| Part Ⅰ:Expression Changes of LPS-mi R-132/212-SIRT1 pathway in Hirschsprung-associated enterocolitisObjective To investigate the expression changes of LPS,mi R-132/212 and SIRT1 in Hirschsprung-associated enterocolitis(HAEC)and their role in the occurrence of HAEC.Methods Intestinal tissue specimens from 64 children with HSCR were collected and divided into two groups based on whether or not they were accompanied by HAEC.One group consisted of 32 postoperative HAEC cases and the other group consisted of 32 HSCR cases without HAEC.The expression of mi R-132/212 and its target gene was detected by RT-q PCR and Western Blot experiments.At the same time,the correlation between mi R-132/212 and SIRT1 was identified using Person correlation analysis,cell transfection,molecular expression regulation and dual luciferase reporter gene assay.Then,the effect of LPS on the expression of mi R-132/212 and its target gene SIRT1 was explored by HT-29 cell culture and transfection.Results The increased expression of mi R-132/212 was found in Hirschsprung-associated enterocolitis.Bioinformatics analysis was performed on mi R-132/212 using prediction websites such as Target Scan,Pic Tar,and mi R-base.The primary downstream target genes of mi R-132/212 is sirtuin1(SIRT1),and the expression of SIRT1 was significantly lower than that of HSCR.There was a negative linear correlation between the expression of mi R-132/212 and SIRT1,and there is a binding relationship between them,confirming that SIRT1 is a common target gene of mi R-132/212.LPS was used to incubate HT29 cells at different concentration levels and the results showed that LPS can regulate the expression of mi R-132/212 and their target gene SIRT1 and participate in the occurrence of Hirschsprung-associated enterocolitis.Conclusion mi R-132/212 and their target gene SIRT1 are involved in the inflammatory process of HAEC,and the results suggest that LPS may regulate the SIRT1 expression through mi R-132/212,which is related to the occurrence of Hirschsprung-associated enterocolitis.Part Ⅱ: LPS mediates NLRP3 inflammasomes to affect cell Pyroptosis via mi R-132/212 in the pathogenesis of Hirschsprung-associated enterocolitisObjective HAEC is an acute intestinal inflammation that complicates HSCR.Cell pyroptosis is closely related to inflammation.This study aims to explore whether LPS can induce pyroptosis in intestinal epithelial cells and then participate in the occurrence of HAEC,and to further study its internal mechanism.Methods RT-q PCR and Western Blot experiments were used to measure the expression of NLRP3,ASC and activated Csapsae-1 in the intestinal tissues of 32 pairs of postoperative HAEC and HSCR children.HT29 cells were incubated with different concentrations of LPS to establish LPS cell damage models.Mi R-132/212 mimics,inhibitor and SIRT1 overexpression plasmids were transfected to regulate the corresponding molecules in the cells.RT-q PCR,Western Blot and Immunofluorescence measured the level of NLRP3,ASC,and activated Csapsae-1.The cell pyroptosis rescue was evaluated by Flow cytometry in vitro,to determine whether LPS may activate Caspase-1 by regulating the mi R-132/212-SIRT1 pathway and mediate intestinal epithelial cell pyroptosis.Results The increased expression of NLRP3,ASC and Caspase-1 were found in HAEC.LPS exposure and mi R-132/212 mimic transfection revealed that both LPS and mi R-132 and mi R-212 can induce increased expression of m RNAs and proteins of cell pyroptosis related genes NLRP3,ASC and Caspase-1.Low expression of mi R-132,mi R-212,or over-expression of SIRT1 can partially reverse the upregulation of cell pyroptosis related genes caused by LPS.Finally,using activated Caspase-1 as a marker of cell pyroptosis,flow cytometry analysis was performed.It was observed that LPS can significantly promote the activity of Caspase-1 in HT29 cells,while mi R-132 and mi R-212 inhibitors or SIRT1 overexpression can reduce this effect.Conclusion LPS can regulate the mi R-132/212-SIRT1 pathway,activate NLRP3 inflammatory bodies,further cause Caspase-1 activation,and mediate intestinal epithelial cell pyroptosis,which is related to the occurrence of Hirschsprung-associated enterocolitis.Part Ⅲ: the mechanism of LPS-mi R-132/212-SIRT1 mediated cell pyroptosis in Hirschsprung-associated enterocolitis in vivoObjective A mouse model of LPS intestinal inflammation was used to further verify whether LPS can further reduce the expression of SIRT1 through mi R-132/212,promote the activation of NLRP3 inflammatory bodies,and mediate cell pyroptosis.Methods The research group used C57 BL / 6 mice(20 ± 2 g,6-8 weeks of age)as the LPS mouse model.E.coli-derived LPS for intraperitoneal injection of mice was used.There were two groups,the LPS group and control group.Mouse models were identified using HE staining and ELISA experiments.The expression levels of mi R-132,mi R-212,SIRT1 and the expression of scoring-related factors NLRP3,ASC and activated caspase-1 were measured by RT-q PCR and Western Blot in the intestinal tissues of LPS mouse models,respectively.Results It was observed that in the LPS group,small intestinal villi were lost during HE staining,the submucosa and lamina propria were separated,while neutrophils were aggregated,and the expression of inflammatory factors IL-18 and IL-1β was significantly higher in the LPS group,suggesting that the mouse model of intestinal inflammation was successfully established.Further detection by RT-q PCR and Western Blot found that mi R-132 and mi R-212 had higher expression levels in the intestine of mice of the LPS group.The level of the target gene SIRT1 showed a significant decrease,the cell pyroptosis-related genes NLRP3,ASC,and the activated Caspase-1 showed higher expression levels in LPS group.Conclusion Further in vivo experiments were performed to verify that LPS may regulate the expression of the target gene SIRT1 through mi R-132/212,activate NLRP3 inflammatory bodies,and mediate intestinal epithelial cell pyroptosis,which is related to the occurrence of Hirschsprung-associated enterocolitis. |
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