The Effect And Mechanism Of Salvia Miltiorrhiza Extract On Non-small Cell Lung Cancer Based On Network Pharmacology

Background:Lung cancer is the cancer with the highest morbidity and mortality in 2020,of which Non-small cell lung cancer(NSCLC)is the most common subtype.A large number of traditional Chinese medicines have been used to treat tumors.Salvia miltiorrhiza,first described in the "Shennong Materia Medica Classic",has the effect of invigorating Qi and activating blood circulation.It is often used to treat diseases such as Zhengjia Jiju.Clinical studies have shown that Salvia miltiorrhiza and its preparation have synergistic and detoxifying effects in combination with chemoradiotherapy.However,its specific mechanism of action is still unclear,Limiting the clinical application and drug research Salvia miltiorrhiza.Objective:The previous project of the National Natural Science Foundation of China(No.81403250)found that Tanshinone ?A sulfonated sodium injection could inhibit the epithelial-mesenchymal transformation of Lewis lung cancer cells by down-regulating the expression of Twist gene.However,due to the comprehensive factors such as too many impurities and strong toxicity of tanshinone IIA sulfonated sodium injection,it is difficult to clarify the material composition and mechanism of action,and Lewis lung cancer cells come from C57BL mice,not human lung cancer cells.Therefore,this study used the method of network pharmacology to analyze and screen the anti-tumor components and possible mechanism of Salvia miltiorrhiza,and took human lung adenocarcinoma cells as the research object to carry out cell experiments and animal experiments.Research contents and Methods:There are three parts to this study.The first part is to screen active ingredients and anti-NSCLC molecular targets through network pharmacology.The active components of Salvia miltiorrhiza were searched through the TCMSP database and screened according to ADME(OB?30%,DL?0.18,Caco-2?-0.4,BBB?-0.3)principle.The results showed that Tan ?A had the highest degree.Swiss Target Prediction and Pharmapper databases were used to predict the action targets of Tan ?A,and Disgenet,GeneCards,OMIM,and DrugBank databases were used to predict the NSCLC disease targets The two data sets were mapped,repeated targets were selected,and the "Tan ?A—NSCLC—Target" data set was constructed and imported into the String database to construct the protein-protein interaction(PPI)Network and Cytoscape 3.5.1 software,and the Network topology parameters were analyzed and calculated through Network Analyzer(Cytoscape built-in plug-in).Obtain network information,including node degree,intermediary centrality,and proximity centrality,and take the median of the above three information as the core target of card value screening.Metascape was used as the core target annotation analysis tool to analyze the main functions and signaling pathways of Tan IIA against NSCLC.The second part is to verify the biological processes and signaling pathways identified by network pharmacology analysis through cell experiments.The method is to use A549 cells as models of NSCLC cell experiment,through the CCK-8 experiment,glucose intake experiment,experiment content of ATP,lactic acid content in the experiment,scratches,Transwell experiments,the projection electron microscope,fluorescence probe detection,flow cytometry,Tan IIA on cell proliferation,aerobic glycolysis metabolism,cell invasion and metastasis,the effect of cell apoptosis.Western Blot,PCR,and ELISA experiments were used to observe the regulation of Tan IIA on the biological processes and signaling pathways identified by network pharmacology.The third part is to explore the anti-tumor effect of Tan IIA through animal experiments.Balb/c nude mice with a subcutaneous transplanted tumor of A549 cells were divided into the model group,cisplatin group,Tan IIA low dose group(Tan IIA-L)and Tan IIA high dose group(Tan IIA-H).,and drug intervention methods were intraperitoneal injection.After 28 days of drug intervention,the effects on body weight,tumor volume,and tumor weight of the nude mice were observed.The pathological tissue and lung metastasis of the tumors were observed by HE staining,and the regulation of the drugs on the above molecular mechanisms was observed by immunohistochemical staining.Results:1.The results of network pharmacology showed that the main biological processes of Tan IIA against NSCLC include cell proliferation,aerobic glycolysis metabolism,cell apoptosis,invasion and metastasis;There were PI3K/Akt/mTOR signaling pathway,HIF-1? transcription factor and Bcl-2/Bax/Cleaved Caspase 3 signaling pathway.2.In the cell experiment,the CCK-8 experimental results show that Tan type ?A on A549 cell toxicity show dose-response relationship,as the drug concentration increases,the cell vitality gradually declines,Tan ?A effect on A549 cells IC50 of 48 h=4.19 ?g/ml.3.In the cell experiment,the experiments related to aerobic glycolysis metabolism showed that compared with the 0.1%DMSO group,the glucose intake,ATP production and lactic acid production of Tan ?A 1 ?g/mL group,Tan ?A 2 ?g/mL group and Tan ?A 4 ?g/mL group were significantly decreased,and the differences were statistically significant(P<0.01).4.In the cell experiment,the results of scratch test and Transwell test,the distance of cell migration and the number of cells transferred to the inferior wall of the chamber decreased significantly in Tan ?A 1 ?g/mL,Tan ?A 2 ?g/mL and Tan ?A 4 ?g/mL groups,and the differences compared with 0.1%DMSO group were statistically significant(P<0.05).5.In the cell experiment,the results of transmission electron microscopy showed that compared with 0.1%DMSO group,A549 cells had different degrees of apoptosis caused by Tan ?A,chromatin was broken and condensed into lumps and clumps.The concentration and volume of the cytoplasm decreased.A large number of small vacuoles or vesicle-like processes appear on the surface of the cell membrane6.In the cell experiment,the results of JC-1 flow cytometry showed that the FL2/FL1 of Tan ?A 1 ?g/mL,Tan ?A 2 ?g/mL and Tan ?A 4?g/mL groups were significantly decreased,and the difference compared with 0.1%DMSO group was statistically significant(P<0.01).7.In the cell experiment,the results of MPTP fluorescence assay of mitochondrial permeability conversion pore showed that compared with 0.1%DMSO group,the average optical density of Tan ?A 1 ?g/mL group,Tan ?A 2 ?g/mL group and Tan ?A 4 ?g/mL group were significantly decreased,and the difference was statistically significant(P<0.01).8.In the cell experiment,Western Blot and ELISA results showed that the protein expressions of PI3K,p-Akt/Akt,p-mTOR/mTOR,HIF-1?,GLUT1,HK2,PKM2,LDHA,MMP-9,VEGF-A and Bcl-2 were significantly decreased And Protein expression of Bax and Cleaved Caspase 3 increased significantly in Tan ?A 1 ?g/mL,Tan ?A 2 ?g/mL and Tan ?A 4 ?g/mL groups.The difference compared with the 0.1%DMSO group was statistically significant(P<0.01).9.In the cell experiment,PCR results showed that compared with the 0.1%DMSO group,the mRNA expressions of GLUT1,HK2,PKM2 and LDHA in Tan ?A 1 ?g/mL,Tan ?A 2?g/mL and Tan ?A 4 ?g/mL groups were significantly decreased,and the differences were statistically significant(P<0.01).10.In the animal experiment,the body weight of nude mice in the cisplatin group was significantly lower than that in the model group,but there was no change in Tan ?A-L group and Tan?A-H group,while no weight loss was observed in Tan ?A-L group and Tan ?A-H group,the difference was not statistically significant(P>0.05).11.In animal experiment,compared with the model group,the tumor volume of nude mice in cisplatin group,Tan ?A-L group and Tan ?A-H group was significantly decreased,with statistical significance(P<0.05).12.The tumor inhibition rate in animal experiments showed that the tumor inhibition rate of cisplatin group was 50.18%,that of Tan ?A-L group was 22.26%,and that of Tan ?A-H group was 37.40%.Tumor weight of nude mice in each group was compared.Compared with model group,tumor weight of cisplatin group,Tan ?A-L group and Tan ?A-H group was significantly decreased,with statistical significance(P<0.01).13.The results of HE staining of lungs in animal experiments showed that tumor cells were metastatic in the lung of the model group.The tumor cells were compact but disordered,and the surrounding fibrous tissue was destroyed.No metastatic cancer cells were found in the experimental group.Microscopically,the lung tissue was mainly normal,with loose structure and orderly arrangement of alveolar cells.14.The results of HE staining of tumors in animal experiments showed that the tumor cells in the model group were in a good growth state and were of different sizes,resembling round or round.The nuclei of the tumor cells were deeply stained,evenly distributed,closely arranged,and showed a cord-like or glandular structure.The cells in the experimental group were diffusely distributed to different degrees,and some tissues showed fragmentary cheesy necrosis or ambiguous phenomenon.15.The results of tumor immunohistochemistry in animal experiments showed that compared with model group,the protein expressions of PI3K,p-Akt,p-mtor,HIF-1?,GLUT1,HK2,PKM2,LDHA,MMP-9,VEGF-A and Bcl-2 were significantly decreased,while the expressions of Bax and Cleaved Caspase 3 were increased in cisplatin group,Tan ?A-L group and Tan ?A-H group(P<0.05).Research Conclusions:Network pharmacology has provided a new research method for the modern study of traditional Chinese medicine.The results of tanshinone ?A on human lung adenocarcinoma A549 cells in vitro and in vivo have verified the prediction results of network pharmacology on the biological process and signaling pathway of tanshinone ?A on non-small cell lung cancer.By inhibiting the aerobic glycolysis of NSCLC,tanshinone ?A further inhibits tumor proliferation,invasion and metastasis,and induces cell apoptosis.The mechanism of its action may be related to the inhibition of PI3K/Akt/mTOR signaling pathway and HIF-1?transcription factor,and the down-regulation of glucose transporter GLUT1 and glycolytic rate-limiting enzymes HK2,PKM2 and LDHA during aerobic glycolysis.The expression of tumor metastasis-related molecules VEGF-A,MMP-9 and anti-apoptotic factor Bcl-2 were further down-regulated,and the expression of pro-apoptotic factor Bax and Cleaved Caspase 3 were up-regulated.In addition,some literatures reported that the drugs that promote blood circulation and remove blood stasis can promote tumor metastasis.The results of lung HE staining in animal experiments showed that tanshinone ?A could inhibit the lung metastasis of subcutaneous transplanted tumors.In combination with the results of immunohistochemistry,tanshinone ?A could inhibit the expression of VEGF-A and MMP-9 protein in tumors.The study indicated that tanshinone ?A as an extract of Salvia miltiorrhiza,a drug for promoting blood circulation and removing blood stasis,could inhibit tumor metastasis.