The Construction Of Chemosensitization Screening Method Based On ~1H NMR And The Study Of Sensitizing Activity

Long-term use of chemotherapy drugs prone to drug resistance,severely limits the clinical application of chemotherapy drugs.The combination of anticancer drugs and chemosensitizer can increase the sensitivity of tumor cells to chemotherapeutic drugs,thereby improving the therapeutic effect of chemotherapeutic drugs.Traditional Chinese Medicine（TCM）has the advantages of novel chemical structure and low toxicity,which is an important source for screening chemosensitizer.The biological activity tracking separation method is a traditional separation method,but it takes time and effort to separate and purify the active compounds one by one and the activity determination.In view of the problems faced in the screening of chemosensitizer,this paper takes colorectal cancer as the research object,establishes a method of nuclear magnetic resonance spectroscopy（1H NMR）combined with biochemical metrology to screen chemosensitization,and takes curcumin as the application object to screen potential sensitizers and their mechanism.The research content of this paper is mainly divided into the following three parts:First of all,this paper establishes a method based on 1H NMR combined with biochemometrics to quickly identify and identify the active ingredients in TCM.The CCK-8 method was used to determine the inhibitory effect of total Curcuma Longa L.extracts on the proliferation of colorectal cancer such as parent cells HCT8 and HCT8/FU resistant cells.Combined with 5-fluorouracil,it can enhance the chemosensitization of colorectal cancer cells to 5-FU,which confirms that Curcuma Longa L.contains active ingredients for chemotherapy sensitization.Before identifying the biologically active ingredients in TCM,column chromatography is used to obtain Curcuma Longa L.fractions of different polarities.Each fraction is divided into two parts:for 1H NMR analysis and for biological activity testing.OPLS-DA was used to study the correlation between plant metabolite profiles and biological activities.Based on the Y correlation coefficient and VIP value,the signal of the active area of the OPLSDA score map was studied,the chemical shifts of the different metabolites in the 1H NMR spectrum were compared,and the structure of the compound was identified by two-dimensional NMR technology.Finally,using multivariate analysis,nine active components of curcumin,demethoxycurcumin,bisdemethoxycurcumin,ar-curcumone,bisacurmol,α-curcuminon,β-curcuminone,ar-curcuminene and ar-curcuminol were identified.According to high performance liquid phase-mass spectrometry（LC-MS）obtain structural information and verify the compounds identified by the above method.Structure-activity relationship analysis,what groups in the structure can significantly improve the activity.Then,HPLC method was established for the determination of the contents of the main differential metabolites curcumin,demethoxycurcumin,and bisdemethoxycurcumin,and the distribution of active compounds in each fraction was quantified,which was helpful to clarify the ’dose-effect’ relationship.Secondly,the biological activity analysis and structure-activity relationship analysis of curcumin,demethoxycurcumin,and bisdemethoxycurcumin,the compounds with the highest content in the turmeric plant.The results showed that lowdose curcumin compounds can enhance the sensitivity of colon cancer cells to 5-FU and significantly inhibit the proliferation of tumor cells;the mRNA levels of resistancerelated genes were detected by qPCR,and the results showed that curcumin compounds can reverse resistance.Medicine;At the same time,it also promotes the depletion of glutathione and the accumulation of reactive oxygen species.It was previously detected that the active fractions in turmeric can lead to the increase of intracellular lactic acid content,which may have a regulatory effect on the expression and function of monocarboxylic acid transporters（MCTs）,and it is speculated that the active components of turmeric will affect the transport of monocarboxylic acid compounds in cells have an impact,so this chapter established an LC-MS method to detect the content of five monocarboxylic acid compounds in the cell.After methodological verification,the established method is accurate and reliable in terms of accuracy,sensitivity,stability and precision.The results showed that curcumin compounds combined with 5-FU can exert cytotoxicity by down-regulating the expression of lactate transporters.Finally,around the cellular metabolic pathways and lactic acid transport related proteins,in-depth study of the mechanism of action of curcumin compounds.Using qPCR to verify the expression and regulation of curcumin compounds on glycolysis pathway-related genes,it was found that there was no significant effect on the mRNA levels of genes SLC16A3,GLUT 1,and LDHA,but they had a certain effect on the expression of genes HK2,PKM2,PDK2,and SLC16A1,especially It is that the expression of SLC16A1 is significantly down-regulated.Early experiments proved that the combination of drugs can affect the content of monocarboxylic acid compounds inside and outside the cell,and inhibit the transport function of MCT1 protein.In order to study the interaction between curcumin compounds and MCT1 protein,this paper uses human embryonic kidney cells 293T to construct an MCT1 protein overexpression system,and uses green fluorescent protein to verify the protein expression.A method based on NMR saturation transfer difference spectroscopy was established to study protein-drug interactions.In this paper,aiming at the identification of chemotherapeutic sensitizers of colorectal cancer in traditional Chinese medicine,a 1H NMR combined biostoichiometry method was constructed to apply to the screening of active ingredients of turmeric,which can quickly discover potential active compounds and improve the efficiency of separation and extraction.Then through further mass spectrometry structure identification and biological activity function verification,eliminate the possibility of false positives,to determine the feasibility of this method.Then,conduct biological activity experiments on the identified curcumin compounds to study the sensitizing activity of chemotherapeutic drugs.Finally,based on the inhibitory effect on lactate transport,the mechanism of targeting the lactate transporter MCT1 was studied,indicating that inhibiting the expression of MCT1 would Increasing the sensitivity of 5-FU provides a theoretical basis for further research on the relationship between the regulation of intracellular glycolysis pathways and sensitization activity.