Study On The Mechanism Of Action Of Hedyotis Diffusa In Treating Pancreatic Neuroendocrine Tumor

Background:Well-differentiated pancreatic neuroendocrine tumor(pNET)is a malignant tumor that originates from pancreatic endocrine cells.Its incidence has been increasing year by year in recent years.Although the current diagnosis and treatment for pNET can benefit many patients,there are certain limitations,such as drug resistance and toxic side effects.The discovery of new drugs and therapies is very important for the treatment of pancreatic neuroendocrine tumors.Chinese medicine has rich clinical experience and a large number of effective drugs and prescriptions for the treatment of malignant tumors.Professor Huangying Tan has been using Hedyotis diffusa as the most commonly used herbal medicine for target disease pNET in clinical practice.For example,in the self-developed "Qizhen Formula to Anti-tumor",Hedyotis diffusa is used as the disease target medicine,which has significantly benefited patients.With a variety of active ingredients,Hedyotis diffusa can treat a variety of tumors such as digestive system tumors,lung cancer,prostate cancer and cervical cancer.It has anti-tumor effects such as inhibiting angiogenesis,inhibiting proliferation and promoting apoptosis,and is the most commonly used anti-tumor Chinese medicine.The effect mechanism of Hedyotis diffusa in the treatment of pNET have not been studied and reported;therefore,the study of the effect mechanism of Hedyotis diffusa in the treatment of pNET is of great scientific value and deserves in-depth study.Objective:This study combines data mining techniques,network pharmacology analysis,mass spectrometry analysis,transcriptomic analysis and in vitro experiments to investigate the effect mechanism of Hedyotis diffusa in the treatment of pancreatic neuroendocrine tumors,providing a theoretical basis and data support for the treatment of pancreatic neuroendocrine tumors with Hedyotis diffusa.Methods:1.The patent of TCM compound for treating pancreatic cancer from inception to May 2022 was searched by national patent database.The prescription database was established with Microsoft Excel software.The ancient and modern medical case cloud platform,IBM SPSS Modeler,and IBM SPSS Statistics software were used to analyze the medication rules of normalized data.2.The active constituents and drug targets of Hedyotis diffusa were obtained from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),and the disease-related targets of pNET were retrieved from GEO,GeneCards,OMIM,PharmGkb and Drugbank databases.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis for common targets of drugs and disease was performed using the R software.3.The decoction and freeze-dried extraction of Hedyotis diffusa were performed.The components were detected on an Ultra Performance Liquid Chromatography/Tandem Mass Spectrometry(UPLC-MS/MS)based detection platform.4.BON-1/QGP-1 cells from a pancreatic neuroendocrine tumor were intervened with the extract of Hedyotis diffusa,and the effects on cell proliferation and migration were assessed using the CCK-8 assay,plate cloning assay,and scratch assay.The effects of the extract of Hedyotis diffusa on cell apoptosis and cell cycle were detected using flow cytometry.The effect of the extract of Hedyotis diffusa on the transcriptomics of BON-1 cells was examined by transcriptome sequencing.The changes in mRNA and protein expression of BON-1/QGP-1 cells interfered by the extract of Hedyotis diffusa were examined by qPCR and western blot assay.Results:1.A total of 55 TCM compound patents for the treatment of pancreatic cancer were included,including 426 Chinese herbs.Their properties were mainly cold and warm;the tastes were mainly bitter,sweet and pungent;drugs were mainly related to liver,spleen,and lung channel tropism.High-frequency single traditional Chinese medicines include Hedyotis diffusa,licorice,atractylodes macrocephala,corydalis rhizoma,scutellaria barbata,radix angelicae sinensis,semen coicis and bupleur radix,etc.High-frequency drug pairs include "atractylodes macrocephala-licorice"and "Hedyotis diffusa-scutellaria barbata",etc.The association rule analysis obtained 27 drug groups.Three core drug groups were obtained by cluster analysis of high-frequency drugs.2.A total of 158 drug targets derived from seven active ingredients in Hedyotis diffusa and 2870 disease-related targets were obtained,resulting in 119 common targets.A total of 2099 items were obtained by GO functional enrichment analysis.A total of 162 signaling pathways were obtained by KEGG enrichment analysis,including PI3K/AKT,mTOR,apoptosis and autophagy signaling pathways,etc.3.300 g of Hedyotis diffusa was taken and repeatedly boiled for 3 times.After mixing,it was filtered and separately packed and lyophilized.A total of 25.85 g of lyophilized powder was extracted,and the paste extraction rate was 8.62%.A total of 1379 compounds were identified,including flavonoids,organic acids,tannins,phenolic acids,quinones,terpenoids,alkaloids,nucleotides and their derivatives,amino acids and their derivatives,lipids,lignans and coumarins.4.The extract of Hedyotis diffusa inhibited the proliferation and migration of BON1/QGP-1 cells,and the IC50 value of BON-1 cells was 1.8 mg/mL,and that of QGP-1 cells was 2.1 mg/mL.The extract of Hedyotis diffusa significantly promoted cell apoptosis and inhibited cell cycle in G0/G1 phase of BON-1/QGP-1 cells.Transcriptomic sequencing showed that the extract of Hedyotis diffusa interfered with the changes of Hippo signaling pathway,PI3K/AKT signaling pathway and mTOR signaling pathway in BON-1 cells.It was found by qPCR that the extract of Hedyotis diffusa could decrease the mRNA expression levels of PI3K,AKT and TAZ in BON-1/QGP-1 cells,and increase the mRNA expression levels of LATS1,CASP3 and CASP9 in BON-1/QGP-1 cells.We used western blot assay technique to find that the extract of Hedyotis diffusa was able to decrease the expression levels of PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR,TAZ,Bcl-2,Cyclin D1,GPX4 proteins and increase the expression levels of Cleaved Caspase-9/Caspase-9,P62,LC3BII/LC3BI proteins.Conclusion:1.The treatment of pancreatic cancer with traditional Chinese medicine compound has an obvious rule.In the treatment of pancreatic malignant tumors,the formula can be combined with cold and warm drugs,and regulate the liver and spleen.The basic goal of treatment is to enhance body function and inhibit tumor growth.Among them,Hedyotis diffusa is the most frequently used herbal medicine for the treatment of pancreatic cancer.This may provide some reference value for the treatment of pancreatic neuroendocrine tumors.2.Network pharmacological analysis showed that Hedyotis diffusa has a target for the treatment of pNET,and it can take effect through the complex network of multi-components,multi-targets and multi-pathways,providing ideas and theoretical basis for subsequent experimental studies.3.The extract of Hedyotis diffusa can inhibit BON-1/QGP-1 cell proliferation and migration,promote apoptosis,and block cell cycle in G0/G1 phase,probably by inhibiting PI3K/AKT/mTOR pathway and Hippo pathway.The extract of Hedyotis diffusa also promoted autophagy and ferroptosis in BON-1/QGP-1 cells.Hedyotis diffusa can treat pancreatic neuroendocrine tumors with multi-targets,multi-pathways and multiple cell death modes.The results provided some theoretical basis and data support for the treatment of pancreatic neuroendocrine tumor by Hedyotis diffusa.