| Purpose:Using the method of network pharmacology,taking Jianpi Anticancer Prescription as the research carrier,and the interaction network of "Jianpi Anticancer Prescription drug target-colorectal cancer disease target" was constructed,and the biological function analysis and pathway enrichment analysis of common targets were carried out to predict the biological mechanism of Jianpi Anticancer Prescription in the treatment of colorectal cancer.Secondly,in vitro experiments were conducted to observe the effects of different doses(low、medium and high of Jianpi Anticancer Prescription containing serum on the proliferation,apoptosis,cell cycle and migration of CT26 WT colorectal cancer cells;Finally,through in vivo experiment,the subcutaneous tumor bearing model of CT26 WT colorectal cancer cells was established to observe the general state of mice,subcutaneous tumor size,tumor inhibition rate and the expression level of proteins related to tumor angiogenesis of mice after different dose of Jianpi Anticancer Prescription intervention(low、medium and high doses);To detect the expression of RAS,ERK,p ERK and downstream related protein of MMP2 and c-myc proteins related to ERK/MAPK signal pathway,and reveal its deep-seated antitumor mechanism of Jianpi Anticancer Prescription,in order to provide experimental basis for the wide clinical application of Jianpi Anticancer Prescription。Method:1 Study on the mechanism of Jianpi Anticancer Prescription(low、medium and high dose)in the treatment of colorectal cancer based on modern network pharmacology:Through literature search and TCMSP(Traditional Chinese Medicine Systems Pharmacology)database,the active components of Jianpi Anticancer Prescription were screened and the target of Jianpi Anticancer Prescription was predicted.Through Gene Cards database and other databases,the colorectal cancer target was screened.The screened drug component target of Jianpi Anticancer Prescription was intersected with the disease target of colorectal cancer,and the protein interaction network was constructed.Go analysis and KEGG pathway analysis were performed on the intersection protein of Jianpi Anticancer Prescription and colorectal cancerto speculate the molecular biological mechanism of Jianpi AnticancerPrescription in the treatment of colorectal cancer,so as to guide the follow-up experimental research.And through experimental research to verify the speculation of network harmacology。2 Effect of serum containing of Jianpi Anticancer Prescription on proliferation and migration of colorectal cancer CT26 WT cells:Twenty SPF rats were divided into groups and given normal saline,Jianpi Anticancer Prescription low、medium and high doses and corresponding dose of capecitabine by gavage to prepare drug containing serum the purchased CT26 WT was subcultured,frozen and resuscitated,and then frozen for subsequent experiments.continuous gavage for 5 days of mice.one hour after the last gavage,the animals were fixed,and the carotid blood of the animals was collected according to the aseptic operation.After standing for 2h,the animals were given 3000 r.min-1,centrifuged for 20 min,the centrifugation radius was 7.5cm,the serum was separated,inactivated at 56℃ for 30 min,filtered and sterilized by 0.22 um filter membrane,sub packed,and stored at-20℃.Observation of the effect of Jianpi Anticancer Prescription on the cycle of Jianpi Anticancer Prescription on the proliferation of CT26 WT cells;MTT assay was used to detect the inhibitory effect of Jianpi Anticancer Prescription on the proliferation of CT26 WT cells;flow cytometry was used to detect whether the containing serum of Jianpi Anticancer Prescription had an effect on the apoptosis of CT26 WT colorectal cancer cell line;Secondly,the cell migration ability was measured by Transwell method of Jianpi Anticancer Prescription on the proliferation of CT26 WT cells.To explore the antitumor effect of Jianpi Anticancer Prescription on the proliferation of CT26 WT cellsin vitro.All experimental data were expressed in(mean±SD).One way ANOVA was used for the comparison between multiple groups,and t-test was used for the comparison between the two groups.The difference was statistically significant with(P<0.05).3 Effect of Jianpi Anticancer Prescription on the expression of VEGF,MVD and PKC in CT26 WT colorectal cancer cell transplantation tumor:Kunming mice,4-6 weeks old,weight ranging from 18 to 20 g,40 in total,CT26 WT tumor cells were subcutaneously injected into tumor cells to establish a mouse intestinal cancer model.After modeling,after the mouse subcutaneous oval mass visible to the naked eye,it indicates that the modeling is successful.The model mice were randomly divided intomodel control group,capecitabine group and Jianpi Anticancer Prescription low,medium and high dose groups.Different intervention measures were given respectively.The intervention time was 2 weeks.Observe the general conditions of mice in each group during the whole experiment(eating,skin condition,activity,weight of mice(low,medium and high dose groups),and so on.).At the end of the second week,after the mice were decapitated and killed,the limbs of the mice were fixed in the supine position with big nails,the tumor tissue was stripped with ophthalmic scissors,the blood around the tumor tissue was cleaned with normal saline,and the tumor inhibition rate of each group(low,medium and high dose groups)was observed;The transplanted tumor cells were stained(low,medium and high dose groups)with TUNEL to detect whether Jianpi Anticancer Prescription could induce apoptosis in vivo;The results of CD34 labeled MVD in tumor tissues were detected by immunohistochemistry of Jianpi Anticancer Prescription(low,medium and high dose groups);Western blot was used to detect the expression of VEGF and PKC in the transplanted tumor tissue of Jianpi Anticancer Prescription.4 Jianpi Anticancer Prescription affects the proliferation and angiogenesis of colorectal cancer through ERK/MAPK signal pathway:Kunming mice,4-6 weeks old,weight ranging from 18 to 20 g,40 in total,CT26 WT tumor cells were subcutaneously injected into tumor cells to establish a mouse intestinal cancer model.After modeling,after the mouse subcutaneous oval mass visible to the naked eye,it indicates that the modeling is successful.The model mice were randomly divided into model control group,capecitabine group and Jianpi Anticancer Prescription low,medium and high dose groups.Different intervention measures were given respectively.The intervention time was 2 weeks.At the end of the second week,after the mice were decapitated and killed,the limbs of the mice were fixed in the supine position with big nails,the tumor tissue was stripped with ophthalmic scissors,the blood around the tumor tissue was washed with normal saline,and the effects of RAS,ERK and p ERK expression in ERK/MAPK signal were detected by Western blot of Jianpi Anticancer Prescription;The effect of c-myc expression was detected by Western blot of Jianpi Anticancer Prescription;The effect of MMP2 expression was detected by Western blot of Jianpi Anticancer Prescription.Results:1 Network pharmacology:In this part,230 chemical components of Jianpi Anticancer Prescription and drug action targets of Jianpi Anticancer Prescription,1313 colorectal cancer targets and 129 intersection targets of Jianpi Anticancer Prescription and colorectal cancer were collected.Through biological function analysis,the results showed that they were mainly related to cell cell proliferation,cell apoptosis and cell angiogenesis of Jianpi Anticancer Prescription.Pathway enrichment analysis found that Jianpi Anticancer Prescription may affect MAPK signal pathway,PI3K-Akt signal pathway TNF mediated signaling pathways are related.The main indicators are VEGF,PKC,RAS,ERK,c-myc,MMP2,and so on.2 Anti tumor effect of Jianpi Anticancer Prescription containing serum on CT26 WT colorectal cancer cells in vitro.2.1 Effect of serum containing of Jianpi Anticancer Prescription on the proliferation of CT26 WT cells:Compared with the control group,the inhibition rate of CT26 WT cell proliferation in different doses of serum containing drugs of Jianpi Anticancer Prescription increased after 24hours(P<0.05).After 48 hours of treatment,compared with 24 hours,the inhibition rate of Jianpi Anticancer Prescription group increased significantly,and the comparison among low,medium and high dose groups was also statistically significant,and the inhibition rate of Jianpi Anticancer Prescription high dose drug containing serum group was the highest of proliferation cell.2.2 Effect of Jianpi Anticancer Prescription containing serum on CT26 WT cell cycle:The ratio of low,medium and high dose Jianpi Anticancer Prescription containing serum on CT26 WT cells in G1 phase of cell cycle was50.22%,52.94% and60.32% respectively.The ratio of G1 phase in capecitabine group was 64.99% of cell cycle,which was significantly higher than 47.56% in the control group(P<0.05).And cell cycle arrest increased with the increase of drug dose.2.3 Effect of Jianpi Anticancer Prescription containing serum on apoptosis of CT26 WT cells:After treatment with Jianpi Anticancer Prescription,12.74% cells were induced inhigh-dose Jianpi Anticancer Prescription group,12.16% cells were induced in medium-dose group,9.46% cells were induced in low-dose group,and 13.82% cells were induced in capecitabine group.Compared with the control group,there were significant differences.2.4 Effect of Jianpi Anticancer Prescription containing serum medicated serum on CT26 WT cell migration:The number of transmembrane cells in different doses of Jianpi Anticancer Prescription containing serum group was(46.00±1.87)、(37.40±1.95)、(37.00±2.83)respectively,which decreased significantly compared with the control group(67.80±8.93)(P<0.05),and the number of transmembrane cells decreased significantly with the increase of drug concentration.3 Effect of Jianpi Anticancer Prescription on general state and the expression of VEGF,MVD and PKC in CT26 WT colorectal cancer cell transplantation tumor.3.1 General state and weight changes of different group:Two weeks after administration,compared with the control group,the mice in the Jianpi Anticancer Prescription group with different doses(low、medium、high)had better mental state,better activity and there was no obvious abnormality in color,especially in the high-dose group of Jianpi Anticancer Prescription.showing that different dose of Jianpi Anticancer Prescription maintained the state and physical strength of mice to a certain extent after treatment.The mice in capecitabine group had poor spirit,more obvious weight loss and dark skin color worsely;In terms of diet,the amount of food in capecitabine group decreased,while in the terms of different dose of Jianpi Anticancer Prescription had a better appatite.In terms of body weight,there was no difference in the body weight of mice in each group before modeling.After treatment,the body weight of each group decreased compared with that of D0 day,especially the mice of control group and capecitabine group(P<0.05).The differences of body weight before and after treatment in different concentration groups of Jianpi Anticancer Prescription is smaller than that in the control group,indicating that capecitabine group has an obvious effect on weight loss of mice,while Jianpi Anticancer Prescription group can stabilize the weight of mice to a certain extent.3.2 Effect of Jianpi Anticancer Prescription on tumor inhibition rate:The growth of transplanted tumor in mice was inhibited by different doses of JianpiAnticancer Prescription,and the average tumor weight in high and medium dose groups was significantly lower than that in the control group(P<0.05),especially in the high dose group of Jianpi Anticancer Prescription.There was no significant difference between the low dose group of Jianpi Anticancer Prescription and the control group.3.3 TUNEL staining results of transplanted tumor tissues of mice in each group:Apoptotic cells detected by TUNEL method showed nuclear pyknosis,brighter fluorescence,round,crescent or irregular.Microscopic results showed that the apoptotic cells in each dose group of Jianpi Anticancer Prescription(low、medium、high)were significantly higher than those in the control group.3.4 Detection results of CD34 labeled MVD in tumor tissues of different groups:The results showed that the expression of CD34 in the control group was(60.67±10.50),and the low,medium and high dose groups of Jianpi Anticancer Prescription were(44.24±8.00)、(39.26±8.57)、(28.83±9.81),which was significantly lower than that in the control group(P< 0.05).The results suggest that Jianpi Anticancer Prescription(low、medium、high)can inhibit the angiogenesis of transplanted tumor mice.3.5 Effect of Jianpi anticancer formula on angiogenesis of transplanted tumor in mice:The results showed that the optical density value of VEGF in the control group was(56.73±5.03),and that in the low,medium and high Jianpi Anticancer Prescription groups were(45.50±7.19)、(32.23±2.33)、(40.59±2.76),which were statistically significant compared with the control group(P < 0.05).It can be seen that the inhibition of Jianpi Anticancer Prescription medium dose group was the most significant.At the same time,for the effect of PKC,after the action of Jianpi Anticancer Prescription(low、medium、high),the expression of PKC decreased significantly(low、medium、high)compared with the control group(P < 0.05),and the inhibition effect of high-dose group was the most obvious.The preliminary results show that Jianpi Anticancer Prescription(low、medium、high)may play an anti angiogenesis role by inhibiting the expression of VEGF and PKC.4 Effect of Jianpi Anticancer Prescription on ERK/MAPK signal pathway and downstream pathway related proteins:4.1 Effect of Jianpi Anticancer Prescription(low、medium、high)on protein expression c-myc:The results showed that the protein expression level of c-myc decreased in varying degrees after treatment with different doses of Jianpi Anticancer Prescription(low、medium、high),which was statistically significant compared with the control group(P<0.05).The decrease in the medium dose group of Jianpi Anticancer Prescription was the most significant.At the same time,there was no significant difference between the medium and high dose groups.4.2 Effect of Jianpi Anticancer Prescription on protein expression of MMP2:The results showed that compared with the control group,the low and medium dose groups of Jianpi Anticancer Prescription could reduce the expression of MMP-2Protein,which was statistically significant(P<0.05).4.3 Effect of Jianpi Anticancer Prescription on the expression of RAS,ERK and pERK proteins in ERK/MAPK signal pathway:Compared with the control group,the low and medium dose groups of Jianpi Anticancer Prescription of pathway can reduce the expression of Ras protein,with statistical difference(P<0.05).At the same time,there was no significant changes in ERK protein content of pathway in each group(low、medium、high),and there was no significant difference between them.However,the phosphorylated form of p-ERK decreased significantly with the increase of the concentration of pathway,especially at medium and high doses of Jianpi Anticancer Prescription.on ERK/MAPK signal pathway and downstream pathway related proteins.Conclusion:1 Based on Network pharmacology,this paper preliminarily explores the main active components and potential molecular mechanism of Jianpi Anticancer Prescription in the treatment of colorectal cancer,mainly involving cell proliferation,cell apoptosis and cell angiogenesis,and MAPK signal pathway,and so on.2 In vitro experiments preliminarily confirmed that the serum containing of Jianpi Anticancer Prescription can inhibit the proliferation of CT26 WT cells,block the cell cycle in G1 phase,promote apoptosis,and inhibit the migration ability of colorectal cancer cells in a dose-dependent manner as well as.3 In vivo experiments show that Jianpi Anticancer Prescription has a good antitumor effectand maintains the state of mice,physical strength and weight of mice to a certain extent;At the same time,Jianpi Anticancer Prescription can inhibit the expression of VEGF,MVD and PKC.4 In vivo experiments further showed that Jianpi Anticancer Prescription may block the expression of Ras and p-ERK of ERK/MAPK signal pathway,reduce the expression of downstream MMP2 and c-myc protein,and finally inhibit the cell proliferation,cell angiogenesis and cell migration of CT26 WT cells. |
PDF Full Text Request