The Role And Mechanism Of MiR-155 In Intestinal Barrier Dysfunction In Sepsis

Objective: Sepsis often lead to intestinal barrier dysfunction,which affects the outcome of the disease.The main purpose of this study was to investigate the role and mechanism of miR-155 in intestinal barrier dysfunction in sepsis.Methods: Part Ⅰ: A total of 129 patients with sepsis admitted to the Critical Care Medical Center of Xinjiang Medical University from November 2021 to April 2023 were selected as the study objects,and healthy physical examination subjects within this time range were selected as the control group(n=129).The general data of the two groups were analyzed,including age,sex,etc.In this study,the expression level of miR-155 in peripheral blood of the two groups was accurately measured and compared by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Intestinal fatty acid binding protein(iFABP),diamine oxidase(DAO)and Interleukin-1β(Interleukin-1β),a key inflammatory factor,were detected in serum by Enzyme-linked Immunosorbent Assay(ELISA),a specific and sensitive technique.The expression levels of Interleukin-1β(IL-1β)and Interleukin-18(IL-18)were quantitatively measured.According to the acute gastrointestinal injury(AGI)diagnostic criteria,the intestinal function of patients with sepsis was classified and scored,and the patients were divided into AGI group(n=56)and non-AGI group(n=73).Logistic regression was used to analyze the relationship between miR-155 and intestinal dysfunction.In this study,receiver operator characteristic curve(ROC)was drawn to systematically evaluate the diagnostic efficacy of peripheral blood miR-155 in sepsis and intestinal damage of sepsis,a pathological injury.All subjects were divided into survival group(n=93)and death group(n=36)according to the 28 d follow-up results.Laboratory detection indexes,APACHEII score,SOFA score,AGI score and so on were compared between the two groups.Cox proportional risk regression analysis was performed for each single factor with differences,then ROC curve was plotted and the area under the curve was calculated.Part Ⅱ: In this study,64 SPF grade male S/D rats were divided into 8experimental groups with 8 rats in each group according to random number table method.The grouping is as follows: sham group,cecal ligation and perforation(CLP)model group,CLP+DMSO group,CLP+ empty carrier(AAV-NC)group,CLP+AAV-miR-155-inhibitor group,CLP+ AAV-miR-155-overexpression(CLP+AAV-miR-155-oe)group,CLP+SIRT1 agonist(SRT1720)group,CLP+SIRT1 inhibitor(EX-527)group.An adeno-associated virus(AAV)vector with miR-155 overexpression and knockdown was constructed,administered by intrabitoneal injection(250μL/ piece),and fed for four weeks to achieve stable expression of AAV virus in vivo.Two hours after modeling,SRT1720 and EX-527 were intraperitoneally injected(10mg/kg).The survival of animals in each group was recorded for 24 h.Blood was collected from the abdominal aorta 24 h after modeling,and ileum tissue was collected from the rats.Hematoxylin-eosin(HE)staining was used to observe pathological injury of ileal tissue.The mRNA levels of miR-155,Silent information regulator 1(SIRT1)and intestinal tight junction proteins were detected by RT-qPCR.Western blot(WB)was used to detect the levels of intestinal tight junction proteins,SIRT1,and pyroptosis-related proteins(NLRP3,caspase-1,ASC,GSDMD-N).Serum levels of iFABP,DAO and inflammatory cytokines IL-1β and IL-18 were determined by ELISA.Immunohistochemistry(IHC)assessed positive expression areas of claudin-1,occludin and SIRT1.The positive expression ratio of pyroptosis-related proteins was detected by Immunofluorescence(IF).The membrane integrity and organelle morphological changes of intestinal epithelium were observed by transmission electron microscopy.Part III: Cultured Caco-2 human colorectal adenocarcinoma cells in vitro,and constructed miR-155-mimic and miR-155-inhibitor stable expression cell lines using lentivirus as carrier.pc DNA-SIRT1 plasmid was used to construct SIRT1 overexpression cell line,and si SIRT1 was used to inhibit SIRT1 expression.The experimental groups were control group,LPS group,LPS+miR-NC group,LPS+ miR-155-mimic group,LPS+miR-155-inhibitor group,LPS+NC group,LPS+ov SIRT1 group,LPS+si SIRT1 group,and LPS+miR-155-inhibitor+si SIRT1 group.The m RNA levels of miR-155,SIRT1 and tight junction proteins were detected by RT-qPCR.The levels of tight junction proteins,SIRT1,Nuclear transcription factor-κB p65(NF-κB p65),Acetyl-p65 and pyrogen related proteins were detected by WB.The levels of cell supernatant iFABP,DAO and inflammatory cytokines(IL-1β,IL-18)were determined by ELISA.Annexin-Alexa Fluo647/PI kit was used to evaluate cell survival.The post-transcriptional regulatory relationship between miR-155 and SIRT1 was identified by dual luciferase gene reporting analysis.Results: Compared with the control group,the expression level of miR-155 in peripheral blood of sepsis group showed a significantly increased trend,and the difference was statistically significant(P<0.001).The level of miR-155 in sepsis patients in AGI group was higher than that in non-AGI group(P < 0.001).The level of miR-155 in non-survival group was significantly higher than that in survival group(P < 0.001).Logistic regression analysis indicated that miR-155 was involved in the occurrence of intestinal damage in sepsis(OR value was 3.156,95% CI was 1.875-5.311,P<0.001).According to the ROC curve,the level of peripheral blood miR-155 can be distinguished between sepsis patients and healthy subjects [area under the curve(AUC): 0.948,sensitivity: 0.922,specificity: 0.919],and AGI patients and non-AGI patients [AUC:0.872,sensitivity: 0.887,specificity: 0.934].In addition to APACHEII score,the level of DAO and iFABP,Cox proportional risk regression analysis indicated that miR-155 expression level was also an independent risk factor affecting the prognosis of sepsis patients.The ROC curve was plotted for each independent risk factor and the area under the curve was calculated.The results indicated that miR-155 levels upon admission exhibited excellent predictive capability in forecasting the 28-day prognosis of sepsis patients,exhibiting an AUC of 0.961,sensitivity of 88.90%,specificity of 94.60%,and a Youden index of 0.835.Specifically,a miR-155 cutoff value of 3.82 was identified.DAO levels also demonstrated robust predictive accuracy in predicting the 28-day prognosis,with an AUC of 0.985,sensitivity of 97.20%,specificity of 92.50%,Youden index of0.925,and a cutoff value of 3.99.In contrast,the AUC values for iFABP and APACHEII scores in predicting the 28-day prognosis were comparatively lower,at 0.654 and 0.673,respectively.These values were accompanied by sensitivities of 61.10% and 77.8%,specificities of 84.90% and 47.3%,and Youden indices of 0.46 and 0.251,respectively.The respective cutoff values were determined to be 418.36 and 11.5.In the second part,compared with sham group,the 24 h survival rate of CLP-induced sepsis animal models decreased.Intestinal villi injury was severe,and chiu’s score was increased.The levels of iFABP,DAO,IL-1β and IL-18 were increased.WB results showed that in the CLP group,the level of pyroptosis-related proteins increased while the expression of claudin-1 and occludin decreased.The IHC results showed that the positive expression area of claudin-1and occludin decreased in CLP group(both P < 0.05).Under transmission electron microscopy,the integrity of intestinal epithelial cell membrane and mitochondrial structure were damaged,and the nucleus was wrinkled.The results of RT-qPCR indicated that the expression level of miR-155 was increased and the expression level of SIRT1 was decreased in the intestinal tissues of the CLP group.The decrease of SIRT1 was more obvious after overexpression of miR-155,and the expression level of SIRT1 was increased after lowering the expression of miR-155.Further studies showed that regulating the expression level of miR-155 could alter the intestinal barrier function and pyroptosis-induced by sepsis.Compared with CLP group,overexpression of miR-155 resulted in further reduction of intestinal tight junction proteins.Intestinal villi injury increased significantly and intestinal pathological score increased.The percentage of positive expression area about intestinal tight junction proteins decreased further.The expression levels of pyroptosis-related proteins were further increased(P<0.05),and the damage of cell membrane integrity and organelles was significantly increased under transmission electron microscopy.Inhibition of miR-155 expression was reversed.The expression level of SIRT1 in intestinal tissues decreased in CLP group,and miR-155 negatively regulated the expression of SIRT1.Overexpression of SIRT1 could improve the pathological injury of intestinal villi,promote the positive percentage of intestinal tight junction proteins and increase the expression level of intestinal tight junction proteins,and reduce the degree of pyroptosis.Ameliorate cell membrane,mitochondria and other organelles damage.Conversely,inhibition of SIRT1 expression was reversed.In the third part,different concentrations of Lipopolysaccharides(LPS)treated Caco-2 cells for 24 h,it was found that with the increase of LPS concentration,cell viability decreased gradually,and the expression level of miR-155 increased gradually(P<0.05).The expression levels of NLRP3,caspase-1,ASC,GSDMD-N and inflammatory factors related to pyroptosis in LPS group were increased compared with those in control group(P<0.05).At the same time,the tight junction of intestinal epithelial cells in LPS group was damaged,and the expression levels of tight junction proteins were decreased(P<0.05).On the contrary,reducing the expression level of miR-155 can significantly reduce the above damaging changes.Target Scan predictions suggest that SIRT1-3 ’-UTR may be a potential target for miR-155.WB results showed that the SIRT1 protein level of Caco-2 cells transfected with miR-155-mimic was significantly decreased compared with miR-NC,while the SIRT1 protein expression level of Caco-2 cells was significantly increased after transfection with miR-155-inhibitor(P<0.05).miR-155-mimic significantly reduced the relative luciferase activity of HEK-293 T cells co-transfected with wild-type(WT)SIRT1-3 ’-UTR,and the effect was significant according to dual luciferase reporter results(P<0.01).However,the mutation of SIRT1-3 ’-UTR in HEK-293 T cells did not significantly change the relative luciferase activity(P > 0.05).Further,WB method showed that SIRT1 could play a role in intestinal injury and pyroptosis of sepsis by regulating the deacetylation level of NF-κB p65 subunit.Overexpression of SIRT1 inhibits the release of inflammatory factors from Caco-2 cells by reducing the level of Acetyl-p65 in the nucleus,alleviates the degree of pyroptosis,and thus makes the cell function more stable.In addition,increasing the expression level of SIRT1 can inhibit the expression of pyroptosis-related proteins,thereby improving intestinal tight junctions.On the contrary,inhibition of SIRT1 expression led to the increase of Acetyl-p65 level in the nucleus and the increase of the release of inflammatory factors,which promoted the occurrence of pyroptosis and aggravated intestinal injury.It should be noted that simultaneously inhibiting the expression of miR-155 and SIRT1 can reverse the protective effect of inhibiting the expression level of miR-155 on cells.Conclusions: miR-155 has certain clinical significance in the diagnosis and prognosis of sepsis and intestinal damage in sepsis.Inhibiting the expression level of miR-155 can effectively alleviate intestinal barrier dysfunction,inflammatory response and cell pyroptosis in sepsis rats.miR-155 reduces the deacetylation of NF-κB subunit p65 by targeting SIRT1,thereby enhancing NF-κB signaling activity and thereby enhancing NLRP3 inflammator-mediated pyroptosis,leading to intestinal barrier impairment in sepsis.Our study provides new insights into the role of miR-155 in regulating the pathogenesis of intestinal barrier dysfunction in sepsis,and may be expected to become a new target for the treatment of intestinal barrier dysfunction in sepsis.