| Objective:In this study,we aimed to explore the clinical experience and academic thought of Professor Xu Li in the treatment of subsolid pulmonary nodules,to find the core drug,and to explore the key active compounds and key targets of anti-cancer activity based on the core prescription.And in vitro experiments,we aimed to verify the anticancer activity of the core prescription,explore the expression level changes of key targets,and provide some guidance for the clinical diagnosis and the treatment and further experimental research.Methods:First,by sorting out the medical records of 100 patients with subsolid pulmonary nodules treated by Professor Xu Li,this paper summarizes the tutor’s "three stages and six differentiations" diagnosis and treatment model for subsolid pulmonary nodules and summarizes the academic ideas,clinical experience,and medication characteristics.Secondly,through the study of medical record data mining,the clinical characteristics of patients with subsolid pulmonary nodules were summarized,and the data mining methods of frequency analysis,hierarchical clustering,Apriori analysis,and Fast Unfolding non-overlapping community discovery were used to find the core drugs and explore the core prescription for a tutor to diagnose and treat subsolid pulmonary nodules.Thirdly,the key active compounds and key targets of core drugs were found by network pharmacology and molecular docking.The web pharmacology approach contains a variety of web platform tools including TCMSP website,ChEMBL website,DisGeNET website,Venny online tools,OmicShare tools platform,KEGG ORTHOLOGY database,etc.The molecular docking method is to obtain the PDB file of the target in the PDB protein database and run the docking program on the compound ligand and target protein in the Schrodinger 2018 software to get the docking data results.With Glide score<-8.0 Kcal/mol as the screening standard,the target and compound combinations with strong molecular docking ability were obtained.Finally,the anticancer activity of the core prescription was verified by A549 lung cancer cells in vitro,and the effects of the core prescription on cell proliferation,apoptosis and metastasis were explored.The protein level of the key targets obtained by molecular docking was detected to verify the possible mechanism of action of the core prescription.Result:In Chapter 1,Professor Xu Li divided the "three stages" of subsolid pulmonary nodules into the low-risk stage,medium-risk stage,and high-risk stage;the "six differentiations" include differentiation of imaging pathological features,differentiation of benign and malignant lesion types,differentiation of clinical malignant probability,differentiation of serum-related indicators,identification of core pathogenesis characteristics,and differentiation of syndrome types.The treatment was based on the principle of "strengthening the body resistance and eliminating the pathogenic factors",with qi-tonifying drugs(ginseng,radix astragali,radix pseudostellariae)and yin-nourishing drugs(herba dendrobii,bulbus lilii,fructus ligustri lucidi),yang-warming herbs(ramulus cinnamomi,fructus psoraleae,and rhizoma curcumae longae),heat-clearing and detoxicating herbs(hedyotis diffusa willd,rabdosia rubescens,humulus scandens,sculellaria barbata,yangtao actinidia root,black nightshade herb,and solanum lyratum thunb),blood-activating and stasis-resolving herbs(salvia chinensis,rhizoma curcumae,and radix notoginseng),phlegm-resolving and mass-resolving herbs(pseudobulb of appendiculate cremastra,radix ranunculi ternati,sun euphorbia herb),toxic drugs(scolopendra,scorpio,taxus chinensis,honeycomb).In Chapter 2,the proportion of subsolid pulmonary nodules in males(27%)was less than that in females(73%),solitary(23%)was less than that in multiple(77%),and pure ground-glass nodules pGGN(39%)were less than mixed ground-glass nodules mGGN(61%).The frequency of symptoms from high to low was dry mouth,poor sleep,cough,expectoration,loose stool,night sweat,chest tightness,and so on.Syndrome frequency from high to low was the deficiency of both qi and yin syndrome,phlegm and blood stasis syndrome,phlegm-heat accumulation,heat toxin accumulation,qi stagnation,and phlegm obstruction.The tongue coating and pulse condition was characterized by the red tongue,thin coating,and thready pulse.Combined with frequency analysis,cluster analysis,and complex network analysis,the core prescription of subsolid pulmonary nodules was determined after discussion with Professor Xu Li as follows:radix astragali 30g,radix pseudostellariae 10g,poria cocos 10g,coix seed 30g,hedyotis diffusa willd 30g,radix ranunculi ternati 10g,pseudobulb of appendiculate cremastra 10g,salvia chinensis 30g,rhizoma curcumae 10g.Combined with the efficacy of traditional Chinese medicine and modern pharmacological studies,the core prescription was divided into the components of strengthening the body resistance "radix astragali,radix pseudostellariae,poria cocos" and the components of anti-cancer and eliminating pathogens "coix seed,hedyotis diffusa,radix ranunculi ternati,pseudobulb of appendiculate cremastra,salvia chinensis and rhizoma curcumae",and the anti-cancer and eliminating pathogens component was named subsolid nodule formula(SSNF).In Chapter 3,the network pharmacology study of SSNF showed that the SSNF enrichment pathway mainly acted on the Apoptosis pathway and PI3K-Akt signaling pathway.Through molecular docking studies,it was found that SSNF was probably composed of active compounds(isoginkgolides,2-hydroxy-3-methylanthraquinone,flavanthrinin,7-O-methylbenzoyl peroxide,etc.).SSNF could bind to key targets(HSP90a,ROS1,PIK3CD,PARP1),regulate the expression level of target proteins,play a role in promoting apoptosis of lung cancer cells through the Apoptosis pathway and PI3K-Akt signaling pathway,and inhibit the progress of subsolid pulmonary nodules.In Chapter 4,in vitro study,A549 lung cancer cells were treated with the aqueous extract of SSNF granules.(1)Compared with the blank control group(0 mg/L),the percentage of cells arrested in the G0/G1 phase in the low,medium,and high dose groups increased with the increase of the dose concentration gradient(P<0.001).(2)The results of apoptosis detection showed that the late apoptosis rate of A549 lung cancer cells induced by low,medium,and high dose groups increased in a dose-dependent manner(P<0.001);(3)In the cell scratch,Transwell invasion and metastasis experiments,it was found that the cell migration rate and cell migration number decreased with the increase of drug dose and concentration(P<0.001).(4)At the same time,we detected cell cycle-related proteins(CyclinE1,CDK2,CDK4),cell apoptosis-related proteins(Bax,Bcl-2,Caspase-3,Caspase-9),and cell metastasis-related proteins(MMP-1,MMP-2,MMP-9)by WB.There was a significant difference in the expression level of each target compared with the blank group(P<0.05).It is suggested that SSNF can inhibit the proliferation of lung cancer cells,promote apoptosis and inhibit metastasis.Compared with the positive group(cisplatin 25 μg/mL),there was no significant difference in cell cycle and apoptosis,scratch,invasion,and metastasis of A549 cells treated with a 60 mg/L high dose of SSNF(P>0.05).It indicated that SSNF in high-dose group can play an anticancer role similar to that of cisplatin,which has also been verified in the detection of related protein levels.Finally,we detected the expression levels of key target proteins(HSP90a,ROS1,PI3K,PARP1)by WB.There was no significant difference in the expression of PI3K between SSNF and A549 lung cancer cells(P>0.05).Compared with the blank group,with the increase in SSNF concentration,the expression of phosphorylated PI3K(p-PI3K)in the middle and high dose groups decreased(P<0.05);the expression of HSP90a and PARP1 in the middle and high dose groups also decreased significantly(P<0.05);the expression of ROS1 in the high dose group decreased(P<0.05).Conclusion:1.Professor Xu Li used the "three segments and six differentiations" model to treat the patients of subsolid pulmonary nodules."Strengthening the body resistance and eliminating the pathogenic factors" is the main method of treatment,and a variety of drugs for strengthening the body resistance and anti-cancer and eliminating the pathogenic factors are combined to treat subsolid pulmonary nodules.2.Professor Xu Li’s core prescription for the treatment of subsolid pulmonary nodules was obtained through the study of medical record data mining:radix astragali 30g,radix pseudostellariae 10g,poria cocos 10g,coix seed 30g,hedyotis diffusa willd 30g,radix ranunculi ternati 10g,edible tulip 10g,salvia chinensis 30g,rhizoma curcumae 10g.In addition,the components for resisting cancer and eliminating pathogenic factors,including semen coix seed,hedyotis diffusa willd,radix ranunculi ternati,edible tulip,salvia chinensis,rhizoma curcumae,were named as SSNF.3.Through network pharmacology and molecular docking studies,it was found that SSNF may bind to key targets(HSP90a,ROS1,PIK3CD,PARP1)by active compounds(isoginkgolide,2-hydroxy-3-methylanthraquinone,flavanthrinin,7-O-methylbenzoyl peroxide,etc.)to regulate the expression level of target proteins.Apoptosis pathway and PI3K-Akt signaling pathway may play a role in controlling malignant potential pulmonary nodules.4.Compared with the blank group,after the A549 lung cancer cells were treated with SSNF,the percentage of A549 cells blocked in the G0/G1 phase increased,the late apoptosis rate increased,and the cell migration rate and cell migration number decreased with the increase of SSNF concentration(P<0.05),which indicated that SSNF had good anticancer activity.Compared with the positive group(cisplatin),in the high dose group(60 mg/L)there was no significant difference in cell cycle,apoptosis,invasion,and migration between the two groups(P>0.05),indicating that high-dose SSNF can play an anticancer role similar to that of cisplatin.WB also confirmed the above conclusion in the detection of related target protein levels.In the multi-target validation,SSNF could not affect the expression of PI3K(P>0.05)but could reduce the expression level of p-PI3K.SSNF also inhibited the expression of HSP90a,PARP1,and ROS1 in A549 lung cancer cells(P<0.05),which verified that SSNF may play an anti-cancer role through multi-target and multi-pathway mechanism,and inhibited the malignant development of subsolid pulmonary nodules.This study verified the key target of network pharmacology and molecular docking research,and provided a basis for further experimental research. |
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