The Extraction Of Triterpenoids From Rhus Chinensis Mill And Its Anti-colorectal Cancer Mechanism By Affecting The Acidic Environment

Objectives:The aim of this paper is to isolate and purify the triterpenoids from Rhus Chinensis Mill?TER?,and to explore its mechanism of anti-colorectal cancer?CRC?by acting on acidic metabolites and microenvironment of colon cancer.Methods:Petroleum ether,Ethyl Acetate,dichloromethane and N-butanol were used respectively to extract 60%ethanol crude extract of Rhus Chinensis Mill;The fraction with the most peaks in 1-20 min was selected for further gradient elutation with dichloromethane-methanol;MTT assay was used for detecting anti-tumor effect of each eluted component;The sensitivity of normal intestinal epithelial HCoEpiC cells and colon cancer SW620 cells to TER were compared with MTT method;Flow cytometry was used to detect apoptosis of SW620 cells;A mouse subcutaneous tumor model with colon cancer cells was established,and treated with different concentrations of TER,5-Fu was used as a positive control.The tumor volume was measured and the tumor inhibition index was calculated.Mouse tumor tissues were analyzed using H&E staining,Immunohistochemical staining and Tunnel staining to observe the morphological changes of tumor tissues,the expression of nuclear proliferating antigen Ki67 and the apoptosis of tumor tissues in mice which were treated with TER.The glucose uptake rate and lactic acid production rate of SW620cells were determined using assay kits.Also,the invasion of SW620 cells was determined using Matrigel invasion assay.The expression of key glycolytic enzymes,H+exchange-related proteins and acid-sensitive ion channel?ASIC?family members in SW620 cells were detected using Western blot assay.Moreover,MMPs expression in SW620 cells were detected.The expression of MMP-9 protein in tumor tissues was also measured.Results:HPLC results showed that triterpenoids components are mainly exists in the dichloromethane elution site,but there were many impurities in it,and the main peak was not obvious.After further gradient elution with dichloromethane-methanol,the sample 9 had a better peak resolution within all the samples obtained,the main peak in sample 9 was also obvious within 1-20 min.The content of betulinic acid was0.528 mg/mL,and the content of betulonic acid was 0.632 mg/mL.The sample 9was chosen as TER for further study.The results of MTT assays showed that TER could inhibit HCT116 and SW620 colon cancer cells effectively.The IC₅₀ value of inhibiting HCT116 cells was 62.27?g/mL,and the IC₅₀ value of inhibiting SW620cells was 53.31?g/mL.However,there was no significant inhibitory effect on normal intestinal epithelial HCoEpiC cells,and the inhibition rate was only 32%at the concentration of 200?g/mL.In vivo experiments also showed that TER could effectively inhibit the growth of mouse tumors.The results of H&E staining showed that TER can cause damage and necrosis in tumor tissues.The results of immunohistochemistry showed that TER down-regulated the expression of nuclear proliferating antigen Ki67 in tumor tissues;Flow cytometry results showed that TER significantly promoted the apoptosis of SW620 cells at the concentration of 100?g/mL,P<0.01.Again,in the tumor tissues of mice treated with varied concentrations of TER,the number of apoptotic cells was reduced using Tunnel staining assay.TER could significantly inhibit glucose uptake and lactic acid production in SW620 cells?at 100?g/mL concentration,P<0.001?,and down-regulate the expression of key glycoproteins GLUT1,PKM2 and LDHA and H⁺exchange-related proteins MCT1and NHE1,while the expression of ASIC2 and the proteins in the calcineurin/activated T cell nuclear factor?NFAT?pathway were also down-regulated.After treating SW620 cells with TER,the expression of MMP-2 and MMP-9 proteins expression was decreased detected with Western blot assay.Meanwhile,the invasion ability of SW620 cells was decreased using Matrigel invasion experiment.The expression of MMP-9 in tumor tissues of mice treated by TER was down-regulated.Conclusion:Our results demonstrated that TER extracted from Rhus Chinensis Mill can effectively inhibit the growth and invasion of CRC cells by targeting the CRC acidic microenvironment and inhibiting the ASIC2-induced calcineurin/NFAT pathway.TER could be an adjuvant therapy for colorectal cancer.