% O2 group （P<0.01）. The fluorescence intensity for autophagy in 1% O2 and 5% O2 groups was significantly increased compared with that in the normoxia group， especially the 5% O2 group. In the presence of 5% O2， compared with the blank serum group， medium-dose and high-dose YHJP groups showed high cell inhibition rate， low autophagy rate， high apoptosis rate （P<0.01）， and low expression of Beclin-1 protein （P<0.05）. Compared with low-dose YHJP group， high-dose YHJP group demonstrated low expression of Beclin-1 protein （P<0.05）. Compared with the blank serum group， the three YHJP groups had low expression of LC3Ⅱ/Ⅰ protein （P<0.05， P<0.01）. Compared with the blank serum group， medium-dose and high-dose YHJP groups showed high expression of p62 protein （P<0.01）. Compared with low-dose YHJP group， high-dose YHJP group showed high expression of p62 protein （P<0.05）. Compared with the blank serum group， high-dose YHJP increased the expression of BNIP-3 and Bax and decreased the expression of Bcl-2 （P<0.01）. The expression of Bax protein in the high-dose YHJP group was increased compared with that in the low-dose YHJP group （P<0.05）. The expression of HIF-1α in the medium-dose and high-dose YHJP groups was decreased （P<0.01） and the expression of p-PI3K/PI3K and p-Akt/Akt in the high-dose YHJP group was increased （P<0.05， P<0.01） compared with that in the blank serum group. The expression of p-Akt/Akt was higher in the high-dose YHJP group than in the medium-dose YHJP （P<0.05）. ConclusionHypoxic microenvironment can significantly promote autophagy and proliferation of colon cancer cells. YHJP can significantly inhibit autophagy and proliferation and promote apoptosis of colon cancer cells in 5% O2 environment by up-regulating the PI3K/Akt signaling pathway.
ABSTRACT
To explore the potential mechanism of frankincense volatile oil in the prevention and treatment of cardiac hypertrophy based on in vitro cell experiment and network pharmacology. METHODS: The anti-hypertrophic effect of frankincense volatile oil was investigated by isoproterenol induced H9c2 cardiomyocytes hypertrophy model. The active chemical components and targets of frankincense volatile oil and targets associated with cardiac hypertrophy were obtained by CNKI, Pubmed, Pubchem databases, etc. String database and Cytoscape 3.8.0 software were used to construct protein-protein interaction network (PPI) and a network of "drug-active component-key target-disease" of frankincense volatile oil in order to screen the key targets of frankincense volatile oil against cardiac hypertrophy. The fluorescent quantitative PCR experiments were performed to verify those key targets. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis of key target genes were performed using David online analysis tool. RESULTS: In vitro cell experiments showed that frankincense volatile oil significantly inhibited the isoproterenol induced increases in cardiomyocytes surface area and protein synthesis, and upregulations of ANP and β-MHC mRNA. A total of 87 active components and 36 ingredient-disease targets of frankincense volatile oil were screened. Network analysis showed that ESR1, NOS3, PTGS2, TNF, MAPK14, and PPARG were key targets. Fluorescence quantitative PCR experiments results indicated that frankincense volatile oil inhibited isoproterenol induced upregulations of ESR1, PTGS2, TNF, and MAPK14 mRNA levels, and downregulations of NOS3, PPARG mRNA levels, respectively. In addition, the GO functional enrichment analysis showed that its biological pathways mainly included lipopolysaccharide-mediated signaling pathway, positive regulation of nitric oxide biosynthetic process, caveola, enzyme binding, etc. The KEGG pathway enrichment analysis included 22 KEGG pathways, which were closely related to VEGF signaling pathway, TNF signaling pathway, sphingolipid signaling pathway and others. CONCLUSION: The active components of frankincense volatile oil may regulate VEGF signaling pathway, TNF signaling pathway, Sphingolipid signaling pathway by acting on ESR1, NOS3, PTGS2, TNF, MAPK14 and PPARG targets, thereby affecting the regulation of lipopolysaccharide-mediated signaling pathway, positive regulation of nitric oxide biosynthetic process, caveola, and enzyme binding, and improving cardiac hypertrophy.