Effects And Mechanism Of MiR-155 On Intestinal Mucosal Injury Following Traumatic Brain Injury

Objective: Intestinal barrier dysfunction,which often following traumatic brain injury(TBI),can affects the outcome of the disease.The purpose of this study was to investigate the effects and mechanism of miR-155 in intestinal mucosal injury after brain injury.Methods: In the first part,49 TBI patients(case group)and 20 healthy people(control group)at the same time were enrolled.The expression of miR-155 in peripheral serum of patients 24 hours after injury and in the peripheral serum of healthy people were detected by RT-qPCR.The serum levels of diamine oxidase(DAO),D-lactic acid(D-LA),endotoxin(ET)and intestinal fatty acid binding protein(I-FABP),which are indices for intestinal mucosal injury,were detected by ELISA.The serum levels of serum amyloid a protein(SAA),which belongs to acute phase protein,were also detected by ELISA.The 49 patients with TBI were divided into the good prognosis group and the poor prognosis group according to the Glasgow Outcome Scale six months after the injury.In the second part,healthy adult male C57BL/6 mice were taken for experiments.TBI model was established by modified free falling method,and miR-155-5p inhibitor was constructed using AAV-DJ virus as vector.Mice were divided into four groups,including Sham group,TBI group,Anti group and negative control(NC)group.In Sham group,bone window was opened,but no impact was performed.The mice in Anti group and NC group were injected with AAV-mmu-mir-155-5p inhibitor or AV-GP-1-NC(DJ)virus transfection solution respectively before suturing scalp after free falling body impact.The changes of serum and intestinal mucosal miR-155,the expressions of clandin-1,occludin and ZO-1 in intestinal mucosa,and the pathological changes of intestinal mucosa(HE staining and immunohistochemical staining)were observed on 7 and 14 day respectively.In the third part,we used Caco-2 human colon adenocarcinoma cells as the research object,and constructed pre-hsa-mir-155 and hsa-mir-155-5p inhibitor using lentivirus as the vector.The cultured cells were divided into four groups: Blank group,,without adding any virus,replacing with fresh cell culture medium;Pre group,adding pre-hsa-mir-155 virus;Anti group,adding hsa-mir-155-5p inhibitor virus;NC group,adding LV3 NC virus.Immunofluorescence was observed on the 5th day after the virus was added,and the cells were collected.The expression of miR-155 and clandin-1,occludin,ZO-1 were detected.Results: In the first part,compared with the control group,the expression of miR-155 increased in serum of the patients in the case group 24 h after injury(P = 0.046),while the expression of DAO?D-LA and SAA increased(all P < 0.05).Compared with the control group,the expression of ET and I-FABP increased in serum of the patients in the case group,but there was no statistical significance(all P > 0.05).Among 49 TBI patients,18(36.7%)had a good prognosis and 31(62.3%)had a bad prognosis.There was no significant difference in the expression of miR-155 between the group with good prognosis and the group with poor prognosis(P = 0.838).Multivariate logistic model showed that high serum DAO level increased the risk of poor prognosis in TBI patients at 6 months [OR = 1.099,95%CI 1.021 ~ 1.183,P = 0.012 ]?In the second part,compared with Sham group,the expression of miR-155 in serum and intestinal mucosa increased in TBI group 7 and 14 days after modeling(all P < 0.05),the expression of clandin-1,occludin and ZO-1 in intestinal mucosa were decreased(all P < 0.05),the injury score of intestinal mucosa increased(all P < 0.05),and the expression of clandin-1,occludin and ZO-1 in intestinal mucosa was also decreased with immunohistochemical staining.Compared with TBI group,the expression of miR-155 in serum and intestinal mucosa decreased 7 and 14 days after modeling(all P < 0.05),the expression of clandin-1 in intestinal mucosa increased(all P < 0.05),the expression of occludin and ZO-1 did not change significantly(all P > 0.05),the injury score of small intestinal mucosa decreased(all P < 0.05).The immunohistochemical staining of small intestinal mucosa indicated that expression of clandin-1 increased,while the expression of occludin and ZO-1 did not change significantly.In the third part,compared with the Blank group,the expression of miR-155 increased in the Pre group(P < 0.001),the expression of clandin-1 and protein decreased(all P < 0.05),while the expression of occludin and ZO-1 did not change significantly(all P > 0.05).Compared with the blank group,the expression of miR-155 decreased in the Anti group(P = 0.002),the expression of clandin-1 and protein increased(all P < 0.05),while the expression of occludin and ZO-1 did not change significantly(all P > 0.05).Conclusion:(1)Intestinal barrier dysfunction is prone to occur after TBI,and the indicators reflecting intestinal epithelial cell damage(such as serum DAO)may be used as biological markers to evaluate the severity and prognosis of patients after TBI.(2)The increased expression of miR-155 in serum and intestinal mucosa after TBI may be involved in the occurrence of intestinal mucosal injury by inhibiting expression of claudin-1.(3)Down-regulation of the expression of miR-155 can increase the expression of claudin-1 in intestinal mucosa and reduce intestinal mucosa damage,which provides a new idea for the prevention and treatment of intestinal barrier dysfunction.