The Effect And Mechanism Of Tianyang Pill Intervention In Liver Cancer Cells And Their Succinylation Modification

Research objectives:On the basis of previous studies related to the glucose metabolism of liver cancer cells and Tianyang pills,the 4D proteomics combined with succinylation modification was further analyzed,and the differential proteomics and succinylation modification results of liver cancer cells after Tianyang Pills intervention were analyzed.The experiment verified the intervention results of Tianyang pills on the succinylation modification level of liver cancer cells.To verify the changes of specific differential protein expression levels,and explore and analyze the effect mechanism of Tianyang pills on the glucose metabolism level of hepatoma cells.Research methods:1.Preparation of Tianyang Pills: Tianyang Pills,an empirical formula proposed by Professor Li Ji from Heilongjiang University of Chinese Medicine,are composed of 20 g of Euphorbia officinalis,15 g of cistanche,15 g of Cyanoyang,15 g of cooked Rehmannia,15 g of Fructus officinalis meat and15 g of Chinese yam.2.Twenty male rats(SPF grade,200 ± 20g)were randomly divided into blank group and Tianyang Pill administration group with 10 rats in each group by random number table method.All rats were fed adaptive feeding for a week,and were given 0.2ml in each group successively for a week starting from the8 th day.The blank group was given equal amount of saline.The rats were killed on the last day,and blood was collected from orbit.3.Cell culture: Hep G2,hepa1-6 and Aml12 cell lines were cultured from the Shanghai Cell Bank of the Chinese Academy of Sciences.4.Detection methods:We detected the succinylated pan antibody of Aml12 cells,Hepa1-6 cells,Hep G2 cells,Hepa1-6 cells and Hep G2 cells interfered by Tianyang Pill,and then analyzed the proteomics and succinylated modification of Aml12 cells,Hepa1-6 cells and Hepa1-6 cells interfered by Tianyang Pill.Differential proteins and differential succinylation modification sites were identified by protein extraction and digestion of cells,TMT labeling,affinity enrichment using succinylation pan-antibodies,liquid chromat-ography and mass spectrometry,and a series of bioinformatic analyses including GO annotation,KEGG pathway enrichment,and cluster analysis.To screen out liver cancer and potential targets after Tianyang pills intervention.The m RNA expression of different proteins in liver cancer and normal tissues was extracted from the TCGA database and the influence of Tianyang pills on the expression level of key proteins in liver cancer cells was verified by Western blot.All experimental data were shown as mean ± standard deviation( ± s),using SPSS 16.0 software.Paired sample t test was used for comparison of paired sample data,and independent sample t test was used for comparison between random samples.P < 0.05 was considered statistically significant.Research results:1.To verify the effect of Tianyang pills on the succinylation modification level of hepatoma cellsSuccinylated panantibody was used to detect the succinylated modification levels of Hep G2 model group,Hepa1-6 model group,AML12 blank control group,Hep G2 Tianyang Pill group and Hepa1-6 Tianyang Pill group by Western blot.It was found that Tianyang pills could significantly reverse the rise of succinylation in mice and human hepatoma cells.2.Analysis of differential proteome and lysine succinylation modified group in hepatoma cellsThrough high-resolution LC-MS/MS analysis,4743 proteins that could be quantified were analyzed,and it was found that 1804 proteins were differentially expressed in liver cancer cells and normal liver cells(P < 0.05),among which 928 proteins were up-regulated and 876 proteins were down-regulated.The up-regulated proteins were mainly concentrated in amino sugars and nucleotide sugars metabolism,while the down-regulated proteins were enriched in oxidative phosphorylation and fatty acid metabolism.Compared with normal liver cells,724 Ksucc sites of 328 proteins were up-regulated and 50 Ksucc sites of 43 proteins were down-regulated in liver cancer cells.Further analysis showed that the proteins with different expressions of succinylation were mainly related to glycolysis and pentose phosphate.In addition,We also identified significant increases in lysine succinylation modification levels of key glycolytic enzymes such as ALDOA,PKM,PGK1,LDH,GPI and HK.3.Analysis of differential proteome and lysine succinylation modification group of hepatocellular carcinoma cells after Tianyang Pill interventionWhen compared with HCC cells,110 proteins were differentially expressed in the administration group,including 46 up-regulated and 64down-regulated proteins.Compared with mouse hepatoma cells in the model group,40 of 37 proteins were up-regulated and 1075 of 438 proteins were down-regulated in the drug administration group.Further GO enrichment and KEGG pathway analysis of the differential proteome and succinylation group showed that the up-regulated proteins of succinylation were less,whereas the down-regulated proteins of succinylation were enriched in glycolysis and other related processes.By analyzing the correlation between the succinylation site and its corresponding protein expression.The succinylation of ALDOA,PGK,LDH,GPI,PKM and other proteins was the most significant.4.To detect the expression of key proteins in liver cancer tissues and the changes of key protein expression levels in liver cancer cells before and after the intervention of Tianyang PillsBy extracting and analyzing the gene expressions of key proteins from the TCGA database,we found that the m RNA levels of ALDOA,PGK,LDH,PKM and GPI were highly expressed in liver cancer tissue,and PKM2 protein was also highly expressed in liver cancer cells by Western blot analysis.After Tianyang pills intervention,the expression level of PKM2 protein in hepatocellular carcinoma cells showed a decreasing trend.Conclusion:1.Tianyang Pill can significantly reverse the phenomenon of increased succinylation modification levels in mouse and human liver cancer cells.2.Compared with normal hepatocytes,hepatoma cells have increased protein expression during amino sugar and nucleotide sugar metabolism and decreased protein expression during oxidative phosphorylation and fatty acid metabolism.The succinylation modification site is upregulated,mainly related to processes such as glycolysis and pentose phosphate.3.After the intervention of Tianyang Pill,the protein expression of liver cancer cells was up-regulated in the process of unsaturated fat acid synthesis,and down regulated in the process of ribosome,and there was a significant down-regulation of succinylated modification sites,which was closely related to the glycolysis pathway.4.Tianyang Pill can significantly reverse the phenomenon of increased PKM2 protein expression level in liver cancer cells.