Analysis of compatibility rules and mechanisms of traditional Chinese medicine for preventing and treating postoperative recurrence of bladder cancer

Xin-Zhe Zhao,Yao Yang,Ying Chen,Li-Hui Zhang,Guo-Wei Zhang*

1College of Chinese Medicine,Hebei University,Baoding,Hebei 071000,China.

Abstract Backgroud:Summarize the formula rule of traditional Chinese medicine for preventing and treating bladder cancer recurrence after operation and explore the molecular mechanism of core medicines.Methods:Literatures collected from CNKI,Wanfang Med Online,CMJD,PUBMED and Elsiver databases were as prescription sources,and association rules and complex system entropy clustering analysis were carried out using the Traditional Chinese Medicine Inheritance Support System (TCMISSV2.5).BATMAN-TCM online analysis tool was used to construct target-pathway-disease correlation map to reveal the potential mechanism of action.Results:A total of 122 prescriptions were eligible for data analysis.The high-frequency traditional Chinese medicines are Poria,Radix et Rhizoma Rhei,Radix Astragali,Herba Hedyotidis Diffusae and Rhizoma Atractylodis Macrocephalae.The high-frequency drug pairs are Rhizoma Atractylodis Macrocephalae/Poria,Poria/Rhizoma Alismatis,Radix Astragali/Rhizoma Atractylodis Macrocephalaeand and Herba Hedyotidis Diffusae/Herba Scutellariae Darbatae..There are 5 groups of drug pairs with high correlation strength.Cluster analysis shows 6 core drug combinations and 3 new prescriptions.In clinical practice,the core compatibility of traditional Chinese medicines for preventing postoperative recurrence of bladder cancer is Poria, Radix Astragali and Herba Hedyotidis Diffusae.The possible signaling pathways are the neuroactive ligand receptor interaction signaling pathway and calcium signaling pathway.Conclusion:Prevention and treatment of postoperative recurrence of bladder cancer mainly use medicines with effects of eliminating dampness and diuresis for removing edema,heat-clearing and detoxifying,and qi-invigorating.The potential mechanism of the compatibility of core drugs may be realized by interfering with the signal pathway of neuroactive ligand receptor interaction and calcium signal pathway.

Keywords:Postoperative recurrence of bladder cancer,Compatibility rules,Mechanism of action,Data mining,Network pharmacology

Background

Bladder cancer is the most common malignant tumor of the urinary system.Postoperative recurrence is the leading cause of death.Bladder cancer is the 10th most common malignancy in the world [1].According to the latest survey report,bladder cancer ranks 6th in the United States oncology population,and the number of male patients are much higher than that of females[2].The incidence of bladder cancer in China in 2013 was 5.46/100,000,accounting for 2.02% of all new tumor cases,ranking 16th in the incidence of malignant tumors [3].In recent years,the incidence and mortality of bladder cancer have been increasing year by year [4].Relapse is a difficult problem for bladder cancer treatment,which brings huge economic pressure to patients and society [5].Clinically,postoperative treatment of bladder cancer urgently requires drugs with low toxicity,high efficiency,easy tolerability,and slight side effect.Modern research through the biological analysis of bladder cancer found several susceptibility genes,including TP53,MTHFR,IL-6,CDKN2A,GSTP1 and then based on this to explore new detection,prevention and treatment of bladder cancer method [6].

The use of traditional Chinese medicine (TCM) has become an indispensable part of the treatment plan for bladder cancer.It plays an important role in enhancing the strengths and avoiding the weaknesses of modern medicine such as preventing metastasis and recurrence after surgery,reducing the harmfulness of radiotherapy and chemotherapy to the body,and improving quality of life [7].This study systematically collated the relevant literatures published in the China Knowledge Network (CNKI),Wanfang Medical Network and Weipu Chinese Biomedical Journal Database,PUBMED and Elsiver in the prevention and treatment of postoperative recurrence of bladder cancer in the past 20 years.The analysis and prediction of the prescription rules and molecular signal pathways clarify the advantages of Chinese medicine on the overall regulation of the body,and provide a reference for the in-depth study of the mechanism of Chinese medicine in the prevention and treatment of postoperative recurrence of bladder cancer.

Materials and methods

Medical record collection

Enter CNKI,Wanfang Medical Network and Weipu Chinese Biomedical Journal Database,PUBMED and Elsiver's advanced search interface,set the subject words as“Bladder Cancer Postoperative Recurrence”and“Traditional Chinese Medicine”in English database,“膀胱癌术后复发”and 中药”in Chinese databases.We set the publication time is from“January 1,1998”to“December 1,2018”.The remaining conditions are the default options.As a result,a total of 81 related literatures were retrieved.

Inclusion criteria

(1) Chinese medicine oral decoction is used for the prevention and treatment of postoperative recurrence of bladder cancer.(2) Symptoms and prescriptions in the literature are clearly recorded and the effect is significant.(3)Those who have a follow-up record take the first prescription,and repeated prescriptions are taken only once.

Exclusion criteria

(1) Those who use non-oral decoctions such as topical Chinese medicine,ear-buried beans,or combined Chinese and western medicine.(2) Non-clinical research such as experience summary,review,cell,animal experiment.(3) Those who has obscure prescription symptoms and drug composition in the literature,and have no obvious treatment effect.(4)Duplicate literature.

Prescription screening and standardized processing

Through the above conditions,a total of 122 prescriptions including 215 drugs were included.In order to ensure reasonable data analysis and the accuracy of traditional Chinese medicine terms,the names of Chinese medicines are standardized according to the“Chinese pharmacology”[8]and“Pharmacopoeia of The People's Republic of China”[9].For example,“Shu Dihuang (Radix Rehmanniae preparata)”and“Sheng Dihuang (Radix Rehmanniae)”are unified into”Dihuang (rehmannia)”,and“Yiren (Semen Coicis)”and“Chao Yiyiren (FiredSemen Coicis)”are unified into“Yiyiren (Semen Coicis)”.Standardize the symptom names,according to the“Differential Diagnosis of TCM Syndromes”[10].

Data entry and verification

The finished prescription is entered into the“clinical collection”system of the“Traditional Chinese Medicine Inheritance Support System”(TCMISS V2.5)software,and a database of traditional Chinese medicine is established,which includes the names of traditional Chinese medicine diseases,the names of western medical diseases,symptoms,treatment principles,and drugs.Data check and data review by professionals with deputy senior titles or above to ensure the accuracy of the information.

Data statistics and analysis

Frequency statistics.In the“statistical report”system,all drugs,symptom statistics,the property,taste and meridian of medicines appear in descending order,and the results were exported separately.

Analysis of association rules for common medicines.

In the“data analysis”system,the number of support levels was set as 16 and the“confidence level”was set at 0.6.And then the medication use mode and rule analysis results were got and exported.

Core combination and new prescription analysis.In the data analysis system,through prediction and debugging,the“correlation degree”was set as 5,and the“penalty degree”was set as 2 to perform entropy cluster analysis for obtain the core drug combination and new prescriptions.The network display was obtained at the same time.

Mechanism analysis.Using the BATMAN-TCM(http://bionet.ncpsb.org/batmantcm/),the action mechanisms of traditional Chinese medicine were analyzed.The core drugs obtained from data mining were entered,with“score cutoff”set to 80 and“adjust P-value”set to 0.1,to analyze the potential signaling pathways for their compatibility.

Results

Drug frequency

As shown in Table 1,among 215 drugs,the top five most frequently used medicines arePoria,Radix et Rhizoma Rhei,Radix Astragali,Herba Hedyotidis DiffusaeandRhizoma Atractylodis Macrocephalae.A total of 13 medicines appear more frequently than 20 times,and 176 medicines less than 10 times.

Table 1 Frequency distribution of high frequency drugs in the prevention and treatment of postoperative recurrence of bladder cancer

TCM symptom statistics

The top type of TCM symptom of bladder cancer recurrence after operation was damp-heat pouring downward syndrome,followed intertwinement of dampness-heat and blood stasis in lower-jiao syndrome and deficiency of spleen and kidney syndrome.The top 5 TCM symptoms were shown in Table 2.

The property,taste and meridial distribution of medicines

As shown in Table 3,cold medicines (35.1%) and sweet medicines (47.4%) are the most frequently used drugs in the four-qi and five-flavor properties,respectively.The meridial distribution proportions of medicines are 17.9% in Liver,and 15.3% in Spleen,respectively.

Table 2 Frequency distribution of symptoms of recurrence prevention and treatment of bladder cancer

Table 3 The frequency distribution of property,taste and meridial of herbs in prescriptions for prevention and treatment of postoperative recurrence of bladder cancer

Analysis of association rules for common drugs

The“Number of Supports”was set to 16,and 8 groups of commonly used medicine pairs were obtained.Among them,the combination ofRhizoma Atractylodis MacrocephalaeandPoria,PoriaandRhizoma Alismatisappeared 24 times,with the highest frequency.The second wasRadix AstragaliandRhizoma Atractylodis Macrocephalae,Herba Hedyotidis DiffusaeandHerba Scutellariae Darbatae,which appears 20 times.The frequency of the drug combination is listed in descending order,and the results are shown in Table 4.The core drug compatibility network display diagram is shown in Figure 1.There are 5 pairs of drug combinations with a confidence level greater than 0.6,of whichRhizoma AlismatisandPoriahave the highest confidence.The rule of medication sorted in descending order of confidence and the results are shown in Table 5.

Table 4 Drug combinations in prescriptions for prevention and treatment of postoperative recurrence of bladder cancer

Table 5 Association rules of drugs in prescriptions for prevention and treatment of postoperative recurrence of bladder cancer

The core groups and new prescriptions analysis

Based on the above-mentioned setting of the correlation between drugs,a total of 127 met the established conditions.Take the drug pair with a correlation coefficient of 0.036 or more,and arrange a total of 11 records in descending order (Table 6).This algorithm extracts 6 pairs of core combinations,as shown in Table 7.The network diagram is shown in Figure 2.Three new prescriptions are shown in Table 8 and their network diagram is shown in Figure 3.

Table 6 Correlation degree of drugs in prescriptions for prevention and treatment of postoperative recurrence of bladder cancer

Table 7 Core drug combinations in prescriptions for prevention and treatment of postoperative recurrence of bladder cancer

Table 8 New prescription combinations for prevention and treatment of postoperative recurrence of bladder cancer

Table 9 Enrichment analysis of compatible diseases of Poria,Radix Astragali and Herba Hedyotidis Diffusae

Mechanism analysis

Based on the statistical results of the above drug frequency,medication pattern,and association rules,three drugs,Poria,Radix AstragaliandHerba Hedyotidis Diffusaeare selected for KEGG pathway analysis.150 diseases are enriched by these 3 drugs,and 12 diseases have more than 5 potential targets(Table 9).There are 108 potential signal pathways and among which 20 pathways have more than 6 potential targets (Table 10).Diseases related to postoperative recurrence of bladder cancer are prostate cancer and non-specific cancer (Table 10).The potential targets,signal pathways and disease networks were shown in Figure 4.

Table 10 Analysis results of KEGG pathway

Discussion

Bladder cancer belongs to the categories of“retention of urine”,“stranguria due to hematuria”,“urinary blood”,“drowning blood”,and“abdominal mass”in TCM.TCM believes that bladder cancer is a result of heat evil,blood stasis and toxin accumulate in the bladder.In addition,most patients were in old age with gradually deficiency in the spleen and kidney [11].Weakened body resistance is the root cause of bladder cancer,and“evil poison”is a necessary condition for the onset of disease.So the clinical treatment methods in TCM should be strengthening the body resistance and resolve toxin [12].Spleen and kidney deficiency worsen after surgery of bladder cancer surgery,making lack of healthy qi and poor resistance to evil qi,which will make cancer relapse.A large number of prospective or retrospective studies have analyzed the predictive factors of bladder cancer recurrence [13,14].Drug resistanceintumor isthe mostdangerousfactor for tumor recurrence [15].The occurrence,development,efficacy,and prognosis ofbladder tumors are related to the body's immune status.Bladdertumorscanescapethebody'simmune surveillance,leadingtorelapseor metastasis [16].

TCM is a common treatment method to prevent recurrence and metastasis after bladder cancer surgery.In order to analyze the regularity and mechanism of TCM to prevent recurrence of bladder cancer after surgery,we use the TCMISSV2.5 software to perform association based on association rules and entropy clustering.We analyzed the KEGG molecular signal pathway of core drugs in BATMAN-TCM to prediction of potential molecular mechanisms of drug compatibility.

Our results showed that high-frequency medicines for the prevention and treatment of postoperative recurrence of bladder cancer are herbs with the role of clearing damp and promoting diuresis includingPoria,Semen Coicis,Rhizoma AlismatisandPolyporus;herbs with role of qi-tonifying includingRadix AstragaliandRhizoma Atractylodis Macrocephalae;hematinic herbs ofRadix Angelicae Sinensis;and herbs with role of heat-clearing and detoxicating includingHerba Hedyotidis DiffusaeandHerba Scutellariae Darbatae.In addition,cold medicines and sweet medicines are the most frequently used drugs in the four-qi and five-flavor properties,respectively.The meridial distribution proportions of medicines are most in Liver and Spleen.For TCM symptoms statistics,the top three are damp-heat pouring downward,intertwinement of dampness-heat and blood stasis in lower-jiao,and deficiency of spleen and kidney.These results are in accordance with the cause and pathogenesis of qi deficiency and the accumulation of evil toxin after bladder cancer surgery.

The top 5 support levels in the medication mode areRhizoma Atractylodis Macrocephalae->Poria,Poria->Rhizoma Alismatis,Radix Astragali->Rhizoma Atractylodis Macrocephalae,Herba Hedyotidis Diffusae->Herba Scutellariae Darbatae,Radix Astragali->Radix Angelicae Sinensis.The top 3 confidence levels areRhizoma Alismatis->Poria,Rhizoma Atractylodis Macrocephalae->Poria,RadixAngelicae Sinensis->Radix Astragali.Combined with the above high-frequency medicines,it can be summarized as the modified Zhu Ling soup (Treatise on Febrile Diseaseswritten by Zhang Zhongjing) and Danggui Buxue Decoction (Inner and Outer Traumatology Confusion written by Li Dongyuan).It was found that the active ingredients in commonly used Chinese medicines can regulate the body's immune function and inflammatory factors to play an anti-tumor effect.For example,carboxymethyl pachymaran inPoriacan significantly improve the immune function of multiple infected mice,up-regulate the expression levels of serum cytokines IL-2 and IFN-α,and down-regulate the expression level of IL-10 [17].Intraperitoneal injection of active ingredients ofRadix Astragalican promote the increase of specific antibody levels.Radix Astragalicomponentsin vivocan improve the immune function of tumor-bearing mices and cyclophosphamide immunosuppressed mices,suggesting thatRadix Astragalihas a wide range of immune enhancing effects [18].ursolic acid,an extractfromHerba Hedyotidis Diffusaecan prevent and cure a variety of tumors,such as liver cancer,prostate cancer,bladder cancer,thymic cancer,colon cancer,lung cancer by inhibiting a variety of targets,such as nuclear transcription factor-κB (NF-κB),B-cell lymphoma-2(Bcl-2),Bcl-xL [19].

Three new prescriptions were obtained based on the complex entropy clustering algorithm.The first prescription areRadix Astragali,Radix Angelicae Sinensis,Radix Salviae Miltiorrhiaeas the core medicines and added withRadix Codonopsis,Rhizoma Atractylodis Macrocephalae,which has the effects of strengthening the spleen and kidney,promoting blood circulation and removing stasis,nourishing qi and nourishing blood.The second prescription is the modified pill of six ingredients with rehmannia,consist ofFructus Corni,Rhizoma Dioscoreae,Rhizoma Alismatis,Radix Rehmanniae,Semen Cuscutae.The whole prescription has the role of nourishing kidney essence and liver blood.In the third prescription,Herba Hedyotidis Diffusae,Herba Duchesneae IndicaeandHerba Solaniare as the core medicines,and added withSemen CoicisandPolyporusto clearing heat and removing wind,anti-cancer and detoxification,clearing damp and promoting diuresis.Then we further analyzed the potential mechanisms of action ofPoria,Radix AstragaliandHerba Hedyotidis Diffusaethrough BATMAN-TCM.Resultsshowed that prostate cancer and non-specific cancer are most closely related to postoperative recurrence of bladder cancer.Studies have shown that traditional Chinese medicine compounds containingRadix Astragali,Semen Coicis,Herba Hedyotidis Diffusaeare effective in treating hormone-independent prostate cancer [20].Clinical studies found Xiaozheng Decoction composed ofRadix Astragali,Semen Coicis,Herba HedyotidisDiffusaecombined with bladder arterial embolization infusion chemotherapy can reduce tumor stage,reduce the incidence of side effects after interventional therapy,and improve the quality of life of patients [21].In order to improve the curative effect and reduce the side effects of chemotherapy,TCM is often used in the adjuvant treatment of cancer chemotherapy [22],which includes TCM prescriptions,TCM ingredients and single Chinese medicine.TCM has multiple targets,multiple effects,low toxicity and high efficiency characteristic for anti-tumor and it is not easy to produce drug resistance [23].Among the enriched signaling pathways,neural active ligand receptor interaction signaling pathways and calcium signaling pathways have the most number of targets.Polysaccharides in Si junzi Decoction (Radix Ginseng,Poria,Rhizoma Atractylodis Macrocephalae,Radix Glycyrrhizae) can increase the intracellular release by promoting Ca2+inflow and increasing the release of Ca2+from the calcium library.Calcium ion concentration promotes cell migration,and also promotes extracel1ular Ca2+influx,which promotes gastrointestinal mucosal repair [24].Nie Ke found thatRadix Astragalihas a significant up-regulation effect of free calcium ion concentration in the gastric wall cells of rats during spleen deficiency [25].The combination of intracellular free calcium ion concentration and CaM [26]is a key to plays a role.The intracellular free calcium ion concentration and CaM are down-regulated during spleen deficiency,which leads to a low energy metabolic state in the spleen deficiency rat.Herba Hedyotidis Diffusaecan promote the release of calcium ions stored in the cell,cause calcium ion depletion,and induce the influx of extracellular calcium ions to increase intracellular free calcium concentration to induce tumor cells apoptosis[27].Neuroactive ligand-receptor interaction signaling pathways are all receptors on the plasma membrane that are related to intracellular and extracellular signaling pathways [28].How this signaling pathway plays its role in bladder cancer is still unclear.

In summary,based on data mining and network pharmacology,this study analyzed the rules and mechanism of TCM for the prevention and treatment of recurrence of bladder cancer.The potential signaling pathways ofPoria,Radix AstragaliandHerba Hedyotidis Diffusaehave been predicted.This study is benefit for providing new directions for the study of drugs for the predicted.This study will help to provide new directions for the further study of drugs for the prevention and treatment of postoperative recurrence of bladder cancer and provide reference for clinical medication.However,as our study mainly focus on data mining,the relevant conclusions still need further research and clinical practice to verify.