The Mechanism Of HSF2 Regulating Intestinal Epithelial Cell Apoptosis Through The Mitochondrial Pathway In Ulcerative Colitis

Part Ⅰ The role of HSF2 in regulating intest-inal epithelial cell layer homeostasis in ulcerative colitisObjective:To observe the correlation between heat shock transcription factor 2(HSF2)and markers of intestinal epithelial cell layer in intestinal mucosal tissue of ulcerative colitis(UC)patients with different disease activities.Then continue to explore the correlation between HSF2 and intestinal epithelial cell markers after changing the HSF2 levels in animals and cells.The aim of this study was to investigate the effect of HSF2 on intestinal epithelial cell layer in UC.Methods:1.Clinical study:15 healthy controls and 15 mild,15 moderate,15 severe UC patients were included.The intestinal mucosa tissues were collected under colonoscopy for RT-qPCR and immunohistochemistry.The gene transcription and protein expression levels of HSF2,ZO-1 and Occludin in the intestinal mucosa tissues of UC patients with different disease activities were detected respectively.2.Animal study:The C57BL/6 wild type(WT)mice and hsf2-/-mice(KO)bred by our research team were used and divided into the WT mouse control group(WT+H2O group,10 males),the HSF2 knockout mouse control group(KO+H2O group,10 males),the WT mouse colitis group(WT+DSS group,10 males)and the HSF2 knockout mouse colitis group(KO+DSS group,10 males)according to whether drink dextran sulphate sodium(DSS).The intestinal mucosal tissues of mice in each experimental group were used for RT-qPCR and western blot to detect the gene transcription and protein expression levels of ZO-1 and Occludin in mice with different HSF2 levels.3.Cell study:HSF2 interference and overexpression lentivirus were used to change the HSF2 levels in Caco-2 cells.The Caco-2 cells were divided into the normal control group(NC group)and the LPS treated groups according to whether LPS was used to induce inflammation.The LPS treated groups included the inflammatory control group(NC+LPS group),the inflammatory HSF2 interference group(shR-HSF2+LPS group),the inflammatory HSF2 interference control group(shR-V+LPS group),the inflammatory HSF2 overexpression group(OE-HSF2+LPS group)and the inflammatory HSF2 overexpression control group(OE-V+LPS group).RT-qPCR and western blot were used to detect the gene transcription and protein expression levels of ZO-1 and Occludin in different HSF2 levels Caco-2 cells.MTS colorimetric method was used to detect the cell proliferation activity.Flow cytometry annexin V-FITC/PI fluorescence double labeling method and immunofluorescence were used to detect the cell apoptosis levels.Results:1.Clinical study:Compared with healthy controls,the gene transcription and protein expression levels of HSF2 increased with the severity of UC,while the gene transcription and protein expression levels of ZO-1 and Occludin decreased with the severity of UC.2.Animal study:The gene transcription and protein expression levels of ZO-1 and Occludin in the mice colitis groups(WT+DSS group and KO+DSS group)were lower than those of the mice control groups(WT+H2O group and KO+H2O group),and the KO+DSS group was lower than the WT+DSS group,but there was no significant difference between the WT+H2O group and the KO+H2O group.3.Cell study:(1)The gene transcription and protein expression levels of ZO-1 and Occludin and cell proliferation activity in Caco-2 cells treated with LPS(NC+LPS group,shR-HSF2+LPS group,shR-V+LPS group,OE-HSF2+LPS group and OE-V+LPS group)were lower than those of the non LPS treated group(NC group).In the LPS treated groups,the OE-HSF2+ LPS group was the highest,and the shR-HSF2+LPS group was the lowest.There was no significant difference in the other three groups(NC+LPS group,shR-V+LPS group and OE-V+LPS group).(2)The apoptosis levels of Caco-2 cells in the LPS treated groups(NC+LPS group,shR-HSF2+LPS group,shR-V+LPS group,OE-HSF2+ LPS group and OE-V+LPS group)were higher than those of the non LPS treated group(NC group).In the LPS treated groups,the shR-HSF2+LPS group was the highest,and the OE-HSF2+LPS group was the lowest.There was no significant difference among the other three groups(NC+LPS group,shR-V+LPS group and OE-V+LPS group).Conclusions:1.The severity of UC was positively correlated with HSF2 gene transcription and protein expression levels in intestinal mucosa,and negatively correlated with ZO-1 and Occludin gene transcription and protein expression levels.With the increase of severity of UC,the damage of intestinal epithelial cell layer was gradually aggravated.2.The HSF2 levels in mice intestinal mucosa of the colitis groups were positively correlated with the gene transcription and protein expression levels of ZO-1 and Occludin.The damage of intestinal epithelial cell layer in the mice colitis groups were more serious than those of the mice control groups,and the knockout of HSF2 gene in colitis group aggravated the damage of intestinal epithelial cell layer.3.The HSF2 levels in Caco-2 cells under inflammatory environment were positively correlated with the gene transcription and protein expression levels of ZO-1 and Occludin as well as cell proliferation activity,and negatively correlated with the level of apoptosis.4.HSF2 may play a protective role in UC by resisting intestinal epithelial cell layer damage.Part Ⅱ The role of HSF2 in regulating mitochondrial pathway apoptosis of intestinal epithelial cell in ulcerative colitisObjective:To observe the apoptosis of intestinal epithelial cells and the activation of mitochondrial pathway in the intestinal mucosa of UC patients with different disease activities.After the HSF2 levels of animals and cells were changed,we continued to observe intestinal epithelial cells apoptosis in the inflammatory environment,and investigate the effects of different HSF2 levels on the activation of mitochondrial pathways.The aim was to explore whether HSF2 regulates intestinal epithelial cells apoptosis through the mitochondrial pathway in UC.Methods:1.Clinical study:Under transmission electron microscopy,the apoptosis of intestinal epithelial cells in the intestinal mucosa of healthy controls,mild,moderate,and severe UC patients was observed.The proteins expression levels of Bax,Cleaved Caspase-9 and Cleaved Caspase-3 in the intestinal mucosa was detected by immunohistochemistry.2.Animal study:Under transmission electron microscopy,the apoptosis of intestinal epithelial cells in the intestinal mucosa of mice in each experimental group(WT+H2O group,KO+H2O group,WT+DSS group and KO+DSS group)was observed.The proteins expression levels of Bax,Cleaved Caspase-9 and Cleaved Caspase-3 in the mice intestinal mucosa was detected by immunohistochemistry.Western blot was used to detect the proteins expression levels of Bax,Cleaved Caspase-9,Cleaved Caspase-3 and cytosolic Cyto-C in the mice intestinal mucosa.3.Cell study:The apoptosis of Caco-2 cells in each experimental group(NC group,NC+LPS group,shR-HSF2+LPS group,shR-V+LPS group,OE-HSF2+LPS group and OE-V+LPS group)was observed under transmission electron microscope.The proteins expression levels of Bax,Cleaved Caspase-9,Cleaved Caspase-3 and cytosolic Cyto-C of Caco-2 cells in HSF2 interference group(NC group,NC+LPS group,shR-HSF2+LPS group and shR-V+LPS group)and overexpression group(NC group,NC+LPS group,OE-HSF2+LPS group and OE-V+LPS group)were detected by western blot.Results:1.Clinical study:Under transmission electron microscope,apoptosis was observed in the intestinal epithelial cells of healthy controls,mild,moderate and severe UC patients.The proteins expression levels of Bax,Cleaved Caspase-9 and Cleaved Caspase-3 in intestinal mucosa increased with the severity of UC.2.Animal study:Under transmission electron microscope,apoptosis was observed in the mice intestinal epithelial cells of each experimental group.The proteins expression levels of Bax,Cleaved Caspase-9,Cleaved Caspase-3 and cytosolic Cyto-C in the mice intestinal mucosa was negatively correlated with the HSF2 levels.3.Cell study:Under transmission electron microscopy,apoptosis was observed in Caco-2 cells of each experimental group.The proteins expression levels of Bax,Cleaved Caspase-9,Cleaved Caspase-3 and cytosolic Cyto-C in the Caco-2 cells was negatively correlated with the HSF2 levels.Conclusions:1.The activation of mitochondrial pathway apoptosis in the intestinal mucosa of patients increased with the severity of UC.2.The activation of mitochondrial pathway apoptosis in the mice colitis groups were higher than those of the mice control groups,and the knockout of HSF2 gene could aggravate the activation of mitochondrial pathway apoptosis in colitis groups.3.The HSF2 levels in Caco-2 cells under inflammatory environment were negatively correlated with the activation of mitochondrial pathway apoptosis.HSF2 interference could promote the mitochondrial apoptosis pathway activation.Conversely,HSF2 overexpression could inhibit mitochondrial apoptosis pathway activation.4.HSF2 may play a protective role in UC by inhibiting mitochondrial pathway to reduce excessive apoptosis of intestinal epithelial cells.Part Ⅲ The mechanism of HSF2 regulating mitochondria permeability transition pore in Caco-2 cellObjective:To explore the changes in mitochondrial membrane permeability of Caco-2 cell inflammation models with different HSF2 levels.The aim was to investigate whether HSF2 plays a protective role in UC by regulating the mitochondria permeability transition pore(mPTP)to inhibit intestinal epithelial cells mitochondrial pathway apoptosis.Methods:Flow cytometry was used to detect the opening degree of mPTP in Caco-2 cells of each experimental group(NC group,NC+LPS group,shR-HSF2+LPS group,shR-V+LPS group,OE-HSF2+LPS group and OE-V+LPS group).The changes of mitochondrial membrane potential were observed by laser confocal microscope.After the Caco-2 cells were treated with mPTP agonist Atr and inhibitor CsA(NC group,NC+LPS group,shR-HSF2+LPS group,OE-HSF2+LPS group,NC+Atr+LPS group,NC+CsA+LPS group,OE-HSF2+Atr+LPS group and shR-HSF2+CsA+LPS group),the above two experiments were performed again.Results:1.The positive rate of mPTP open cells in the LPS treated groups were higher than those of the non LPS treated groups.In the LPS treated groups,the shR-HSF2+LPS group was the highest,and the OE-HSF2+LPS group was the lowest.There was no significant difference among the other three groups(NC+LPS group,shR-V+LPS group and OE-V+LPS group).2.The mitochondrial membrane potential in the LPS treated groups were lower than those of non LPS treated groups.In the LPS treated groups,the shR-HSF2+LPS group decreased the most,and the OE-HSF2+LPS group decreased the least.There was no significant difference among the other three groups(NC+LPS group,shR-V+LPS group and OE-V+LPS group).3.There was no significant difference in the positive rate of mPTP open cells between the NC+CsA+LPS group and the OE-HSF2+LPS group,the NC+Atr+LPS group and the shR-HSF2+ LPS group.The positive rate of mPTP open cells in the OE-HSF2+Atr+LPS group was lower than that of the NC+ATR+LPS group.The positive rate of mPTP open cells in the shR-HSF2+CsA+LPS group was higher than that of the NC+CsA+LPS group.4.There was no significant difference in mitochondrial membrane potential between the NC+CsA+LPS group and the OE-HSF2+LPS group,the NC+Atr+LPS group and the shR-HSF2+LPS group.The mitochondrial membrane potential in the OE-HSF2+Atr+LPS group was higher than that of the NC+Atr+LPS group.The mitochondrial membrane potential in the shR-HSF2+CsA+LPS group was lower than that of the NC+CsA+LPS group.Conclusions:1.The HSF2 levels in Caco-2 cells were negatively correlated with mitochondrial membrane permeability.HSF2 interference could promote the opening of mPTP and decrease the mitochondrial membrane potential,which was similar to the effect of mPTP agonist Atr.On the contrary,HSF2 overexpression could inhibit the opening of mPTP and reduce the decline of mitochondrial membrane potential,which was similar to the effect of mPTP inhibitor CsA.2.HSF2 could resist the over-opening of mPTP which induced by Atr,and could reverse the increase of mitochondrial membrane permeability.3.HSF2 may play a protective role in UC by inhibiting the opening of mPTP and reducing the activation of mitochondrial pathway apoptosis.