| Objective: Sophora flavescens radix is the dried root of the legume plant Sophora flavescens Ait.It was originally published in the ‘Shennong Materia Medica’ and is mostly grown in sandy or hillside grasslands.It is distributed throughout the country.In recent years,as wild resources of Sophora flavescens radix have been greatly reduced,artificial cultivated products have gradually become the main source of clinical medicine.Modern pharmacological studies have found that the alkaloids in Sophora flavescens radix have good anti-cancer activity.Among them,Sophoridine has been independently developed by our country as its preparation "Sophoridine Injection".In recent years,it has been reported that sophoridine has the activity of inhibiting colorectal cancer,but the specific mechanism of action is not yet clear.In this study,we investigated the pharmaceutical identification of radix Sophora flavescens and deeply explored the potential target and specific mechanism of sophoridine,the main ingredient of radix Sophora flavescens against colorectal cancer,with a view to the resource utilization of radix Sophora flavescens and anti-colorectal cancer drug development provide reference.Methods: Pharmacological identification of radix Sophora flavescens:(1)Combined with Chinese Flora,Chinese Digital Herbarium,the fourth national survey of traditional Chinese medicine and literature analysis,the pharmacological characteristics and resource distribution of Sophora flavescens were investigated.Study on the molecular effect of sophoridine on colorectal cancer:(1)Using HCT116,SW480 and RKO colorectal cancer cell models,the effects of different concentrations of Sophoridine on cell cloning ability were detected by plate clone formation experiments,and CCK-8(Cell Counting Kit-8)was used to detect different concentrations of Sophoridine effect of proliferation rate.(2)Colorectal cancer SW480 and RKO cells were treated with Sophoridine at different concentrations,and Western blot was used to detect the expression level of apoptosis marker proteins,Bcl-2(B-Cell CLL/Lymphoma 2)and Bax(Bcl-2 Associated X Protein).Flow cytometry was used to detect the apoptotic ratio,and studied the effects of Sophoridine on thecolorectal cancer cell apoptosis.(3)Western Blot and IF(Immunofluorescence Staining)were used to detect the expression level of cell autophagy marker proteins,LC-3(Microtubule Associated Protein 1Light Chain 3)and p62(Sequestosome 1)after treatment with Sophoridine,and the effect of Sophoridine on the colorectal cancer cell autophagy was observed.(4)Colorectal cancer SW480 and RKO cells were treated with Sophoridine at different concentrations,and detect the expression level of cell cycle related proteins,p27(Cyclin-Dependent Kinase Inhibitor 1B)and Cyclin D1(G1/S-Specific Cyclin)by Western Blot.Flow cytometry was used to detect the cell ratio of each cell cycle,and the effect of Sophoridine on the colorectal cancer cell cycle was observed.Research on the potential targets of sophoridine against colorectal cancer:(1)Using network pharmacology to predict the potential target of sophoridine in colorectal cancer.The pharm Mapper database was used to predict the potential targets of sophoridine,and the target set was obtained.Mapping potential drug targets use colorectal cancer-related genes downloaded from the dis Ge NET database.And use Cytoscape software to build a Drug-Target Network(D-T).Obtain the protein interaction list from the String database and build a Protein-Protein Interaction Network(PPI).Biological functions and pathway enrichment are constructed by the Clue GO plugin of Cytoscape software.(2)Detection the binding ability between Sophoridine and MAPKAPK2(MAP Kinase-Activated Protein Kinase 2)by CETSA(Cell Thermal Transformation Analysis)and DARTS(Drug Affinity Response Target Stability).Molecular docking was used to simulate the binding mode of sophoridine and MAPKAPK2.The mechanism of sophoridine against colorectal cancer by acting on potential targets:(1)Ectopic expression of MAPKAPK2 protein by transfecting the MAPKAPK2 plasmid in colorectal cancer SW480 and RKO cells.The expression of MAPKAPK2 was detected by Western Blot.After over-expressing MAPKAPK2,plate clone formation experiments,CCK-8 detection experiments,Western Blot,IF,and flow cytometry experiments were used to detect the effects of MAPKAPK2 on Sophoridine regulating colorectal cancer cell proliferation,apoptosis,autophagy and cell cycle.(2)After using si RNA(Small Interfering RNA)to silence endogenous MAPKAPK2 ofcolorectal cancer SW480 and RKO cells,apply plate clone formation experiment,CCK-8detection experiment,Western Blot,IF and flow cytometry experiments to detect the effects of silenced MAPKAPK2 on Sophoridine regulating colorectal cancer cell proliferation,apoptosis,autophagy and cell cycle.(3)Based on the gene expression profile of colorectal cancer patients in the GEO database,the correlation between MAPKAPK2 and the survival rate of clinical patients and the occurrence of colorectal cancer was analyzed.GSEA(Gene Set Enrichment Analysis)was used to analyze the correlation between MAPKAPK2 and the biological process of colorectal cancer cells.Results: Pharmacological identification of radix Sophora flavescens:(1)Through literatures and field investigations on plant morphology and medicinal traits of Sophora flavescens,the standards for identification of Sophora flavescens traits were summarized,the large-scale distribution of wild resources of Sophora flavescens in China and the distribution of large-scale artificial cultivation bases were counted,and the geographical environment suitable for the growth of Sophora flavescens.Study on the molecular effect of sophoridine on colorectal cancer:(1)The results of plate clone formation experiments showed that Sophoridine significantly inhibited cell cloning ability,and CCK-8 test showed that sophoridine inhibited the proliferation rate of colorectal cancer cells in a dose-dependent manner..(2)Western Blot results showed that Sophoridine significantly decreased the expression of Bcl-2 and increased the expression of Bax.Flow cytometry results showed that Sophoridine significantly induced colorectal cancer cell apoptosis.(3)The results of Western Blot and IF experiments showed that different concentrations of Sophoridine significantly reduced the expression of p62 and increased the expression of LC-3,and promoted colorectal cancer cell autophagy.(5)Western Blot results showed that Sophoridine significantly increased the expression level of p27 and inhibited the expression level of Cyclin D1.Flow cytometry results showed that Sophoridine significantly increased the number of cells in the G0/G1 phase,while reducing the number of cells in the G2/M phase,and has a certain blocking effect on the colorectal cancer cell cycle.Research on the potential targets of sophoridine against colorectal cancer:(1)Combining the predicted by network pharmacology with pathway enrichment analysis,it was found that the MAPK signaling pathway is important in the process of Sophoridine regulating colorectal cancer.MAPKAPK2 may be the potential target for Sophoridine in colorectal cancer.(2)CETSA and DARTS experiments show that Sophoridine can directly bind to MAPKAPK2 protein.Molecular docking simulation results show the best binding mode of Sophoridine and MAPKAPK2,and Sophoridine can specifically bind to the ATP pocket of MAPKAPK2.Western Blot have detected that Sophoridine inhibits phosphorylation at Thr222 of MAPKAPK2.The mechanism of sophoridine against colorectal cancer by acting on potential targets:(1)After overexpression of MAPKAPK2 protein in colorectal cancer SW480 and RKO cells,the results of plate clone formation experiments,CCK-8 detection experiments,Western Blot,IF,and flow cytometry experiments showed that MAPKAPK2 can significantly restore Sophoridine-regulated colorectal cancer cell proliferation,apoptosis,autophagy,and cell cycle.(2)After silencing endogenous MAPKAPK2 of colorectal cancer SW480 and RKO cells with si MAPKAPK2,the results of plate clone formation test,CCK-8 detection experiment,Western Blot,IF and flow cytometry experiments showed that the regulation of si MAPKAPK2 on colorectal cancer cell proliferation,apoptosis,autophagy and cell cycle is similar to that of sophoridine,and even enhances the effect of sophoridine.(3)The analysis of survival information of colorectal cancer patients in the GEO database shows that when the MAPKAPK2 transcription level is high,the patient’s survival time is significantly shortened,and when the MAPKAPK2 transcription level is low,the patient’s survival time is significantly extended.And the transcription level of MAPKAPK2 in colorectal cancer tissues was significantly upregulated compared to normal tissues.GSEA results show that MAPKAPK2 is related to genes that promote cell proliferation and cell cycle,and genes that are related to inhibition of apoptosis and cell autophagy.Conclusion: This study summarizes the standard for identification of traits of Sophora flavescens,and statistics of the large-scale distribution of wild resources of Sophoraflavescens and the distribution of large-scale artificial cultivation bases in China,which provide a reference for the development and utilization of Sophora flavescens resources.Using molecular biology experiments and network pharmacology,the main anti-cancer active ingredient in Sophora flavescens,sophoridine,is the anti-colorectal cancer target of MAPKAPK2.Sophoridine inhibits MAPKAPK2 through targeted binding,regulates the proliferation,apoptosis,autophagy and cell cycle of colorectal cancer cells,thereby inhibiting the occurrence and development of colorectal cancer. |
