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OBJECTIVE: Tumor is one of the major diseases that threaten human health, chemotherapy is the most important means for the treatment of tumors. However, the generation of multidrug resistance of tumor cells (multidrug resistance, MDR) is a major cause of failure in cancer chemotherapy. How to reverse multidrug resistance problems to be solved to improve the efficacy of chemotherapy. Lead to MDR for many reasons, including the P-glycoprotein-mediated multidrug resistance is the most in-depth study. P-glycoprotein as a target for the development of multidrug resistance reversal agents. A variety of traditional Chinese medicine extracts prepared in this experiment, high expression of P-glycoprotein is characterized by the use of Caco-2 cells, the establishment of Caco-2 cell high-throughput screening method, screening can effectively inhibit P-glycoprotein from different Chinese herb extracts \The outer pump \And preliminary validation of several positive drug inhibition of P-glycoprotein. Methods: (1) cultured Caco-2 cells, Rho-123 as a fluorescent probe to establish a high-throughput screening of inhibitors of P-glycoprotein medicine Caco-2 cell model. Initial screening of the positive Chinese medicine inhibit P-glycoprotein and Rho-123 fluorescence detection of intracellular volume changes; (2) screening obtained several positive Chinese herb extracts on P-glycoprotein inhibition verify: Rho -123 accumulation of experimental evaluation of drug inhibition of the outer pump function of the P-glycoprotein (multifunctional microplate reader two means of detection and flow cytometry); MTT method. detection of drugs combined with ADM on cell proliferation impact; the intracellular ADM volume of flow cytometry drugs. Results: (1) the establishment of Caco-2 cell model of P-glycoprotein inhibitors from traditional Chinese medicine in a high-throughput screening, initial screening of extracts from 17 Chinese herbs P-glycoprotein inhibitors, and to determine to L1, D2, S3 extract the object of the study follow-up experiments; (2) found by MTT different concentrations L1, D2, S3 extract after the Caco-2 cells, cell inhibition rate increased with the increase in the concentration, and when L1 of, D2 extract at the concentrations LT.; 0.125μg/mL and S3 extract at a concentration of LT; 0.0625μg/mL when not on cell proliferation have a significant impact, non-cytotoxic dose. (3) Rho-123 accumulates experiment proved L1, D2, S3 extract can increase the fluorescence intensity of intracellular Rho-123. Multifunctional microplate detection found that with the Chinese medicine extract concentration increased, the fluorescence intensity within the cell also increases, when the L1, D2 concentration of 62.5ng/mL, 125ng/mL, can be significantly increased cell accumulation within Rho-123 (P-LT; 0.05), S3 under 31.25ng/mL, 62.5ng/mL concentration, can be very significant increase in the accumulation of intracellular Rho-123 (P LT; 0.01). Flow cytometry showed 125ng/mL the L1 intracellular fluorescence intensity of Rho-123 compared to negative role of the D2 extract significantly enhanced 62.5ng/mL concentration of S3 also increase the intracellular fluorescence intensity of Rho-123 . (4) the MTT assay found 62.5ng/mL, 125ng/mL L1 D2, 31.25,62.5 ng / mL S3 extract objects with the combined effects of anticancer drugs ADM significantly increased ADM inhibition of cell proliferation, increased chemosensitivity. (5) 125ng/mL of L1, D2, after 62.5ng/mL S3 extract of cells and increase in the amount of accumulation of intracellular ADM. Conclusion: Caco-2 cell model is a high-throughput screening of inhibitors of P-glycoprotein medicine effective model. L1, D2, S3 extracts significantly inhibited the Caco-2 cells of the P-glycoprotein, can significantly improve the sensitivity of the Caco-2 cells to anticancer drugs. Tip L1, D2, S3 may be multidrug resistant P-glycoprotein-mediated effective reversal agents.
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