Research On The Reversal Effect And Mechanism Of Jianpi Jiudu Recipe On Drug Resistance Of The Colorectal Cancer Cell HCT8/5-FU

Objectives:This study aims to verify the effect of Jianpi jiedu（JPJD）recipe on reversing the drug resistance of colorectal cancer cell line HCT8/5-FU to5-FU in vitro experiment.The specific molecular mechanism of its reversal of drug resistance was discussed based on the metabolomics research strategy.Methods:1.The HCT8/5-FU cells were divided into four groups as follows:control,5-FU group,JPJD group and 5-FU+JPJD group.The proliferation ability of colorectal cancer cells in each group was detected by CCK8 assay,the cell cycle and apoptosis rate of each group were detected by flow cytometry.The activity of P-gp transporter and the accumulation of intracellular chemotherapeutic drugs were measured by rhodamine 123experiment.2.Differential metabolites were screened by LC-MS non-targeted metabolomics technology,and then the differential metabolites were confirmed according to public databases such as Metlin and HMDB,as well as self-built databases.Then,pathway annotation was performed by integrating HMDB database and KEGG compound database.The main metabolic pathways involved in the reversal of 5-FU resistance of HCT8/5-FU cells by JPJD recipe were analyzed.3.The quantitative ELISA,Microwell plate assay,colorimetry assay and fluorescence staining were performed to determine the changes of key transporters,enzymes and products in the most important metabolic pathways.To elucidate the molecular mechanism of JPJD recipe in reversing the drug resistance of colorectal cancer cell line HCT8/5-FU to5-FU.Results:1.The results of cell biological function test suggest that:（1）JPJD recipe can inhibit the proliferation of HCT8/5-FU cells in a concentration-dependent manner.796.16μg/ml JPJD recipe has a cell inhibition rate of20%,which can be used as a non-toxic dose;A series of concentration gradients of 5-FU individually or in combination with JPJD recipe acted on HCT8/5-FU cells,the IC₅₀ value of HCT8/5-FU cells was 87.63μg/ml and 385.1μg/ml respectively,it was calculated that the reversal fold（RF）of JPJD recipe on HCT8/5-FU cells was 4.39;（2）The results of flow cytometry showed that the apoptosis rate and the proportion of cells in G2/M phase treated with 5-Fu+JPJD recipe were significantly higher than those in 5-FU individually（P<0.05）;（3）The results of rhodamine 123assay showed that the accumulation of rhodamine 123 in HCT8/5-FU cells was lower than that in parental cells HCT8,and the difference was statistically significant（P<0.001）.However,JPJD recipe treatment could increase the accumulation of rhodamine 123 in HCT8/5-FU cells,and the difference was statistically significant（P<0.01）.2.Results of metabolomics analysis showed that:（1）Compared colorectal cancer drug resistant cell line HCT8/5-FU with the parent cell line HCT8,the differential metabolites were mainly enriched in three drug resistance-related pathways:ABC transporters,m TOR signaling pathway,and Glutathione metabolism;（2）Compared the 5-FU group of HCT8/5-FU cells with the 5-FU+JPJD recipe group,the differential metabolites are mainly enriched in the three drug resistance-related pathways,Glutathione metabolism,m TOR signaling pathway and Apoptosis.Among them,glutathione metabolic pathway has the smallest P value,so we believe that JPJD recipe can reverse the resistance of colorectal cancer drug-resistant cells to 5-FU mainly by affecting the glutathione metabolic pathway.3.Based on the results of metabolomics experiments to explore the mechanism of JPJD reversing 5-FU resistance in HCT8/5-FU cells,the corresponding experimental results showed that:（1）the expression level of X-CT in HCT8/5-FU cells was significantly higher than that in parental HCT8 cells,and JPJD recipe treatment could significantly reduce X-CT expression in HCT8/5-FU cells;（2）The determination of total GSH content in HCT8/5-FU cells showed that compared with the control group,the GSH content in JPJD group,5-FU group and 5-FU+JPJD group decreased significantly,while the GSH level in 5-FU+JPJD group decreased obviously compared with 5-FU group（P<0.05）;（3）The results of cytoplasmic ROS content in HCT8/5-FU cells showed that compared with the control group,the ROS level in JPJD group,5-FU group and 5-FU+JPJD group increased significantly,while the ROS level in 5-FU+JPJD group increased obviously compared with 5-FU group（P<0.05）.Conclusions:1.JPJD recipe could reverse 5-FU resistance in HCT8/5-FU cells.2.JPJD recipe may reverse the drug resistance of HCT8/5-FU cells by inhibiting the expression of X-CT protein and down-regulating the level of GSH in HCT8/5-FU cells,resulting in the increase of intracellular 5-FU concentration and ROS level,inducing tumor cell apoptosis and cell cycle arrest.