Study On The Molecular Mechanism Of Dot1L Involved In Mitosis Regulation And DNA Damage Repair In Osteosarcoma Cell

Purpose:Anemarrhenae Rhizoma（AR）,a traditional Chinese herb,refers to the dried rhizome of Anemarrhena asphodeloides Bge.It was first recorded in the"Shennong’s Herbal Classic of Materia Medica"and mainly produced in Hebei and Shanxi.It has the effects of heat-clearing,purging fire,nourishing yin,and moisturizing dryness.According to the records in Chinese Pharmacopoeia,the herb possesses the functions of external contraction of febrile,high fever with vexation and thirst,lung heat with dry cough,bone-steaming and tidal fever,internal heat washing-thirst,and constipation caused by intestinal dryness.AR is a medicine of enhancing renal function,which can nourish the kidney yin and reduce excess heat.For the first time,our research team found and reported that the hypoglycemic effect of AR was significantly enhanced after salt processing.After years of continuous research,the hypoglycemic effect of AR was verified from many aspects.This study is based on the previous research work.It is intended to analyze the saponins,flavonoids of AR before and after salt processing,and analyze and screen the blood components from the perspective of serum pharmacochemistry;Network pharmacology was used to study the possible synergism effection between active ingredients and the biological pathway of key targets,and combined with metabolomics to explain the mechanism of AR and salt-processed Anemarrhenae Rhizoma（SAR）for the treatment of type 2diabetes mellitus（T2DM）,and to explore the mechanism of"multi-component-multi target-multi pathway"of AR in the treatment of T2DM.Material and Method:.Material:Anemarrhenae Rhizoma（Sichuan New Lotus Chinese Medicine Decoction Co.,Ltd.,batch no.1510038）was identified as the dried rhizome of Anemarrhena asphodeloides Bge.by Professor Haibo Yin of Liaoning Traditional Chinese Medicine University.Formic acid（LC-MS grade）was purchased from Merck KGa A（Darmstadt,Germany）,acetonitrile（LC-MS grade）was bought from Thermo Fisher Scientific Corp.（MA,United States）.Other reagents were at analytical grade,and were obtained from Tianjin Kermol Chemical Reagent Co.,Ltd.（Tianjin,China）.Ultrapure water was purified by a Milli-Q water purification system（Millipore,Billerica,MA,USA）.Waters Xevo G2-XS Mass Spectrometer（Massachusetts,USA）.Multiskan MK3 enzyme labeling instrument（Semefield Technology Co.,Ltd.）,Ins R and m TOR antibodies were purchased from Cell Signaling Biotechnology Co.,Ltd;SPF SD rats were purchased from Liaoning Changsheng Biotechnology Co.,Ltd.Hep G-2 Cells were purchased from Dalian Bergolin Biotechnology Co.,Ltd.Method:1 Based on UPLC-Q-TOF-MS/MS to analyze differences of saponin and flavone in AR before and after salt processing.Macroporous adsorption resin was used to enrich the total saponins and flavonoids in the 75%ethanol extract of AR and SAR.UPLC-Q-TOF-MS/MS technology was used in combination with UNIFI database to qualitatively analyze the chemical components of total saponins and flavonoids in AR and SAR,and the differential chemical components of AR before and after salt processing were systematically analyzed.2 Analysis of blood components of AR and SAR in rats based on UPLC-Q-TOF-MS/MS technology.UPLC-Q-TOF-MS/MS technology was used to establish the identification method for the chemical components of crude and SAR in the serum of normal rats and type II diabetes model rats,and the UNIFI database was used to extract the blood components and their relative peak areas.The peak areas of the blood components of AR before and after salt processing were compared and analyzed as a whole,to find out the difference components and content change trend of the blood components,and to explore the active components that play a role in the efficacy of AR,further analysis of the pharmacological substance basis of AR after salt processing.3 Based on the network pharmacology and experimental validation to explore the mechanism of AR and SAR in the intervention of T2DM.The chemical components of AR were collected by TCMSP and related literature,and establish a sub database.According to the principle of drug properties,oral bioavailability（OB≥30%）and drug similarity（DL≥0.18）,and the results of chemical composition studies in vitro and in vivo were combined to obtain the active ingredients.The basic information of each component is found in Pub Chem database,the chemical component name is unified,and its Pub Chem ID and 2D structure diagram are downloaded and saved.The obtained chemical composition map was uploaded in SDF format to Swiss Target Prediction database to obtain its corresponding component target,and the Uniport name database was used to unify the protein name as the gene name,and the"medicine-compound-target-disease"network was constructed through Cytoscape 3.7.2.The potential mechanism of AR in the treatment of T2DM was discussed through KEGG and GO enrichment analysis.Based on the results of network pharmacology,the Insulin Signaling Pathway was identified as the key pathway for hypoglycemic action.Hep G-2 cells were used as the research object,and molecular biology methods were used to verify the regulatory effect of AR and SAR on the core pharmacological targets Ins R and m TOR on this pathway,further elucidating the hypoglycemic mechanism of AR and SAR.4 Study on the mechanism of AR and SAR in the treatment of type II diabetes based on metabonomics.UPLC-Q-TOF-MS/MS technology and QI platform were used to analyze the effects of alcohol extracts of AR and SAR on endogenous substances in T2DM rats;SIMCA-P 14.0 was used to analyze the obtained data for multivariate statistical analysis.With VIP>1,t-test P<0.05 and Max Fold Change>2 as the standards,the differential metabolites in urine and plasma of rats in each group were analyzed,and the differential variables with callback effect on diabetes biomarkers were selected to analyze the metabolic pathways affected by the screened differential metabolites.In combination with the results of previous network pharmacology research,the core target proteins of AR and SAR on hypoglycemic metabolic pathway were found,and then the conjoint analysis was carried out.Result:1 Based on UPLC-Q-TOF-MS/MS to analyze differences of saponin and flavone in AR before and after salt processing.The saponins and flavonoids were identified in the negative ion mode.In this mode,21 saponins and 7 flavonoids were identified in AR and SAR.The total number of saponins and flavonoids did not change much after salt processing of AR.But compared from quantitative changes,salt processing can transform some saponins and flavonoids,and the saponins and flavonoids belonging to its effective hypoglycemic components is higher than that of AR.2 Analysis of blood components of AR and SAR in rats based on UPLC-Q-TOF-MS/MS technology.A method for the determination of medicinal components in serum of AR and SAR extract was established.A total of 21 components were identified.They were pantothic acid,neomangiferin,trans-N-（4-hydroxyphenethyl）-ferulinamide,mangiferin,baohuoside I,timosaponin G,timosaponin D,timosaponin C,timosaponin BⅤ,timosaponin BⅢ,timosaponin H₂,timosaponin AⅢ,hinokiresionol,timosaponin I₂,Markogenin,gitogenin,timosaponin F,Diosgenin,hexadecanoic acid,epi-Smilagenin,and octadecanoic acid.By analyzing the metabolic reactions of the blood components,it was found that the components of AR were mainly involved in theⅠmetabolic reactions of reduction,oxidation and dehydration,and theⅡmetabolic reactions of glucuronidation,acetylation,glycosylation and methylation.Finally,by comparing the degree of each component into the blood,it was found that the content of neomangiferin,mangiferin,timosaponin AⅢ,timosaponin BⅢand timosaponin F in the serum of the SAR group was higher than that of the AR group.3 Based on the network pharmacology and experimental validation to explore the mechanism of AR and SAR in the intervention of T2DM.The traditional Chinese medicine system pharmacology（TCMSP）database was used to screen the active ingredients,and the ingredients without corresponding targets were removed,and 16 active ingredients of AR were obtained.A total of 213targets corresponding to the active ingredients were screened from TCMSP and Swiss databases.By searching OMIM,Dis Ge NET and Drug Bank databases,1972diabetes-related targets were obtained,of which 62 overlapped with the targets of the active ingredients of AR.The core components included Timosaponin A-Ⅲ,Anemarsaponin G,Mangiferin,diosgenin,etc.GO enrichment obtained 596 results,including 552 results of biological process,32 results of cell composition,and 12results of molecular function.KEGG pathway enrichment showed 69 related pathways,which were predicted to act on multiple signaling pathways such as insulin resistance,insulin signaling pathway,AMPK signaling pathway and so on.Molecular docking results showed that the core targets and active ingredients had good binding ability.Finally,Ins R and m TOR targets in the insulin resistance pathway were selected for in vitro verification experiments.Compared with the control group,the relative expression level of Ins R in the Hep G-2 cells of the model group was significantly decreased,while the relative expression level of m TOR was increased（P<0.05 or0.01）.After drug treatment,compared with the model group,the relative expression of Ins R m RNA in the AR and SAR groups was significantly increased（P<0.05 or P<0.01）,and the relative expression of m TOR m RNA was significantly decreased（P<0.05）.The improvement in the SAR group was better than that in the AR group.Compared with the normal group,the expression level of m TOR protein in the model group was significantly up-regulated（P<0.01）,and the expression level of Ins R protein was significantly down-regulated（P<0.01）.Compared with the model group,the expression level of m TOR protein was significantly down-regulated（P<0.01）,and the expression level of Ins R protein was significantly up-regulation（P<0.01 or P<0.05）in the AR,SAR and positive control groups.The results showed that the regulation effect of SAR on m TOR protein was similar to that of AR.However,the regulation effect of SAR on Ins R protein was significantly better than that of AR（P<0.05）.4 Study on the mechanism of AR and SAR in the treatment of type II diabetes based on metabonomics..A total of 22 differential compounds were identified in the urine of diabetic rats.After the intervention of AR and SAR,a total of 17 differential metabolites were adjusted,including 12 differential metabolites regulated by both AR and SAR."These include L-Valine,D-Fructose,Pantothenate,Xanthosine,2’,3’-Cyclic AMP,Arachidonate,3’,5’-Cyclic GMP,Isocitrate,Pyruvate,OPC6-Co A,4-fumarylacetoacetic acid,L-Dopa,However,Thymine,7-Methyluric acid,L-Tyrosine,Arginine,and D-Ribitol-5-P were only regulated by SAR.Subsequently,Metabo Analyst 5.0 was used to perform pathway enrichment,and it was found that 10pathways were involved in the regulation of AR and SAR after treatment."These include phenylalanine,tyrosine,and tryptophan biosynthesis,α-nitrous acid metabolism,tyrosine metabolism,arachidonic acid metabolism,pyruvate metabolism,tricarboxylic acid cycle,glycolysis/gluconeogenesis,arginine biosynthesis,arginine and proline metabolism,and pyrimidine metabolism."The analysis of blood of diabetic rats showed that there were 20 biomarkers as the callback group after treatment.Among them,Lyso PC（16:0/0:0）,taurine,1-heptadecanoyl-sn-glycerol-3-phosphocholine,and phosphoethanolamine in the SAR group recovered to more similar levels to those in the control group.The 20metabolites were involved in a total of 14 metabolic pathways,including bile acid biosynthesis,glycerophospholipid metabolism,tryptophan metabolism,sphingolipid metabolism,taurine and hypotaurine metabolism,arachidonic acid metabolism,pentose and glucuronic acid interconversion,and fatty acid biosynthesis.Conclusion:1.The contents of timosaponin AⅢ,timosaponin BⅢ,timosaponin F,mangiferin and other components were increased after the salt processing of AR,and their contents also increased in the blood of both normal and diabetes rats.Timosaponin AIII,timosaponin BIII,timosaponin F,and mangiferin are the material basis for the enhanced hypoglycemic effect of SAR.2.AR and SAR can regulate the core targets（Ins R and m TOR）in the insulin signaling pathway,affect gluconeogenesis and protein production,thereby achieving hypoglycemic goals.3.AR and SAR can regulate metabolic pathways such as bile acid biosynthesis,sphingolipid metabolism,arachidonic acid metabolism and so on.;SAR also exhibits regulatory effects on metabolic pathways such as glycerophospholipid metabolism,glycolysis/gluconeogenesis,linoleic acid metabolism,fatty acid biosynthesis,and pyrimidine metabolism and so on.This explains the enhanced hypoglycemic effect of SAR from the perspective of metabolic pathways.