ABSTRACT
AIM:To investigate the change of intestinal flora distribution and its relationship with interleukin -23(IL-23)/IL-17 axis in ulcerative colitis(UC)patients.METHODS:The fresh fecal samples from 20 patients with ac-tive UC and 20 healthy controls were collected.The distribution of the flora was analyzed by direct smear and traditional bacterial culture.The changes of bacteria were detected by real-time PCR.The hemoglobin,albumin,erythrocyte sedimen-tation,and C-reactive protein levels were tested routinely.Both normal and damaged mucosal tissues of UC patients were examined and obtained by colonoscopy,and further assessed by Mayo scoring,Baron grading and HE staining.The expres-sion of IL-17 and IL-23 was observed by immunohistochemistry and Western blot.RESULTS:(1)The degree of flora im-balance in active UC patients was higher than that in the healthy controls(P<0.05).(2)The results of aerobic culture showed that the number of Escherichia coli in the UC patients was significantly lower than that in the normal controls(P<0.01),while Enterococcus was increased obviously(P<0.01).The results of anaerobic culture revealed that the numbers of Bacteroidetes,Bifidobacterium bifidum and Lactobacilli in the UC patients were significantly decreased(P<0.01).(3) Quantitative analysis of target bacteria showed that the relative quantification of Escherichia coli,Bacteroidetes,Bifidobacte-rium bifidum and Lactobacilli in the UC patients was significantly lower than that in the normal subjects,and the number of Enterococcus was significantly increased(P<0.01).(4)Compared with control group,no significant change of hemoglo-bin in the UC patients was ovserved,albumin was significantly decreased(P<0.05), but erythrocyte sedimentation and C-reactive protein levels were elevated obviously(P<0.01).(5)The Mayo score, Baron grade, and histopathological score were all increased(P<0.01).(6)High IL-17 and IL-23 expression levels were detected in the UC patients(P<0.01).(7)Correlation analysis showed that the average absorbance values of IL -17 and IL-23 expression were positively correlated with Baron grade(r=0.717,P=0.02;r=0.849,P=0.016)and pathological score(r=0.660, P=0.03;r=0.675,P=0.032).Meanwhile, the average absorbance value of IL-23 expression was negatively correlated with the number of Escherichia coli(r =-0.699, P =0.025), and positively correlated with Enterococcus(r =0.872, P =0.010).Furthermore,the average absorbance value of IL-17 expression was positively correlated with Enterococcus(r=0.764,P=0.046),and both of them were not correlated with other bacteria.CONCLUSION: Obvious flora imbalance exists in active UC patients,changed intestinal microflora is closely related with the degree of inflammation.IL-23/IL-17 axis,as a key factor in the development of UC,may be related to the changes of intestinal microflora.The interaction be-tween intestinal microflora and IL-23/IL-17 axis plays an important role in the pathogenesis of UC.
ABSTRACT
This study is to observe the effect of N-(3-phenylallylidene)-6-fluoro-1, 8-(2, 1-propoxy)-7-(4-methylpiperazin-1-yl)-quinolin-4(1H)-one-3-carbonyl hyarazine (FQ16) on apoptosis of hepatocarcinoma SMMC-7721 cells in vitro. With different concentrations of FQ16 at different times used to treat SMMC-7721 cells in vitro, the proliferation of the cells and the inhibition effect of FQ16 on the cell proliferation were examined by MTT assay. Cell apoptosis was determined by Hoechst 33258/PI fluorescence staining, TUNEL and agarose gel electrophoresis method. The effect of FQ16 on topoisomerase II activity was measured by agarose gel electrophoresis using Plasmid pBR322 DNA as the substrate. Mitochondrial membrane potential (MMP, delta psi m) was measured by high content screening image system. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression changes of Bcl-2 mRNA and Bax mRNA. The caspase-9, caspase-8, caspase-3, p53, Bcl-2 and Bax protein expressions were detected by Western blotting analysis. The results showed that the cell proliferation was inhibited by FQ16 at 0.625 - 10 micromol L(-1) in a time-dose dependent manner. Treatment of SMMC-7721 cells with different concentrations of FQ16 for 24 h increased the percentage of the apoptosis cells obviously (P<0.05), the typical ladder DNA in apoptotic cells and a concomitant dissipation of the mitochondrial membrane potential. Compared with control group, FQ16 influenced obviously DNA topoisomerase II activity, stimulated DNA cleavage and inhibited DNA reunion mediated by topoisomerase II. In addition, FQ16 (3 - 7.39 micromol L(-1)) increased mRNA expression of Bax and protein expression of p53, Bax, caspase-9, caspase-3, separately, and induced cytosolic accumulation of activities caspase-9 and caspase-3, whereas the mRNA and protein expression of Bcl-2 decreased with no change of caspase-8. Therefore it can be concluded that the effects of inhibited topoisomerase II and mitochondrial-dependent pathways were involved in FQ16 induction of apoptosis of SMMC-7721 cells.