Clinical Data Mining And Experimental Study On Core Drug Effective Monomer In Differentiation And Treatment Of Colorectal Cancer By Professor Xu Zhen-ye

Objective: To study Professor Xu Zhenye’s clinical reasoning and choice of medication in colorectal cancer(CRC)treatment from different aspects using data mining tools and techniques as a reference for traditional Chinese medicine(TCM)-based CRC treatment.On this basis,an in vitro experiment was performed on ursolic acid(UA),an effective chemical compound found in the antidote core ingredient of Professor Xu’s clinical prescriptions,to observe the inhibitory effects of UA on colon cancer(CC)cells and identify its target in this process.This study revealed the mechanism of action of UA on CRC cells and provided a new target for TCM-based CRC treatment.Methods:Theoretical research: Using the method of literature review to systematically review the research progress of Chinese and Western medicine in colorectal cancer;This paper summarized Professor Xu Zhenye’s academic thoughts on differentiation and treatment of colorectal cancer,and summarized the relevant application of data mining in TCM.Clinical study: A retrospective clinical study was performed on Professor Xu’s patients who received his outpatient services during January 2016 and March 2021 and met the following inclusion criteria: 1)diagnosed with CRC based on pathological and imaging findings,2)with at least two follow-ups after diagnosis,3)showing improvements in clinical symptoms of CRC,4)More than 1 month of chemotherapy had been completed at the time of inclusion.Demographic and clinical characteristics were analyzed,including the stage of CRC upon diagnosis,age,sex,frequency of consultation,clinical symptoms,tongue diagnosis,pulse diagnosis,differentiation of symptoms and signs for classification of syndromes,and prescriptions and medications.Data preprocessing was performed before including any patients in the study to ensure that the demographic and clinical data such as basic information,clinical symptoms,differentiation of symptoms and signs for classification of syndromes,affected sites,pathological factors,and clinical medication were arranged in a uniform,standardized,and normalized fashion.Following that,relevant data were recorded in Excel spreadsheets.The software SPSS 25 was used for frequency,cluster,and factor analysis.The data mining workbench IBM SPSS Modeler 18 was employed to generate association rules from the prescriptions and medications for CRC treatment.Further data mining was conducted to analyze clinical characteristics such as clinical symptoms,TCM types of syndrome differentiation,affected sites,pathological factors,and prescriptions.Through in-depth analysis,this study summarized Professor Xu’s clinical reasoning and choice of medication in clinical differential diagnosis and treatment of CRC.In vitro experiment: 1)Ursolic acid,quercetin and olenolic acid,the common monomers of the core drug of antidote,were screened by CCK8 to inhibit SW480 cells,and ursolic acid showed a better inhibition effect among the three.2)The effects of UA on HCT116 and SW480(human CC cell lines)were analyzed using an in vitro experiment.The CCK8 assay was carried out to evaluate the viability of UA-treated CC cells.Based on the UA concentrations determined in the preliminary experiment,the HCT116 cells were treated with 10,20,and 30 μM UA,respectively,and the SW480 cells were treated with 5,10,and 15 μM UA,respectively.After UA treatment for 24,48 h,the Transwell and scratch assays were undertaken to observe the effects of UA on cell migration,while its effects on cell apoptosis were measured using flow cytometry.3)Candidate genes with differential expression in UA-treated human CC cells were identified by querying the GEO Data Sets based on TMT proteomics technology and advanced bioinformatics.4)The candidate gene RPLP1 was analyzed to determine its functions and expression levels in the HCT116 and SW480 cells,respectively.The HCT116 and SW480 cells were assigned to five groups,namely con,short hairpin RNA(sh RNA)-NC,sh RNA-57068,sh RNA-57069,and sh RNA-57070,to measure cell viability with the CCK8 after being treated with a control reagent and different sh RNAs,respectively.Besides,the HCT116 and SW480 cells were treated respectively and divided into Con(Blank),con(PI),con(FITC),sh RNA-NC,and sh RNA-57068 groups to evaluate cell apoptosis and cell cycle via flow cytometry.As to cell migration and invasion,the Transwell and scratch assays were conducted following treatment with sh RNA-NC and sh RNA-57068,respectively.5)The effects of UA treatment combined with RPLP1 knockdown on the HCT116 and SW480 cells were examined.The HCT116 and SW480 cells were divided into four groups,including NC,RPLP1-KD,UA,RPLP1+UA,to observe cell proliferation using the CCK8 and colony formation assays,detect cell apoptosis by flow cytometry and measure cell migration and invasion by the Transwell and scratch assays.Results: Clinical study results: A total of 87 CRC patients were included in this study,including 51(59%)males and 36(41%)females.The eight symptoms most commonly seen in the CRC patients were torpid intake,lack of strength,loose stools,emaciation,restless sleep,abdominal distention,sore waist,and dry throat.There were 10 highly frequent syndrome-differentiation types,including spleen vacuity and essence depletion,spleen vacuity and dampness and stagnation,liver-Qi stagnation and spleen deficiency,vital Qi deficiency and blood stasis,spleen-kidney Yang deficiency,spleen vacuity and essence depletion accompanied with dampness,vital Qi deficiency and toxin accumulation,lung-spleen vacuity,spleen vacuity and Qi stagnation,and Qi-Yin deficiency,with spleen vacuity and essence depletion most commonly occurring in the CRC patients.Dampness and stasis were the most important pathological factors in the CRC patients,present in 50.179% and 39.068% of all patients.Spleen,kidney,and bowel involvement was largely detected in the CRC patients,representing 96.057%,41.219%,and 24.373% of all cases,respectively.Based on the analysis of 279 prescriptions,10 Chinese herbal medicines(CHMs)were most frequently prescribed,including Astragalus membranaceus(AM),Atractylodes macrocephala Koidz.(AMK).,Galli gigerii endothelium corneum(GGEC),Poria cocos sclerotium(PCS),Oldenlandia diffusa(OD),Smilax china(SC),Fructus akebiae(FA),Polygonatum sibiricum(PS),Cayratia japonica(CJ),and Gynostemma pentaphyllum(GP).Clinically,AM was shown as the core ingredient in these prescriptions.The following combinations had Qi-nourishing and spleen-invigorating effects: AM and AMK,AM and PCS,AMK and PCS;AM and SC,as well as AM and OD,were demonstrated to invigorate the spleen and clear toxins;AM and GGEC were used as a Qi-nourishing,spleen-invigorating and digestion-promoting formula to improve the strongly correlated gastric function and gastric emptying kinetics.The CRC patients were usually prescribed with three different four-ingredient formulas with relatively high support values and Qi-nourishing,spleen-invigorating,detoxifying and stagnation-diminishing benefits,including 1)AM,AMK,PCS,and SC,2)AM,AMK,PCS,and OD,and 3)AM,AMK,PCS,and GGEC.The top five combinations with high confidence values were 1)Cnidium rhizome(CR),GGEC,and AM,2)CR,OD,GGEC,and AM,3)CR,GGEC,AMK,and AM,4)CR,GGEC,PCS,and AM,and 5)Pseudostellaria heterophylla,PS,PCS,and AMK.The support and confidence analysis suggested strong associations between each of the five combinations with the spleen vacuity and essence depletion syndrome-differentiation type.According to the cluster analysis,the CHMs used for CRC treatment and the symptoms observed in the CRC patients were classified into six categories.In terms of the four properties of CHMs,the prescriptions contained28.11% of neutral ingredients,representing a proportion higher than those with other properties.Among the five tastes(i.e.,pungent,sweet,sour,bitter and salty),sweet herbs contributed to the highest proportion(54.56%)of all prescribed CHMs.The proportion of spleen-invigorating ingredients was higher than those offering other health benefits.Ten prescriptions with common factors were obtained from the factor analysis of clinical medication.Experimental results: This study indicated that UA could inhibit the proliferation and promote the apoptosis of HCT116 and SW480 cells.Based on proteomics technology,advanced bioinformatics,and the GEO Data Sets,it was found that the RPLP1 expression level was reduced in UA-treated CC cells.Further,RPLP1 knockdown was demonstrated to have associations with reduced proliferation,increased apoptosis,and modulated migration and invasion of CC cells.The combined use of UA and RPLP1 knockdown was shown to enhance the inhibitory effects on CC cell proliferation,induce cell apoptosis and inhibit cell migration and invasion.The clinical study and in vitro experiment revealed that RPLP1 had a high expression level in CC cells and demonstrated that RPLP1 knockdown could produce inhibitory effects on CRC cell growth.Conclusions:Professor Xu Zhenye has made good use of invigorating the spleen and nourishing sperm,inhibiting cancer and detoxification in the diagnosis and treatment of colorectal cancer,which has expanded a new idea for enlightening the syndrome differentiation and treatment of colorectal cancer with traditional Chinese medicine.Hedyotis diffuser occupies a core position in Professor Xu Zhenye’s antidote for the treatment of colorectal cancer.Through screening a variety of effective monomers of Hedyotis diffusa,UA showed a strong inhibitory effect on colon cancer cells.RPLP1 is a new target of UA to inhibit colon cancer.RPLP1 knockdown can inhibit the proliferation,migration and invasion of colon cancer cells and promote their apoptosis,but has no significant effect on the cell cycle of the tumor.The combined intervention of UA and RPLP1 knockdown can significantly inhibit the proliferation,migration and invasion of colon cancer cells and promote their apoptosis,but it has no obvious effect on the tumor cell cycle.These results suggested that UA could inhibit the growth of colon cancer by regulating the expression of RPLP1,and the molecular mechanism of UA should be further studied.