Mechanisms Of Heat Shock Transcription Factor 2 Regulating Mitophagy In Ulcerative Colitis

Objective:The level of mitophagy in the intestinal epithelial cells was measured under conditions of different severities of ulcerative colitis(UC).The correlation between the level of mitophagy in the intestinal epithelial cells and UC was judged at the clinical level.At the animal and cell level,explore the potential mechanisms of heat shock transcription factor 2(HSF2)affecting the activation of NLRP3 inflammasome by regulating mitophagy in intestinal epithelial cells.This study aimed to further clarify the role of HSF2 and mitophagy in UC and lay a new theoretical foundation for finding new therapeutic targets in UC.Methods:This study was divided into three parts:clinical,animal and cell.Part I:Clinical level:1.According to the consensus on the diagnosis and treatment of inflammatory bowel disease in China,90 UC patients were enrolled in the study,including 30 mild UC,30 moderate UC and 30 severe UC patients,and 60 volunteers who were screened for colon cancer were selected as healthy controls.Colonoscopy was performed in each group,and the mucosal sample was collected from the fixed site.2.The morphology of mitochondria and mitophagy of intestinal epithelial cells were observed by transmission electron microscopy.Immunohistochemistry and RT-PCR were used to detect changes in the PARL/PINK/Parkin pathway,a mitophagy pathway,and immunofluorescence was used to detect the level of reactive oxygen species in the intestinal mucosa to preliminarily determine the correlation between mitophagy in intestinal epithelial cells and the severity of UC and to explore the potential role and mechanism of intestinal epithelial cells mitophagy in UC.Part ?:Animal level1.C57BL/6 mice were selected for animal experiments.The hsf2-/-knockout mouse was used.The mice were divided into a wild-type group(WT group,20 mice)and a gene knockout group(KO group,20 mice)according to whether the gene was knocked out.The level of hsf2 was measured by RT-PCR and Western blotting.Then,the model was established with DSS.The above two groups were randomly divided into a water-drinking wild-type group(WT+H2O group,10 mice),a DSS-drinking wild-type group(WT+DSS group,10 mice),a water-drinking gene knockout group(KO+H2O group,10 mice)and a DSS-drinking gene knockout group(KO+DSS group,10 mice).The WT+H2O and KO+H2O groups drank distilled water continuously for 7 days,the WT+DSS and KO+DSS groups drank distilled water containing 3%DSS continuously for 7 days.On the eighth day of modelling,the mice were sacrificed to collect colon samples.2.The DAI score,degree of colon shortening and gross appearance of the lesions of mice in each group were evaluated.The histopathological score of mice in each group was evaluated by HE staining.3.The mitochondrial morphology and mitophagy level in intestinal epithelial cells in the intestinal mucosa of mice were observed by transmission electron microscopy.4.RT-PCR,immunohistochemistry,western blotting and immunofluorescence were used to detect the expression of the PARL/PINK/Parkin pathway,reactive oxygen species and NLRP3 inflammasomes in the intestinal mucosal tissues of mice in each group.5.The expression levels of IL-1beta and IL-18 in the serum of mice in each group were detected by ELISA.6.The correlation between hsf2 and the above indicators at the animal level were evaluated,and the potential mechanisms were explored.Part ?:Cell level:1.Lentiviral transfection was used to overexpress or knock out HSF2 in Caco-2 cells to change HSF2 gene and protein expression levels;Caco-2 cells were stimulated with LPS and ATP to simulate the inflammatory environment.Based on the above treatment,the cells were divided into the normal control group(NC),control with inflammation stimulation group(NC+LPS+ATP),overexpression of HSF2 with inflammation stimulation group(OV-HSF2+LPS+ATP),overexpression of HSF2 negative control with inflammation stimulation group(OV-NC+LPS+ATP),knockout of HSF2 with inflammation stimulation group(shR-HSF2+LPS+ATP),and knockout of HSF2 negative control with inflammation stimulation group(shR-NC+LPS+ATP).2.The differences in mitochondrial morphology and mitophagy levels in each group of cells were observed by transmission electron microscopy.3.RT-PCR,immunohistochemistry,Western blotting and flow cytometry were used to detect the expression of PARL/PINK/Parkin,reactive oxygen species and NLRP3 inflammasomes in each group of cells.The expression levels of IL-1beta and IL-18 in each group of cells were detected by ELISA.Results:Part ?:Clinical Level:1.Compared with the healthy controls,there were more mitochondria with abnormal morphology in the intestinal epithelial cells of UC patients,and the level of mitophagy increased significantly.At the same time,with the increase of the severity of UC disease,the number of mitochondria with abnormal morphology and the level of mitophagy in the intestinal epithelial cells increased gradually.2.Compared with the healthy controls,the level of ROS in the intestinal mucosa of UC patients increased significantly,the gene and protein expression of PARL decreased,and the gene and protein expression of PINK/Parkin increased.Meanwhile,with the increase of the severity of UC disease,the level of ROS in the intestinal mucosa of UC gradually increased,the gene and protein expression of PARL gradually decreased,and the gene and protein expression of PINK/Parkin gradually increased.The difference between groups was statistically significant.Part ?:Animal level:1.The colitis model in mice was successfully induced with DSS.Compared with each group,the KO+DSS group had the highest DAI score and the most obvious degree of colon shortening.HE staining indicated that the KO+DSS group had the most significant destruction of colonic tissue structure,the most severe inflammatory reaction and the highest pathological score.The difference between the groups was statistically significant.2.The level of mitophagy in intestinal epithelial cells of mice increased after drinking DSS.The level of mitophagy in intestinal epithelial cells of the KO+DSS group was lower than that of the WT+DSS group.The difference between groups was statistically significant.3?In the DSS model,the gene and protein expression of PARL were downregulated and those of PINK/Parkin were increased,the level of reactive oxygen species in intestinal mucosa was increased,and the expression of the NLRP3 inflammasome was upregulated.The expression of the mitophagy pathway was relatively lower in the KO+DSS group,while the level of reactive oxygen species and the expression of the NLRP3 inflammasome were higher in the KO+DSS group,than in the WT+DSS group.The difference between groups was statistically significant.4.In the DSS model,the serum levels of IL-1beta and IL-18 in mice increased.Serum levels of IL-1beta and IL-18 were higher in the KO+DSS group than in the WT+DSS group.The difference between groups was statistically significant.Part III:Cell level1.The gene and protein levels of HSF2 were regulated by lentivirus.2.Mitophagy in Caco-2 cells was induced by LPS and ATP.Transmission electron microscopy showed that there was more obvious mitophagy in the HSF2 overexpression with inflammation-stimulated group(OV-HSF2+LPS+ATP)than in the control with inflammation-stimulated group(NC+LPS+ATP)and the HSF2 overexpression negative control with inflammation-stimulated group(OV-NC+LPS+ATP).Conversely,the mitophagy level in the knockout of HSF2 with inflammation-stimulated group(shR-HSF2+LPS+ATP)was lower than that in the control with inflammation-stimulated group(NC+LPS+ATP)and the knockout of HSF2 negative control with inflammation-stimulated group(shR-NC+LPS+ATP).These results suggest that HSF2 is positively correlated with the level of mitophagy in intestinal epithelial cells.3.Overexpression of HSF2 inhibited PARL gene and protein expression,promoted PINK/Parkin gene and protein expression,reduced intracellular ROS,inhibited NLRP3 inflammasome activation,and reduced IL-1beta and IL-18 expression levels.Conversely,knockout of HSF2 increased PARL gene and protein expression,inhibited PINK/Parkin gene and protein expression,increased intracellular ROS,and promoted NLRP3 inflammasome activation.The expression levels of IL-1beta and IL-18 were increased.Conclusions:1.In UC patients,the numbers of damaged mitochondrial and mitophagy in intestinal epithelial cells increased with the severity of UC disease,and ROS in intestinal mucosa also increased.PARL/PINK/Parkin signaling pathway may play an important role in mitophagy of UC intestinal epithelial cells.2.Low levels of hsf2 lead to decreased mitophagy in intestinal epithelial cells,increased ROS and expression of the NLRP3 inflammasome and pro-inflammatory cytokines in a DSS-induced colitis mouse model.These results suggest that hsf2 is positively correlated with the level of mitophagy in intestinal epithelial cells at the animal level,affecting cellular ROS,thus regulating the degree of activation of the NLRP3 inflammasome and ultimately changing the levels of pro-inflammatory cytokines such as IL-1? and IL-18.Secondly,hsf2 may regulate mitochondrial autophagy in mouse intestinal epithelial cells through the PALL/PINK/Parkin pathway,and play a role in regulating intestinal inflammation in DSS mouse colitis model.3.HSF2 is positively correlated with the level of mitophagy in Caco-2 cells and promotes mitophagy in Caco-2 cells through the PARL/PINK/Parkin pathway,which promotes the clearance of damaged mitochondria in Caco-2 cells through mitophagy under inflammatory conditions,thus reducing intracellular ROS and inhibiting the degree of NLRP3 activation,ultimately reducing the release of pro-inflammatory cytokines,and inhibiting the inflammatory response.