The Preventive Effects And Mechanism Research Of "Coptis Chinensis And Dried Ginger" For Inflammatory Bowel Disease And Related Colon Cancer

ObjectiveInflammatory bowel disease is a chronic relapsing inflammatory bowel disease,mainly includes Crohn’s disease(CD)and ulcerative colitis(UC),the main symptoms are abdominal pain,diarrhea,mucus and purulent blood.About half of the severe cases need operation cure,and are often associated with serious complications.Sustained chronic inflammation can also increase the risk of colorectal cancer,seriously affect the patients’ life quality.Recent studies had shown that the risk of colorectal cancer with intestinal inflammation is closely relative to the severity and duration.Coptis chinensis has a prominent advantage in treatment of gastrointes--tinal tract inflammation and colon cancer.But coptis chinensis belong to bitter and cold medicine and long-term use can bring some side-effects.The compatibility of"coptis chinensis and dried ginger" in TCM clinical medicine embodies the the integration of typhoid fever and blast prescription,which can ease the long-term application experience medicine for the treatment of chronic diseases have a series of adverse reactions.In this research,we adopted DSS induced colitis in mice model,macrophage RAW264.7 cells and HCT116 and HT29 colon cancer cell line as the research objects,to explore the preventive effects and mechanism of "coptis chinensis and dried ginger" for inflammatory bowel disease and its effects and mechanism of promoting apoptosis of colon cancer cells.Methods1.The main chemical composition research of "coptis chinensis and dried ginger"According to the 1:1 proportion of coptis chinensis and dried ginger,400g herbs were soaked in cold water for 1 h with 8 times amount water.After 1 h micro boiling reflux,pour out the liquid medicine,and then plus seven times the amount of water,boiling reflux 30 min,pour out the liquid medicine,merging two liquids,filters,the reduced pressure concentration,freeze drying,weighing producing weight is 31.8g.Taking 1 g of the above said all producing,dissolved with 20.96 ml ddH20,centrifuged with 14000 rpm for 30 min,taken the supernatant which contains 0.6 g/ml technical solution.According to the literature,establish two herbs coptis chinensis and dried ginger chemical composition database.Establish "coptis chinensis and dried ginger" liquid ms(UPLC/Q-TOF-MSS)analysis method,and using the method of"coptis chinensis and dried ginger" chemical composition were analyzed.According to the chromatog--raphic retention time,accurate molecular weight,molecular fragment peaks,reference substance information and self-built two flavor formula medicinal chemistry information database,the main source of chromatographic peak of belonging and identification,and quantitative determination of berberine in aqueous extract and the content of 6-gingerol.2.The prevent i ve effects and mechan i sm reseach of "copt i s ch i nens i s and dr i ed ginger" for DSS induced colitis in mice45 C57BL/6 male mice were randomly divided into blank group,model group,low dose Chinese traditional medicine(1.2 mg/(kg · d))group,middle dose Chinese traditional medicine dose(2.4 mg/(kg · d))group and high dose Chinese traditional medicine(4.8 mg/(kg · d))group.Chinese medicine groups preventive drug lavage after 7d(blank group and model group by gastric equivalent physiological saline),continues to give corresponding intervention at the same time,the model group,Chinese medicine groups mice were given 3%DSS(Dextran Sulfate Dodium)aqueous solution free drink,change once every 2d,continuous drinking 7d,blank group mice were given free drinking tap water.Disease Activity Index(DAI)scores were record daily.The mice were sacrificed on day 7,and the colons(from the ileocecal junction to the anal verge)were removed and the length was measured.Colons were collected for HE staining,ELISA,immunofluorescence and Western blot.Pathological and histological was calculated.The level of IL-6 and TNF-α in the plasma and the phosphorylation level of NF-κB,JAK2 and STAT3 were detected.3.The preventive effects and mechanism reseach of "coptis chinensis and dried ginger" for LPS induced RAW264.7 cell inflammatory reactionDetected the killing effect of 0,0.125,0.25,0.125,1,2,4,8mg/ml "coptis chinensis and dried ginger" water extract for macrophage RAW264.7 cell by MTT assay.Preventively gave "coptis chinensis and dried ginger" aqueous extract(0,0.5,1,2 mg/ml)for 1 h and then gave LPS stimulation for 8 h,detected the levels of IL-6 and TNF-α in medium by ELISA.Western blot detected the phosphorylation levels of STAT3,and immunofluorescence method detected P-STAT3 and nf-kappa B expression level in RAW264.7 cells.4."Coptis chinensis and dried ginger" on the function and mechanism of HT29 and HCT116 colorectal cancer cell lineDetected the killing effect of 0,1,2,4,6,8,15 mg/ml "coptis chinensis and dried ginger" water extract for HT-29 and HCT116 cell by MTT assay.Respectively gave "coptis chinensis and dried ginger" water extract(0,1,1.5 mg/ml)for HT-29 and HCT116 cells for 48 h,flow cytometry instrument detected the apoptosis rate by PI/Annexin V-FITC double staining method,Western blot method detected Cleaved Caspase3 protein expression level and the level of STAT3 protein phosphorylation.Results1.The main chemical composition research of "coptis chinensis and dried ginger""Coptis chinensis and dried ginger" medicine is established on the analysis of fluid mass combination method,and use the method successfully analysed the chemical composition of "coptis chinensis and dried ginger"medicine.According to the chromatographic retention time,accurate molecular weight,molecular fragment peaks,reference substance information and self-built two flavor formula medicinal chemistry information database,the main source of chromatographic peak of belonging and identification,ownership and identified 28 main chromatographic peak。At the same time,the quantitative detection of the concentration of berberine was 54.43 mM,6-gingerol drug concentration was 2.2 mM.2.The preventive effects and mechanism reseach of "coptis chinensis and dried ginger" for DSS induced colitis in miceAfter 2 days of drinking 3%DSS,the DAIs of model group animals were higher than blank group;Compared with model group,the DAI of low dose Traditional Chinese medicine(TCM)group were lower after drinking 3%DSS,but the difference only in 2,4,6 days were statistically significant;The DAI of middle dose TCM group were lower than the model group except the fourth day after drinking 3%DSS,and continued until the end of the experiment;The DAI of high dose TCM group drinking 3%DSS is lower than the model group from the 2nd day and continued until the end of the experiment.The colon length of model group mice significantly reduced than blank group(P<0.01);Compared to model group,low and medium dose TCM group colon length increased(P<0.05),high dose TCM group colon length increase dramatically(P<0.01).Model group colon tissue pathology score signifycantly higher than blank group(P<0.01);Compared to model group,low dose TCM group colon histopathologic grading has no obvious change,when middle dose TCM group colon tissue pathology score lower than model group(P<0.05)and high dose TCM group had a significantly lower colon histopathological grade(P<0.01).In model group,mice plasma IL-6 levels rised sharply than blank group(P<0.01);Compared with model group,each dose TCM group mice plasma IL-6 levels were significantly lower than model group(P<0.01).Compared with the blank group,model group plasma TNF-α content significantly increased(P<0.01);Compared with model group,each dose TCM group mice plasma TNF-αlevels were significantly lower than model group(P<0.01).The NF-κB,JAK2 and STAT3 protein activation levels in colon tissue of model group mice were significantly higher than that of blank group(P<0.01);The phosphorylation levels of colon epithelial cells NF-KB protein in Low and high dose TCM groups mice were significantly lower than that of model group(P<0.01).Compared with model group,the phosphorylation levels of colon epithelial cells JAK2 protein in low and high dose TCM group were significantly reduced(P<0.01),and the phosphorylation levels of colon epithelial cells STAT3 protein in each dose TCM group were significantly reduced(P<0.01).3.The prevent i ve effects and mechan i sm reseach of "copt i s ch i nens i s and dr i ed ginger" for LPS induced RAW264.7 cell inflammatory reactionDetected by MTT assay showed that the IC50 of "coptis chinensis and dried ginger"water extract for RAW264.7 cells for 36h is 3.79 mg/ml,ELSIA result showed that "coptis chinensis and dried ginger "water extract could significantly suppress LPS induced the RAW264.7 cells secretion of inflammatory cytokines of IL-6 and TNF-a.Western blot and immunofluorescence showed"coptis chinensis and dried ginger "can significantly inhibit the activation of the NF-κB and STAT3 protein in RAW264.7 cells induced by LPS.4."Coptis chinensis and dried ginger" on the function and mechanism of HT29 and HCT116 colorectal cancer cell lineDetected by MTT assay showed that "coptis chinensis and dried ginger"water extract can significantly inhibit HT29 and HCT116 cell vitality,and the IC50 of 48h were 5.648 mg/ml and 2.43 mg/ml.Flow cytometry result showed that"coptis chinensis and dried ginger" water extract could significantly promote the apoptosis rate of HT29 and HCT116,Western blot results show that "coptis chinensis and dried ginger" could significantly promote the expression of Cleaved Caspase3 and depress the phosphorylation levels of STAT3 protein in HT29 and HCT116 cells.Conclusion1."Coptis chinensis and dried ginger" could significantly inhibit symptoms and signs of DSS induced colitis in mice model,improve local pathological damage,the inhibit plasma inflammatory factors levels.Its mechanism may be through inhibiting partial colon activation of nf-kappa B signaling pathway and the JAK2/STAT3 signal pathway in DSS induced colitis in mice model.2."Coptis chinensis and dried ginger" can significantly inhibit LPS induced RAW264.7 cell inflammatory factor TNF alpha and IL-6 secretion,which may be through inhibiting the nf-kappa B and JAK2/STAT3 signaling pathways in RAW264.7 cells induced by LPS.3."Coptis chinensis and dried ginger" medicine can significantly inhibit proliferation of HT29 and HCT116 colon cancer cell line and promote apoptosis of HT29 and HCT116 colon cancer cell line,which is probably by inhibiting phosphorylation of STAT3 in HT29 and HCT116 colon cancer cell line.