To Explore The Effect Of Yangyin Huayu Jiedu Formula On Autophagy And Apoptosis Of Colon Cancer Cells Based On PI3K/Akt Signaling Pathway

ObjectiveIn this study,transcriptome RNA-seq sequencing technology was used to analyze the differential genes and pathways in HCT-116 colon cancer cells under normoxia and hypoxia,and combined with cell experiments,the effects of different oxygen concentrations on proliferation and autophagy of colon cancer cells and their mechanism were explored.Based on phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt）signaling pathway,the effect of Yangyin Huayu Jiedu Formula on autophagy and apoptosis of colon cancer HCT-116 cells under hypoxia was further investigated.To establish a tumor-bearing mouse model of colon cancer CT26 cells,and to explore the anticancer effect of Yangyin Huayu Jiedu Formula on CT26 tumor-bearing mice and the mechanism of autophagy and apoptosis.Methods1.To explore the effect of hypoxia on autophagy and proliferation of colon cancer HCT-116 cells and its mechanism based on transcriptomicsMTS assay was used to detect the cell proliferation under three different oxygen concentrations:the normoxia group（20%O₂/5%CO₂）,the 1%hypoxia group（1%O₂/94%N₂/5%CO₂）and the 5%hypoxia group（5%O2/90%N₂/5%CO₂）.RNA was extracted from HCT-116 cells in normoxia group and 5%hypoxia group,and Illumina Novaseq6000 platform was used for transcriptome RNA sequencing analysis.fastp,Hi Sat2and String Tie software were used for quality control and quality assessment of raw sequencing data,and RSEM software was used to quantitatively analyze the expression levels of different genes.DESeq2 software screened Differentially expressed genes（DEGs）according to the standard.GO（Gene Ontology）database was used to classify genes according to different biological processes,and Goatools software was used to enrich GO functions.KOBAS software was used to perform Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis of the screened differentially expressed genes,and the top 10significantly enriched KEGG pathways were screened out.Furthermore,the autophagy staining detection kit（MDC method）was used to detect the fluorescence expression of autophagy and the rate of autophagy cells in the three different oxygen concentration groups.Western blot analysis The autophagy-related proteins microtubule-associated protein（LC3Ⅱ/LC3Ⅰ）,programmed death receptor-1（Beclin-1）,hypoxia-inducible factor-1α（HIF-1α）and pathway proteins phosphatidylinositol 3-kinase（PI3K）,phosphorylated phosphatidylinositol3-kinase（p-PI3K）,serine-threonine protein in different groups were detected by blotting Kinase（Akt）and phosphorylated serine-threonine protein kinase（p-Akt）were detected.2.To investigate the effects of Yangyin Huayu Jiedu Formula on autophagy and apoptosis of colon cancer HCT-116 cells and its mechanism under hypoxiaHCT-116 cells were treated with Yangyin Huayu Jiedu Formula in 5%hypoxic microenvironment,and divided into normal group,blank serum group,low-,medium-,and high-dose Yangyin Huayu Jiedu Formula groups.MTS assay was used to detect the proliferation of cells in each group.MDC method combined with flow cytometry was used to detect the changes in the proportion of autophagy-positive cells in each group.Indirect immunofluorescence was used to detect the protein expression of LC3Ⅱin each group under hypoxia.Flow cytometry was used to detect the total apoptosis rate of each group after drug intervention.Western The autophagy proteins LC3Ⅱ/LC3Ⅰ,Beclin-1,ubiquitin-binding protein p62 and apoptosis-related proteins Bcl-2/adenovirus E1B（BNIP-3）,Bcl-2 associated X protein（Bax）,B-cell lymphoma-2（Bcl-2）,and activated aspartate proteolytic enzyme 3（C）in different groups were detected by blotting leaved Casepase-3),hypoxia-inducible factor HIF-1αand pathway proteins PI3K,p-PI3K,Akt,p-Akt.3.Animal experiment to explore the anti-cancer effect and mechanism of Yangyin Huayu Jiedu Formula on colon cancer CT26 tumor-bearing miceA total of 60 specific pathogen-free Babl/c male mice were divided into normal group（n=10）and model group（n=50）.CT26 cells in logarithmic phase were selected,and the cell concentration was adjusted to 2×10⁷/m L with PBS solution,and 0.2 m L CT26 cell suspension was inoculated under the armpit of the right arm to establish the model.Then 50 mice in the model group were randomly divided into model group,low-,medium-,and high-dose Yangyin Huayu Jiedu Formula groups and positive drug group.The rats in the normal group and the model group were given normal saline by gavage,the rats in the positive drug group were given oxaliplatin suspension（5 mg/kg）by gavage,and the rats in the low-,medium-and high-dose Yangyin Huayu Jiedu Decoction treatment groups were given Yangyin Huayu Jiedu Formula concentrated solution by gavage at doses of 9 g/kg,18 g/kg and 36 g/kg,respectively.During 14 days of continuous administration,the general condition of mice in each group was observed and the tumor growth of tumor-bearing mice was recorded.After 14days of drug administration,the body weight of the mice in each group was weighed,the eyeballs of the mice were removed to collect blood for routine blood test（red blood cells,white blood cells）,the spleen tissue of each group was weighed,the tumor tissue was stripped for preparation,and the tumor inhibition rate of each group was calculated.Hematoxylin-eosin（HE）staining was used to detect the pathological changes of tumor tissues in each group.Immunohistochemistry was used to detect the expression of Ki67 protein in tumor tissues.The expressions of apoptosis proteins Bcl-2,Bax,BNIP-3,Cleaved Casepase-3,hypoxia-inducible factor HIF-1α,autophagy protein Beclin-1and pathway proteins PI3K,p-PI3K,Akt and p-Akt in tumor tissues of each group were detected by Western-Blot.Results1.MTS assay showed that both 1%hypoxia group and 5%hypoxia group could promote the proliferation of HCT-116 cells（P<0.01）,and the proliferation rate was the fastest in 5%hypoxia group.The transcriptome RNA sequencing results of the normoxia group and the 5%hypoxia group showed that the experimental samples were selected reasonably,and 215significantly differentially expressed genes were obtained.The functional annotation of up-regulated genes included 50 GO entries,and the functional annotation of down-regulated genes included 46 GO entries.KEGG pathway enrichment analysis showed that among the top ten KEGG pathways,PI3K-Akt signaling pathway was the most significantly enriched and the number of genes was the largest.Therefore,PI3K-Akt signaling pathway was selected for the next pathway protein verification.MDC staining results showed that compared with the normoxia group,the fluorescence intensity of autophagy in 1%hypoxia group and 5%hypoxia group was significantly enhanced,and the rate of autophagy was significantly increased（P<0.01）.WB results showed that compared with the normoxia group,the p-PI3K/PI3K and p-Akt/Akt in the 5%hypoxia group were significantly decreased（P<0.01）.The expressions of Beclin-1,LC3Ⅱ/LC3Ⅰand HIF-1αwere significantly increased in 1%hypoxia group and 5%hypoxia group（P<0.05）,especially in 5%hypoxia group.Therefore,we selected 5%hypoxia environment for subsequent drug intervention..2.MTS results showed that compared with the blank serum group,the proliferation inhibition rate of HCT-116 cells in the Yangyin Huayu Jiedu Formula group increased significantly（P<0.01）;MDC staining showed that the rate of autophagy in Yangyin Huayu Jiedu Formula treatment group was significantly decreased（P<0.01）;Flow cytometry showed that the apoptosis rate of the treated group was significantly increased（P<0.01）;Indirect immunofluorescence assay showed that the LC3Ⅱfluorescence expression was significantly decreased in the drug administration group under hypoxic environment.Western blot analysis showed that the expression of autophagy protein Beclin-1 and LC3Ⅱ/LC3Ⅰin the treatment group was significantly decreased（P<0.05）,and p62 protein expression increased（P<0.01）,and the expressions of pro-apoptotic proteins BNIP-3 and Bax increased（P<0.01）and decreased expression of anti-apoptotic protein Bcl-2（P<0.01）,and the expression of hypoxia factor HIF-1αwas significantly decreased（P<0.01）,p-PI3K/PI3K and p-Akt/Akt increased significantly（P<0.05）3.During the process of colon cancer model establishment and administration,the mental state and appetite of the mice in the model group and the oxaliplatin positive drug group were worse than those in the normal group and the Yangyin Huayu Jiedu Formula group.Compared with the model group,the tumor tissue of the Yangyin Huayu Jiedu Formula group and the positive drug group grew relatively slowly.After 14 days of drug administration,compared with the normal group and the model group,the body weight of the positive drug group was significantly reduced（P<0.01）,while there was no significant change in Yangyin Huayu Jiedu Formula group.Compared with the model group,the tumor weight of the Yangyin Huayu Jiedu Formula group and the positive drug group was significantly reduced（P<0.05）,and the tumor inhibition rate was significantly increased.Compared with the normal group and the model group,the number of red blood cells and white blood cells in the positive drug group decreased significantly（P<0.05）,spleen weight also decreased significantly（P<0.05）;HE staining showed that the tumor tissue in the model group had dense cells,vigorous growth,closely arranged cells,and clear outline.Compared with the model group,the tumor tissue in each dose group of Yangyin Huayu Jiedu Formula and the positive drug group had sparse cell growth,apoptosis,and some cells became small or swollen and necrotic.Compared with the model group,the expression of Ki67 protein in tumor tissues of the Yangyin Huayu Jiedu Formula group and the positive drug group was significantly decreased（P<0.05）;Compared with the model group,the expressions of pro-apoptotic proteins BNIP-3,Bax and Cleaved Casepase-3 in the Yangyin Huayu Jiedu Formula group and the positive drug group were significantly increased（P<0.05）,and the expression of anti-apoptotic protein Bcl-2 was significantly decreased（P<0.05）,the expression of autophagy protein Beclin-1 was enhanced,and hypoxia factor HIF-1α（P<0.01）and p-PI3K/PI3K and p-Akt/Akt expressions were significantly decreased（P<0.05）.ConclusionsHypoxia may promote the proliferation and survival of HCT-116 cells by increasing the level of autophagy,and the mechanism may be related to the down-regulation of PI3K-Akt signaling pathway,which leads to cytoprotective autophagy.Yangyin Huayu Jiedu Formula can significantly reduce the autophagy ability of colon cancer HCT-116 cells under 5%hypoxia,inhibit cell proliferation and promote cell apoptosis.The mechanism may be related to the up-regulation of PI3K/Akt signaling pathway and the destruction of cytoprotective autophagy.Yangyin Huayu Jiedu Formula can inhibit the tumor growth of CT26 tumor-bearing mice and promote autophagy and apoptosis of CT26 cells.The mechanism may be related to the autophagy and apoptosis caused by the down-regulation of PI3K/Akt signaling pathway.Yangyin Huayu Jiedu Formula can promote apoptosis by inhibiting autophagy in vitro and promote autophagy in vivo by interfering with different types of tumor cells,it may be due to the different stages of tumor development in vivo and in vitro and the different degrees of drug stimulation,which affect the autophagy-apoptosis Dynamic equilibrium,produce complex bidirectional effects,and inhibit the growth of colon cancer cells.