Anti-proliferation And Antioxidant Effects Of Nrf2/ARE Pathway In Colon Cancer Cells And DSS Colitis Mice Models By Matrine,Oxymatrine And A TCM Decoction

Objective:The objective of this study is to explore the anti-proliferationand antioxidant effects of QYJD and its main compounds MT,OMT in human colorectal cancer cells andto study the anti-oxidative stress activitiesof QYJD in dextran sulfate sodium(DSS)colitis mice models focusing on the activation of Nrf2/ARE signaling pathway.Methods:QYJD was provided by Kangmei Pharmaceutical Co.Ltd.It is an aqueous extract of a Chinese herbal and freeze-dry into powder.MT(10mg/ml)and OMT(10mg/ml)were purchased from SIGMA.QYJD was test by HPLC to confirm the content of MT and OMT.The cell viabilities were tested by MTS.HT29 cells cloning efficiency were tested in the soft agar growth assay.Transcriptionaland translational levels of treatments on Nrf2/ARE pathway were studied by assessing expression of Nrf2 related proteins and RNAs(Nrf2,HO1,KEAP1 and NQO1)in HT-29 cells by Western blot and Quantitative Real-time PCR(qPCR).HepG2-C8 cells were used for AREs luciferase reporter assay.Annexin-v-PI staining was used for apoptosis analysis.In vivo study,mice were treated with 1.2%DSS and randomized to receive either a control diet or a 1,2,and 4 g/kg QYJD diet during the study period.On day 42 of the experiment,half the colon was processed for scraping the epithelial cells and the other half was processed for histopathological examination and further evaluations.Results:We found that QYJD treatment effectively inhibited the proliferation of LS-174t and HT-29 cells in a dose-and time-dependent manner.Furthermore,MT can also inhibit the proliferation of HT-29 cells while OMT treatment did not show such an effect.Specifically,only QYJD treatment inhibited colony formation of HT29 cells in the soft agar growth assay.We also found that QYJD and MT treatment induced luciferase activity in the ARE luciferase reporter assay on HepG2-C8 cells.Nrf2 and HO-1 protein expressions in HT29 cells were up-regulated by QYJD and MT treatment.No regulating actions showed in the OMT treatmentQYJD and MT up-regulate the Nrf2(Fig.4A),HO-1(Fig.4C)mRNA expressions in this study.The mRNA levels of Nrf2 increased 1.8,2.1,3.2 fold in QYJD 1.25,2.5,5 mg/ml treatment and 1.3,1.7,2.2 fold in MT 0.2,0.4,0.8 mM treatment.The HO1 mRNA levels increased 1.3,1.9,5.5 fold in QYJD 1.25,2.5,5 mg/ml treatment and 1.0,1.4,3.1 fold in MT 0.2,0.4,0.8 mM treatment.Additionally,5mg/ml QYJD up-regulate the NQO1 mRNA expression 3.0 fold.All treatment did not show a regulating action on KEAP1 mRNA expressions.In vivo study,colonic shortening,disease activity index(DAI)change and splenomegaly were also attenuated by administration of QYJD.As a similar results with vitro study,QYJD up-regulate Nrf2,HO-1 RNA expressions in the epithelial cells of DSS mice models.What's more,QYJD reduced the inflammation and Nrf2 expressions in parattin-embedded tissue slides.Conclusions:Our results suggest that the anti-tumor and the antioxidant effect of QYJD and Matrine in vivo and the anti-inflammatory action of QYJD in vitro could,at least in part,involve the Nrf2/ARE pathway.