Active Components Of Cortex Fraxini Inhibiting Glycolysis Of Liver Tumor And Its Mechanism

Patients’ lives have been threatened by liver tumors,particularly liver cancer.Hepatocellular carcinoma is responsible for more than 90% of primary liver malignancies,with a 5-year survival rate of fewer than 30%.In 2020,liver cancer accounted for 9% of the total number of cancer patients in China,ranking fifth,while the fatality rate was 13%,ranking second in cancer fatalities in the country.The therapy options and therapeutic outcomes for liver cancer are currently restricted.Expanding liver cancer treatment techniques and developing effective medications for the prevention and treatment of liver cancer are critical.Natural products derived from plants have the advantages of complex and novel structure,diverse activities and low toxicity and side effects,and have become an important source of new drug research and development.Common sources of Cortex fraxini are the dry branch bark or trunk bark of Fraxinus rhynchophylla Hance,Fraxinus chinensis Roxb.,Fraxinus szaboana Lingelsh.or Fraxinus stylosa Lingelsh.It mainly has the functions of clearing away heat and dampness,relieving asthma and relieving cough,and improving eyesight.At present,its pharmacological research mainly focuses on antibacterial,anti-inflammatory and anti-tumor aspects.Previous research has discovered that the active ingredients in Cortex fraxini affect the binding and interaction of enzymes involved in liver cancer metabolism,as well as the glucose and lipid metabolism of the disease.Cortex fraxini is widely distributed,abundant in resources,has a long history of medicinal use,and has significant development and utilization potential.Metabolic reprogramming is a hallmark of malignant tumors.The "Warburg effect"(aerobic glycolysis)is a well-known reprogramming of glucose metabolism in liver cancer.It not only provides the necessary energy and substrate for liver cancer cells to proliferate rapidly,but it also produces lactic acid,which modifies the tumor microenvironment,allowing cancer cells to evade immune detection and develop resistance to chemotherapy medicines.Using the "Warburg effect" as a starting point,screening anti-tumor medications from Cortex fraxini could be a useful strategy for increasing the anti-cancer drug resource library,improve liver cancer treatment strategies,and overcome cancer cell drug resistance.Objective: Screening the active components of Cortex fraxini against liver tumor and finding their target proteins,analyzing the anti-tumor mechanism of the active components of Cortex fraxini,and providing new ideas for the treatment of liver cancer and the development and application of Cortex fraxini.Methods: The extracts of Cortex fraxini with various polarity were produced using the ethanol percolation method and the system solvent extraction method in this work.Based on the survival rate of Hep G2 cells,the CCK-8method was used to screen the active sites of Cortex fraxini against liver tumors.LC-MS/MS was used to analyze the chemical components of the active site,and the anti-tumor active components in the active site of Cortex fraxini were screened using the survival rate of Hep G2 cells,glucose consumption,and lactate production as indicators.Bioinformatics was used to evaluate differentially expressed genes associated to glycolysis in liver tumorpatients,and virtual molecular docking and surface plasmon resonance(SPR)technology were used to find the direct targets of active substances.To investigate the alterations in the glycolysis pathway of liver tumor cells following target gene silencing,small interfering RNA(si RNA)was utilized to silence target genes.To uncover differentially expressed genes and other pathways influenced by target gene silencing,researchers used transcriptome analysis and high-throughput sequencing techniques.RT-PCR and WB techniques were used to confirm the screening results,and the kit additionally detected glycolysis-related metabolic markers.After active components were added,high-throughput sequencing technology was employed to examine the transcriptome of Hep G2 cells.The expression variations of glycolysis-related genes and proteins were determined using RT-PCR and WB technologies,and Hu H7 cells were employed to confirm the experimental results.The anti-liver tumor mechanism of active components of Cortex fraxini has been thoroughly investigated.A tumor-bearing mice model was constructed with H22 cells,and the effects of active components in Cortex fraxini on tumor formation in mice were examined in vivo.Kits were used to determine the effects of active ingredients on liver and kidney function in mice,HE staining was used to examine the changes of tumors and organ tissues in each group of mice.Immunohistochemistry and WB were used to examine the effects of active ingredients on the expression of glycolysis-related proteins in tumor tissues.The in vivo efficacy of Cortex fraxini’s active ingredients on liver tumors was systematically studied,providing a foundation for further research,development,and application of Cortex fraxini’s anti-cancer effect.The main findings of this study include:1.Four types of extracts with different polarities,named petroleum ether extract,ethyl acetate extract,n-butanol extract and water extract,were obtained from the ethanol extract of Cortex fraxini by systematic solvent extraction.The active sites were screened according to the survival rate of Hep G2 cells,and it was found that the ethyl acetate site had the strongest anti-tumor activity.28 components were identified from the ethyl acetate extract by LC-MS/MS combined with literature reports,10 of which were determined with reference substances,named 4-Methylumbelliferone,scopolin,scopoletin,oleuropein,ligustroflavone,fraxin,esculin,esculetin,umbelliferone,isofraxidin.Using cell survival rate,glucose consumption and lactic acid production as indicators,oleuropein and esculetin were screened as the active components to inhibit glycolysis,among which oleuropein was the most active.2.1362 differentially expressed genes related to glycolysis were screened from the transcriptional sequencing data of liver cancer patients,of which 41 genes may play a key role in the glycolysis of liver cancer.Among the 41 genes,23 genes named ABL1,BIRC5,CDC20,CDC42,DHX9,DNAJC9,E2F1,EIF3 B,ERBB3,FUBP1,GPI,HMMR,HSPB1,ILF3,ME1,PPIA,PSMA5,PIBP1,PTTG1,RHOA,RUVBL1,STIL,TACC3 were associated with the overall survival of patients with liver cancer.12 proteins including ABL1,BIRC5,CDC20,E2F1,EIF3 B,ERBB3,FUBP1,GPI,ME1,PPIA,RHOA,STIL,had strong virtual binding to oleuropein and are potential targets of oleuropein.SPR validated oleuropein’s direct binding to GPI and confirmed that GPI was oleuropein’s direct binding target with a binding Kd of 107 μM.3.GPI si RNA was designed and screened,and it was confirmed that si RNA could significantly inhibit the expression of GPI protein at 48 h and 72 h after transfection of Hep G2 cells.GPI interference can prolong the time for liver tumor cells to reach the growth plateau,and reduce the glycolytic metabolic flux of liver tumor to a certain extent.GPI interference can inhibit the expression of glycolysis-related genes such as PFKL,ENO1,ENO3,PKM2,LDHA,etc.,and also has a certain tendency to inhibit the expression of related proteins.After GPI interference,transcriptome analysis revealed that1298 genes were significantly up-regulated and 540 genes were significantly down-regulated,affecting biological functions such as liver tumor cytokine secretion,secretion and transport,cell metabolism,signal transduction,and other factors.It has an impact on the glycolysis,pyruvate metabolism,HIF-1signaling,AMPK signaling,glucagon signaling,insulin resistance,and insulin signaling pathways.After GPI interference,the expressions of PYGM and PFKFB4 increased dramatically,suggesting that PYGM-mediated glycogenolysis and PFKFB4-mediated fructose-6-phosphate conversion may provide metabolic substrates for the glycolysis pathway.GPI interference and combined with PYGM and PFKFB4 inhibitors can help reduce the glycolysis metabolite flux,and significantly inhibit the gene expression and protein expression of PFKL,ALDOA,PGK1,PGAM1,ENO1,PKM2,and LDHA.4.Oleuropein inhibits Hep G2 cell migration and colony formation and inhibits hepatocellular carcinoma cell proliferation.Transcriptome analysis after oleuropein intervention in cells identified 1494 up-regulated genes and856 down-regulated genes affecting glycolysis/gluconeogenesis,central carbon metabolism in cancer,AMPK signaling pathway,insulin signaling pathway,insulin resistance,thyroid hormone signaling pathway and other signaling pathways.To a certain extent,it has similar pharmacological effects with the glycolysis inhibitor 2-DG.Oleuropein can inhibit the gene expression of PFKL,ALDOA,PGK1,PGAM1,ENO1,ENO3,PKM2,LDHA,and lead to the down-regulation of PFKL,ALDOA,GAPDH,PGK1,ENO1,PKM2,LDHA protein expression,and has the effect of inhibiting glycolysis,may also has potential PYGM and PFKFB4 inhibitor-like effects.5.Oleuropein has anti-tumor effect in vivo.The tumor inhibition rate of oleuropein was 59.5%,the tumor inhibition rate after oleuropein combined with the inhibitors was 64.2%,the tumor inhibition rate of the chemical drug positive control drug adriamycin was 80.6%,and the tumor inhibition rate of Chinese patent medicine positive control drug Huaier granule was 46.2%.The results of liver and kidney function index detection and HE staining showed that adriamycin affected the liver and kidney function of mice to a certain extent,but oleuropein showed no toxic side effects.The results of immunohistochemistry showed that oleuropein could affect the expression of GPI and PYGM in vivo,and combined with the dual inhibitor more significantly inhibited the expression of PYGM and PFKFB4.Oleuropein inhibited the protein expression of ALDOA,GAPDH,PGK1,PKM2 and LDHA,decreased glucose uptake and lactate generation in tumor tissues,but had no effect on blood glucose levels in mice.Conclusion: Oleuropein is one of the active components of anti-liver tumor in Cortex fraxini,and its direct target is GPI,the key enzyme of glycolysis.The expression trend of GPI in liver tumor cells affects a variety of physiological functions and signal transduction.Oleuropein can regulate the glycolysis pathway of liver tumor through GPI.Oleuropein has anti-liver tumor activity in animals,can inhibit glycolysis,and without toxic side effects.The active ingredient in Cortex fraxini,oleuropein,binds well to the upstream protein GPI of glycolysis,and inhibit glycolysis through GPI to achieve anti-tumor effect,which gives scientific support for Cortex fraxini’s anti-tumor action.It was also suggested that oleuropein could be developed and utilized as a promising drug for the prevention and treatment of liver cancer.