Study On Molecular Mechanism Of Compound Gegen Decoction Against Colon Cancer And Optimization Of Its Reduced Formulas

Objectives:This study aims to explore the molecular mechanism against colon cancer and the reduced formulas’optimization of Compound Gegen Decoction composed of 14traditional Chinese medicines.Through UPLC-MS/MS detection conbined with literature review,the chemical constituents of the water extract of Compound Gegen Decoction were obtained;The potential targets and action pathways against colorectal cancer of Compound Gegen Decoction were predicted by network pharmacology;Colon cancer HCT116 cell model in vitro and HCT116 cell derived tumor-bearing BALB/c nude mouse model in vivo were developed to explore the pharmacodynamic effect and its molecular mechanism of compound Gegen Decoction against colon cancer;The pharmacodynamic effect against colon cancer and subacute toxicity experiment in vivo of Compound Gegen Decoction and its reduced formulas were evaluated and compared to find the better reduced formula.Thus,this study provided scientific data supporting the treatment of colon cancer by Compound Gegeng Decoction and its optimizated reduce formula.Methods:1.Detection of chemical components of the water extract of Compound Gegen DecoctionThough UPLC-MS/MS technique to establish the analytical method for the extract samples of Compound Gegen Decoction.Agilent SB-C18 column and ESI source,QQQ scanning adopts MRM mode were used to detect the chemical components of the extract.2.Network pharmacology predicts the gene target,pathway and mechanism of the inhibitory effect of Compound Gegen Decoction on colorectal cancerThrough TCMSP database platform and literature reports,the five traditional Chinese medicines in Compound Gegen Decoction were screened according to OB≥20%and DL≥0.1,and the prediction target of active substance components was obtained through protein library Uni Prot,and then GO function enrichment and KEGG pathway enrichment analysis were performed through gene expression database（GEO）,GEO2R analysis,David analysis,STRING database and Cytascape3.9.1 software,and obtain the key genes related to colorectal cancer disease in the protein-protein interaction network.3.In vitro experimentThe colon cancer HCT116 cells were treated with the Vehicle（Saline）or various concentrations of water extractions from Compound Gegen Decoction.（1）MTT method and cell clone formation test were used to detect the effect of Compound Gegen Decoction on the proliferation of HCT116 cells.（2）The effect of different concentrations of Compound Gegen Decoction on the morphology of HCT116 cells was observed and detected.（3）Wound healing test and Transwell method were used to detect the effect of Compound Gegen Decoction on HCT116 cell migration.（4）ROS detection kit was used to explore the effect of Compound Gegen Decoction extraction on the oxidative metabolism of HCT116 cells.（5）Western Blot assay was performed to detect the level of ferroptosis-related protein GPX4,PTGS2,ACSL4 and IL-6,JAK2,p-JAK2,STAT3,p-STAT3,MMP2,TWIST protein in HCT116 cells.4.In vivo experimentThe HCT116 cell-derived tumor bearing BALB/c nude mouse model was established,and the control group（10 m L/kg of normal saline）and the administration group（1.0 g/kg/d of Compound Gegen Decoction）were set up.（1）After intragastric administration of Compound Gegen Decoction or its vehicle,the body weight and the length and width of tumor were measured and the tumor volume was calculated every three days,and the effect of Compound Gegen Decoction on tumor growth was evaluated.（2）24 h after the 23^rdintragastric administration,the tumors and organs were dissected and weighed,and body,heart,liver,spleen,lung and kidney were weighed and the organ index were calculated.（3）Western Blot assay was used to detect the level of ferroptosis related protein SLC7A11,GPX4,PTGS2,ACSL4 and IL-6,STAT3,p-STAT3,MMP2,TWIST,MMP7 protein in the tumors of the control group and the treatment group.5.Reduced formulas optimizationIn the experiment,the colon cancer cell line HCT116 was selected as the cell model.According to whether it has the effect of clearing heat and detoxifying or tonifying spleen and stomach,the original formula（Gegen,Huangqin,Huanglian,Huangbai,Qinjiao,Zexie,Binglang,Cangzhu,Baizhu,Danggui,Baihuasheshecao,Baitouweng,Muxiang,Shenggancao）was divided into Three Tastes-Deletion（Compound Gegen Decoction without the three traditional Chinese medicines of Qinjiao,Zexie and Binglang）,Qin Ze Bing group（Qinjiao,Zexie and Binglang）;According to functions of clearing heat and detoxification and return to the large intestine or lung meridian combined with the function of warming spleen and stomach and return to the large intestine or lung meridian combination Three Tastes-Deletion was divided into Five Tastes（Gegen,Huangqin,Huanglian,Baihuasheshecao,Muxiang）and Six Tastes（Huangbai,Cangzhu,Danggui,Baitouweng,Baizhu,Shenggancao）;According to the cold property and clearing heat and detoxifying function of Huangqin and Huanglian medicine combined with Muxiang with the functions of warming up the spleen and stomach,Five Tastes was divided into Three Tastes（Huangqin,Huanglian,Muxiang）,Two Tastes（Gegen,Baihuasheshecao）and Erhuang（Huangqin,Huanglian）;Including Vehicle group,basic on above,cells were divided to 7 groups.（1）MTT assay and cell cloning assay were used to detect the effects of different drugs on the proliferation of HCT116 cells.（2）The morphological changes of HCT116 cells under different concentrations of drugs were detected by cell morphology observation.（3）Wound healing test and Transwell method were used to detect the effect of different drugs at 0.0625 mg/m L on HCT116 cell migration.（4）KM mice were given saline and different drugs by intragastric administration at a dose of 3.2 g/kg/d for consecutive 14 days,and their body weight was measured and animal status were observed and recorded daily;24 h after the 14^thadministration,mice were weighed,and then were sacrificed,and heart,liver,spleen,lung and kidney were dissected,observed and weighed,and the viscera index was calculated for subacute toxicity experiment.Results:1.Results of UPLC-MS/MS detection of chemical constituents of Compound Gegen DecoctionA total of 265 compounds were detected from the extracts of Compound Gegen Decoction,including 89 flavonoids,63 terpenoids,50 phenolic acids,21 alkaloids,10lignans and coumarins,5 quinones,3 tannins and 24 other compounds.2.Network pharmacology predicts the potential target and action pathway of Compound Gegen Decoction in the treatment of colorectal cancerA total of 98 active components of five drugs in Compound Gegen Decoction were collected,and 20 common targets with colorectal cancer were obtained,among which prostaglandin endoperoxide synthase 2（PTGS2）was related to most active components;GO enrichment analysis obtained 63 items（significantly enriched 51items）,involving extracellular space,affecting the activity of serine-type endopeptidase through signal transduction and inflammatory reaction;The enrichment analysis of KEGG pathway obtained 17 main entries（significantly enriched 13entries）,mainly involving IL-17 signal pathway,tumor necrosis factor signal pathway,tyrosine metabolism pathway,Toll-like receptor signal pathway,ovarian steroid production pathway,IL-6/STAT3 signal pathway,etc.3.Experimental results in vitro（1）The results of MTT test showed that Compound Gegen Decoction had obvious inhibitory effect on the proliferation of HCT116 cells in a concentration and time dependent manner,and the IC50 of 48 h was about 0.7737 mg/m L.The results of crystal purple-dyeing cell clone formation experiment showed that compared with normal saline,0.05 mg/m L（P<0.05）and above of Compound Gegen Decoction could significantly reduce HCT116 cell clones,and dramatically reduced clones to few clones at 0.125 mg/m L（P<0.001）and above.（2）The observation of cell morphology showed that the cell morphology became round,the cell gap increased,and the number of cells decreased at the concentration of 0.0625 mg/m L and higher,and the relative number of round cells was positively correlated with the concentration.No apoptotic cells and apoptotic bodies were observed.（3）The results of wound healing test and Transwell test showed that Compound Gegen Decoction had significant inhibitory effect on the migration of HCT116 cells（P<0.05,P<0.01,P<0.001）.（4）The detection results of reactive oxygen species showed that 0.125 mg/m L of Compound Gegen Decoction significantly increased the content of intracellular reactive oxygen species（P<0.05）,resulting in the oxidative accumulation and cell death of HCT116cells.（5）Western Blot results showed that Compound Gegen Decoction significantly down-regulated the levels of ferroptosis-related protein GPX4（P<0.05,P<0.01,P<0.001）,significantly up-regulated the levels of ferroptosis-related protein PTGS2（P<0.05,P<0.01）and ACSL4（P<0.05,P<0.001）,significantly decreased IL-6（P<0.05,P<0.01,P<0.001）protein level and significantly decreased p-JAK2/JAK2（P<0.05）,p-STAT3/STAT3（P<0.001）,significantly reduced MMP2（P<0.05,P<0.01）,TWIST（P<0.05,P<0.001）protein level.4.In vivo experimental results（1）Compound Gegen Decoction significantly inhibited the growth of xenograft tumor derived from HCT116 cells in vivo;Compared with control group,Compound Gegen Decoction significantly inhibited the size and growth rate of HCT116cell-derived xenograft transplanted tumors in vivo（P<0.05）.（2）There was no significant difference in the index of each organ between the Compound Gegen Decoction group and control group.（3）Western Blot results showed that Compound Gegen Decoction significantly down-regulate the levels of ferroptosis-related protein SLC7A11（P<0.01）,GPX4（P<0.05）and up-regulated the levels of ferroptosis-related protein PTGS2（P<0.01）,ACSL4（P<0.05）,and significantly decreased IL-6（P<0.001）protein level and significantly down-regulated p-STAT3/STAT3（P<0.05）,and significantly down-regulated downstream factors:MMP2（P<0.01）,TWIST（P<0.05）,MMP7（P<0.001）protein level.5.Reduced formula optimization results（1）The results of MTT and crystal purple-dyeing cell clone formation experiment showed that Three Tastes-deletion group significantly inhibited the proliferation of HCT116 cells in a time-dependent and concentration-dependent manner,similar to that of Compound Gegen Decoction.Five Tastes,Three Tastes and Erhuang group significantly inhibited the proliferation of HCT116 cells,but the inhibitory effect was significantly lower than that of Compound Gegen Decoction.Two Tastes or Qinzebing showed no inhibition effect on the proliferation of HCT116 cells.（2）Compared with the control group,for Three Tastes-deletion group at 0.03125mg/m L,the cell shape began to turn round and the number of cells decreased in a concentration-dependent manner like those of the original formula,while this effective concentration was lower than the dose of 0.0625 mg/m L of the original formula;While other groups have no obvious changes in morphology.（3）The results of wound healing test showed that Three Tastes-deletion group had a significant inhibitory effect on the migration of HCT116 cells（P<0.05）,similar to that of Compound Gegen Decoction.（4）Compared to control group,Three Tastes-deletion,Five Tastes and Erhuang slightly slowed down the weight growth rate of mice without significantly difference,and Three Tastes group slightly enhanced the weight growth rate of mice without significantly difference,while Compound Gegen Decoction significantly slowed down the weight growth rate after 10-days’intragastric administration（P<0.05,P<0.01）and significantly increased spleen index.Conclusions:1.Using UPLC-MS/MS technology combined with literature comparison,265compounds were detected in the water extract of Compound Gegen Decoction.2.Compound Gegen Decoction can significantly inhibit the proliferation and migration of HCT116 cells,which may be caused by down-regulating the level of GPX4 protein,and up-regulating the level of PTGS2 and ACSL4 protein to induce cell ferroptosis,and by inhibiting the IL-6/JAK2/STAT3 pathway and down-regulating the level of its downstream factors MMP2 and TWIST protein.3.Compound Gegen Decoction can significantly inhibit the growth of xenograft tumor derived from HCT116 cells in vivo.Its mechanism may be through down-regulating the level of SLC7A11,GPX4 protein,and up-regulating the level of PTGS2 and ACSL4 protein to induce cell ferroptosis,and through inhibiting the IL-6/JAK2/STAT3 pathway and down-regulating the level of its downstream factors SLC7A11,MMP2,MMP7 and TWIST protein to inhibit tumor growth and development.4.The inhibitory effect of Three Tastes-deletion group on colon cancer HCT116cells is not significantly different from that of Compound Gegen Decoction.The effect of other reduced formulas of Compound Gegen Decoction is lower than that of Compound Gegen Decoction.Three Tastes-deletion and other formulas did not significantly affect the weight increase rates of KM mice,but long-term irritation administration of Compound Gegen Decoction（here 3.2 g/kg/d）could significantly slow down the rate of weight growth and increase spleen index.Thus,Three Tastes-deletion shows higher safety than Compound Gegen Decoction with similar pharmacodynamic effects against colon cancer.