| Objective:1.To investigate the effect of nitidine chloride(NC)on the treatment of colon colitis induced by DSS and its relation to miR-31.2.Explore the molecular mechanism of the protective effect of nitidine chloride on DSS induced mouse colitis by targeting miR-31.Methods:1.1% DSS(Dextran Sulfate Sodium)was used to induce mouse colon colitis.Thirty C57BL/6 male mice were randomly divided into four groups: normal control group(Control group,n = 7),DSS group(DSS group,n = 8),DSS + nitidine chloride group(7.27mg/kg)(DSS+NC group,n = 8)and nitidine chloride group(NC group,n = 7).DSS was given in drinking water,while nitidine chloride was applied through oral gavage.The modeling lasts for 3 weeks.The mouse in DSS group and DSS + nitidine chloride group were given 1% DSS for the first week,then water the second week followed by 1% DSS the third week.Nitidine chloride in DSS + nitidine chloride group and nitidine chloride group was given by oral gavages during the third week.After the animal experiment,(1)The colitis related disease activity index(disease activity index,DAI)were observed;(2)the colon of the mouse were taken,part of them were kept at-80? at once for miRNA and protein expression test,the rest of them were used for histopathological purpose.After measuring the length of the colon,the colons were fixed with neutral formalin,embedded with paraffin,and processed to slides;(3)colonic histopathological scores were recorded by H&E staning;(4)at the same time,the mouse spleen was taken and weighted to estimate the change of spleen index;(5)qRT-PCR was used to detect the expression of miR-31 in colonic tissue;(6)the expression of protein SATB2,NF-?B,COX-2,Bax and Bcl-2 in colonic tissue were detected by Western Blot;(7)immunohistochemistry(IHC)was used to detect the expression of SATB2,NF-?B p65 and COX-2 protein in mouse colon tissues;(8)in situ end-transferase labeling TUNEL was used to detect apoptosis of mouse colon tissue.2.HCT116 cells were transfected with miR-31-5p mimic and miR-31-5p inhibitor using liposome transient transfection,and then grouped into: normal control group(Control group),nitidine chloride group(NC group),miR-31-5p mimic group,miR-31-5p mimic+NC group,miR-31-5p inhibitor group,miR-31-5p inhibitor+NC group.The final concentration of NC was 10 mM.The expression of NF-kB,SATB2,Bax and Bcl-2 protein in 6 groups of cells was detected by Western Blot.Results:1.During the modeling process,the body weight of the control group and the NC group did not decrease,and no occult blood was observed;the body mass of the DSS group decreased significantly,and the occult blood began to be observed on the second day,and the bloody stool appeared in the third week.The DAI scores of the DSS group were higher than those of the control group(P<0.01).The body mass and blood in the DSS+NC group were not different from that of the control group.Compared with the DSS group,the body mass and blood in the DSS+NC group were lighter than those of the DSS group(P<0.01).2.The colon was cut along the longitudinal line and the length was measured.The results showed that compared with the control group,the colon length of the DSS group was significantly shorter(P<0.01).the colon length of the NC group was not different from that of the control group.The colon length of the DSS+NC group was not different from that of the control group.Compared with the DSS group,the colon length of the DSS+NC group was significantly increased(P<0.01).3.The colonic H&E staining results of the four groups of mice showed that the colonic tissue crypts of the control group and the NC group were neatly arranged,the epithelium was intact,and the colonic mucosa was intact.In the DSS group,under the microscope,the infiltration of obvious inflammatory cells in the mucosa and submucosa were observed.The intestinal wall was thickened,the crypts were disordered,and the epithelium and crypt were damaged.The damage of colonic mucosa in the DSS+NC group was lighter than the DSS group,and the colonic mucosa was almost intact.The histopathological scores of each group are as follows: the histopathological scores of DSS group were significantly higher than those of the control group(P<0.01).The histopathological scores of DSS+NC group were not different from that of the control group.4.The spleen weight and spleen index in the DSS group increased significantly compared with the normal control group(P<0.01).After treatment with NC,both spleen mass and spleen index of DSS+NC group were not different from that of the control group;Compared with the DSS group,spleen mass and spleen index of DSS+NC group decreased(P<0.05).5.The results of qRT-PCR showed that compared with normal control group,the expression of miR-31 in colonic tissue of DSS group was elevated than that of normal control group(P<0.01),the expression of miR-31 in DSS+NC group was not different from that of the control group.Compared with DSS group,the expression of miR-31 was decreased after treatment with nitidine chloride(P<0.05).6.Western Blot showed that compared with the control group,the expression of NF-kB p65 and COX-2 in the colon tissue of the DSS group was higher than that of the control group(P< 0.01),the expression of SATB2 was lower than that of the control group(P< 0.01),the ratio of Bcl-2/Bax in the DSS group was lower than that in the control group(P<0.01);After treatment with NC,the expressions of NF-kB p65 and COX-2 in DSS+NC group were significantly decreased(P<0.05),the expression of SATB2 was significantly increased(P<0.05),and the ratio of Bcl-2/Bax was significantly increased(P<0.01).7.Immunohistochemistry results showed that SATB2,NF-kB and COX-2 were expressed in colon tissue of each group.In the DSS group,the expression of NF-kB p65 was significantly increased in the nucleus(P<0.01),the expression of COX-2 in cytoplasm was significantly increased(P<0.01);the expression of SATB2 in the DSS group was lower than that in the normal control group(P<0.01),which was mainly expressed in the cytoplasm.After treatment with NC,the expression of NF-kB and COX-2 in DSS+NC group was significantly decreased(P<0.01);the expression of SATB2 was significantly increased(P<0.01).8.The TUNEL results of mouse colon tissue apoptosis showed that the positive rate of apoptotic cells in the DSS group was significantly increased compared with the control group(P<0.01).After the treatment with NC,the apoptosis of the DSS+NC group was significantly reduced.(P<0.01).9.After miR-31 knocked down or overexpressed,Western Blot was used to detect SATB2 protein expression.Compared with the control group,the expression of SATB2 was decreased after overexpression of miR-31-5p(P<0.01).After administration to NC,the expression of SATB2 was higher than that of miR-31-5p mimic group;When miR-31-5p was inhibited,SATB2 expression was increased compared with control group(P<0.01);when treated with NC after inhibition of miR-31-5p,SATB2 expression was also higher than that of the control group(P<0.01).Expression of NF-kB protein: compared with the control group,NC inhibited the expression of NF-kB(P<0.05),and the expression of NF-kB in the miR-31-5p mimic group increased(P<0.05).After NC treatment,the expression of NF-kB was lower than that of miR-31-5p mimic group(P<0.01);In the miR-31-5p inhibitor + NC group,the expression of NF-kB was lower than that of the control group(P<0.01).Bcl-2/Bax ratio expression results: Compared with the control group,the Bcl-2/Bax ratio decreased in the NC group(P<0.05),and the Bcl-2/Bax ratio in the miR-31-5p mimic group increased(P<0.05).After NC treatment,the ratio of Bcl-2/Bax in miR-31-5p mimic+NC group decreased(P<0.01);in the miR-31-5p inhibitor +NC group,the Bcl-2/Bax ratio was significantly decreased(P <0.01).Conclusion:1.Nitidine chloride has obvious therapeutic effect on DSS induced mouse colitis,and its anti-inflammatory mechanism is related to the down-regulation of miR-31 expression.2.Nitidine chloride relieved inflammatory response through targeting miR-31 to reduce the apoptosis of epithelial cells. |
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