| Objective:To clarify the predictive value of Heat Shock transcription Factor 2(HSF2)in mucosal healing of Ulcerative Colitis(UC)by testing HSF2 levels in feces and intestinal mucosa of UC with different Mayo endoscopic subscore(MES).To explore the mechanism of HSF2 in the mucosal damage repair of UC.Recombinant expression of HSF2 protein and verify its biological activity.To be aimed at providing a new noninvasive index for predicting the mucosal healing of UC,explaining the mechanism of HSF2 in mucosal healing and laying a foundation for antibody preparation.Methods:This study is divided into three parts.Part one1.Fecal and mucosal samples were collected from 12 UC patients with MES=0;15 with MES=1,14 with MES=2,10 with MES=3 and 10 in health controls.The concentrations of Fecal HSF2 and Fecal Calprotectin were detected by ELISA.The expression level of HSF2 in the colon mucosa was detected by immunohistochemistry.The correlations between Fecal HSF2 levels and MES,mucosal HSF2 levels and MES,Fecal HSF2 and Fecal Calprotectin levels were compared by Pearson.correlation analysis.2.A total of 231 follow-up UC patients were included in the study.The fecal samples were collected in early morning and colonoscopy was performed in the next day for MES scoring.The concentrations of Fecal HSF2 and Fecal Calprotectin were detected by ELISA.MES ?lwas made as the evaluation standard for mucosal healing.The concentration 1.8ng/ml of Fecal HSF2 was used as a cut-off value to predict mucosal healing.The specificity and sensitivity,positive predictive value and negative predictive value of Fecal HSF2 predicting mucosal healing were calculated by the diagnostic test evaluation method.The predictive accuracy was evaluated byROC curve and contrasted with Fecal Calprotectin.Part two1.In the animal experiment of DSS induced chemical colitis mice treated by methylprednisolone..(1)60 male SPF C57BL/6 mice were equivalently and randomly divided into normal control group,DSS model group,MP treatment group and Placebo group.The DSS module group,MP group and Placebo group were drinking of 3%DSS solutions for 6 days.MP group and Placebo group were given MP or HPMC solution by gavage respectively from day 7 to day 12.The normal control group and DSS model group were drinking deionized water in day 7 to day 12.All the mice were sacrificed and colon samples were collected in 13rd day.(2)The DAI,colon damage score and histopathology score were recorded.(3)The transcription and expression level of HSF2 and TGF-p in the colonic tissue samples of each group were detected by PCR and immunohistochemistry.2.The expression of HSF2 in HT-29 cells was interfered by RNA interference and plasmids transfection.The concentrations of IL-1? and TNF-? in supernatant were detected by ELISA after induced by LPS.The expression and phosphorylation level of ERK,P38 and JNK were detected by Western Blot.3.The expression of HSF2 in HT-29 cells was interfered by RNA interference and plasmids transfection.The concentrations of TGF-? in supernatant were detected by ELISA after induced by LPS.The expression and phosphorylation level of Smad2/3 were detected by Western Blot.Part three1.A pMAL-c2X-HSF2 prokaryotic expression plasmid was constructed.HSF-MBP fusion protein was induced by IPTG and purified through amylose column.HSF2 recombinant protein was identified by coomassie brilliant blue staining and Western-Blot after cleavage MBP label using factor Xa.2.The HSF2 recombinant protein binding to the heat shock transcription element HSE was identified by electrophoretic mobility shift assay.The transcription level of HSP70 was detected by PCR after co-culture HSF2 recombinant protein with HT-29 cells.Results:Part one1.The concentration of Fecal HSF2 in the normal control group and ulcerative colitis patients with MES= 0,1,2,3 were(0.64±0.09,1.30±0.35,1.84±0.46,2.38±0.57,3.38±0.42)ng/ml respectively.The concentration of Fecal Calprotectin in the normal control group and ulcerative colitis patients with MES= 0,1,2,3 were(31.90±11.89,181±113.31,490.53±171.12,916.85±371.77,1320.20±286.09)ug/g.The concentration of Fecal HSF2 and Fecal Calprotectin were increasing in the group of UC patients with MES=0,1,2 and 3 compared with the normal control group.The difference between the groups were statistically significant(P<0.01).2.The levels of Fecal HSF2 and Fecal Calprotectin were positive correlation with MES score(r=0.81,r=0.85).The levels of Fecal HSF2 were positive correlation with Fecal Calprotectin were positive correlation(r=0.91).3.HSF2 was expressed in fossae glandular cells and stromal cells in colonic mucosal tissue of UC.The immunohistochemistry staining positive cells proportion of HSF2 in colonic mucosal tissue of UC with MES = 0,1,2,3 were(25.35±5.20,41.83±13.54,54.21±16.54,66.98±16.96)%respectively.The expression of HSF2 increased significantly with increasing score of MES.The difference between the groups were statistically significant(P<0.01).The expression of HSF2 was positive correlation with MES score(r=0.74).4.The concentration 1.8ng/ml of Fecal HSF2 and 181 ug/g of Fecal Calprotectin were used as a cut-off value to predict mucosal healing.The sensitivity of Fecal HSF2 and Fecal Calprotectin were(67.8%,75%),specificity were(80.9%,80.9%),positive predictive value(67.1%,82.9%)and negative predictive value(81.5%,92.4%)respectively.The clinical value of specificity and negative predictive value of Fecal HSF2 to predict mucosal healing was better than sensitivity and positive predictive value.5.The AUC of Fecal HSF2 to predict mucosal healing was 0.919(95%[CI]:0.846-0.992,P<0.0001)and Fecal Calprotectin was 0.958(95%[CI]:0.910-1.00,P<0.0001).Both AUC were greater than 0.9 that indicated the Fecal HSF2 had a high accuracy to predict mucosal healing of UC.Part two1.In the animal experiment of DSS induced chemical colitis mice treated by methylprednisolone,the DAI,colon damage and pathology score of DSS model group,MP treatment group and Placebo group increased significantly compared with normal control group.Among them,the index of MP treatment group was lower than DSS model group and Placebo group.The transcription and expression of HSF2 and TGF-? in colonic mucosal tissue were increased and decreased synchronously in DSS model group and MP treatment group.2.HSF2 siRNA interference dramatically decreased the expression level of HSF2 in HT-29 cells.The phosphorylation level of ERK,P38 and JNK increased significantly in HSF2 siRNA compared with the negative control group.The concentrations of IL-1? and TNF-a in the supernatant of HT-29 cells induced by LPS increased in HSF2 siRNA compared with the negative control group.On the contrary,HSF2-FLAG plasmid transfection markedly increased the expression level of HSF2 in HT-29 cells.The phosphorylation level of ERK,P38 and JNK decreased significantly in HSF2-FLAG plasmid transfection compared with the Blank Vector group.The concentrations of IL-1? and TNF-? in the supernatant of HT-29 cells induced by LPS reduced as well.3.The phosphorylation level of Smad2/3 decreased in HSF2 siRNA compared with the negative control group.The concentrations of TGF-? in the supernatant of HT-29 cells induced by LPS reduced in HSF2 siRNA compared with the negative control group.On the contrary,the phosphorylation level of Smad2/3 increased significantly in HSF2-FLAG plasmid transfection compared with the Blank Vector group.The concentrations of TGF-? in the supernatant of HT-29 cells induced by LPS increased as well.Part three1.ApMAL-c2X-HSF2 prokaryotic expression plasmid was constructed successfully.Lots of HSF-MBP soluble recombinant protein was induced by IPTG and purified through amylose column.A molecular weight correct HSF2 recombinant protein was identified by coomassie brilliant blue staining and Western-Blot after cleavage MBP label using factor Xa.2.The HSF2 recombinant protein could bind to the heat shock transcription element HSE identified by EMSA.The transcription level of HSP70 increased significantly in HT-29 cells co-culture with HSF2 recombinant protein.These results confirmed that HSF2 recombinant protein had biological activity.Conclusions:1.Fecal HSF2 concentration maybe used as a high accuracy evaluation index for predicting the mucosal healing of UC.The clinical value of specificity and negative predictive value of Fecal HSF2 to predict mucosal healing is better than sensitivity and positive predictive value.2.The transcription and expression of HSF2 and TGF-? in colonic mucosal tissue are increased and decreased synchronously in DSS model indicates that HSF2 may regulate TGF-? expression promoting mucosal repair.3.HSF2 inhibits pro-inflammatory cytokines expression induced by LPS via down-regulation phosphorylation of ERK,P38 and JNK pathways in HT-29 cells.What's more,HSF2 promotes TGF-? expression via up-regulation phosphorylation of Smad2/3 pathways in HT-29 cells.HSF2 can promote the mucosal repair of ulcerative colitis by inhibiting the inflammatory response and promoting the expression of the mucosal repair factor.4.Successfully expressed and purified human HSF2 recombinant protein with biological activity.It lays a good foundation for the preparation of HSF2 polyclonal antibody and ELISA kit to detect Fecal HSF2 in a large scale cohort. |
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