| ?Objective?Spinal cord injury(SCI is a common clinical refractory disease,there is no particularly effective treatment,so there is an urgent need to study the pathogenesis and new treatment of ASCI.miR-136-5p and IKK?/NF-?B As a gene expression regulator,it plays an important role in the deve lopment of spinal cord injury.However,it is unclear how miR-136-5p an d IKK?/NF-?B affect spinal cord injury and their relationship in spinal co rd injury.Need to study and explore.This topic applies miR-136-5p mim etic(miR-136-5p inhibitor)and constructed lentiviral miR-136-5p on astroc ytes,which is confirmed by luciferase assay IKK? is a direct target gene of miR-136-5p;in vivo and in vitro studies on the molecular mechanism of miR-136-5p expression of inflammatory cytokines in the spinal cord i njury model of NF-?B signaling pathway through target IKK?,thus miR-136-5p targeted therapy for spinal cord injury provides new clues.Accordi ng to bioinformatics analysis,it is predicted that miR-136-5p has a bindin g site with IKK?,so we speculate that miR-136-5p is involved in the pro gression of spinal cord injury,and its mechanism may be related to IKK?.The aim of this study was to explore the role of miR-136-5p and IKK?in the course of acute spinal cord injury and the relationship between th em and spinal cord injury,and to clarify the regulatory mechanism of mi R-136-5p and IKK? in spinal cord injury.The treatment of spinal cord in jury brings new approaches and methods.?Method?In vitro experiments: SD rat astrocytes were cultured in vitro to esta blish an IL-17 injured cell model.A luciferase reporter plasmid containing IKK? was obtained: a sequence mutating the binding site and binding sit e of IKK? was cloned into the pGL3 reporter plasmid.Astrocytes were c o-transfected with pGL3-KK?-3'UTR-Wild/Mut and miR-136-5p inhibitor.The results showed that after up-regulating the expression of miR-136-5p,the results showed that the luciferase activity of the experimental group containing wild-type IKK? was significantly increased.This result indicate s that in astrocytes,the IKK? gene is miR-136-The target of 5p.In vivo experiments: Compared with the sham group,the SCI + LVmiRNA-NC group,the SCI + LV-sponge group,and the LV-miRNA-136-5p group BBB score(Basso,Beattie,Bresnahan Scale)scores significantly decreased 1 day after modeling(p<0.05),indicating that the SCI animal model was successfully established.The expression of IKK? and p-NF-?B protein was detected by Western Blot in injured spinal cord segment.Th e expression level of each experimental group increased gradually with dif ferent time(1d,3d,7d)after model establishment,and the expression lev el of 7d in overexpression group increased.Highly significant;double im munofluorescence results showed that: the number of GFAP-positive astroc ytes in SCI + LV-sponge group was lower than that in injury group,andthere was statistical difference;GF-miRNA-136-5p group GFAP positive staining star glue The number of cytoplasmic cells was higher than that o f the injured group,and there was statistical difference.The levels of infl ammatory factors in the experimental group were increased,compared wit h the control group(P < 0.05),and the overexpression group was 7 days after modeling.The increase of inflammatory factors was the most obvio us;it decreased in the order of overexpression group,model group and in hibition group.The above results suggest that miRNA-136-5p in rat astroc ytes can promote acute spinal cord injury through target IKK? regulation of NF-?B signaling pathway.Inflammatory response in rats.In vivo studies of miR-136-5p regulate NF-?B signaling through the t arget IKK? regulates the expression of inflammatory cytokines in rats wit h spinal cord injury.SD rat spinal cord injury model was prepared by Al len's method and relevant in vivo experimental studies were performed.R andomized SD rats were divided into sham operation group,spinal cord i njury group(SCI)+ lentiviral miRNA negative control group(SCI + LVmiRNA-NC),spinal cord injury group(SCI)+ inhibitory lentivirus Plasmi d miR-136-5p(SCI + LV-sponge)and spinal cord injury group(SCI)+ o verexpressed lentiviral plasmid miR-136-5p(LV-miRNA-136-5p).On the 7t h day before modeling,each spinal cord injured SD rat was injected with200 ul of lentivirus solution through the tail vein,and the normal group and the virus-negative group SD rats were injected with the same amoun t of normal saline.The rats in each group were evaluated for function on the first day after modeling.Spinal cord specimens were taken for histol ogical examination after perfusion with 4% paraformaldehyde on the 1st,3rd,and 7th day after modeling.Spinal cord specimens were taken after perfusion with normal saline,and IL-1? and IL-6 were observed by ELIS A.Expression levels of inflammatory factors such as TNF-? and IKK? an d NF-?B proteins.?Result?The 3'UTR regions of IKK? and miR-136-5p can bind to each other.After up-regulating the expression of miR-136-5p,the results showed that the luciferase activity of the experimental group containing wild-type IK K? increased significantly,and the result was shown in the star.In glial cells,the IKK? gene is the target of miR-136-5p.In vivo experiments: Basso scoring(Basso,Beattie,Bresnahan Scale)scores 1 day after model establishment,SCI + LV-miRNA-NC group,SCI+ LV-sponge group,LV-miRNA-136-5p group compared with sham group Significant decrease(p<0.05),indicating that the SCI animal model was successfully established.The expression of IKK? and p-NF-?B protein wa s detected by Western Blot in injured spinal cord segment.The expression level of each experimental group increased gradually with different time(1d,3d,7d)after model establishment,and the expression level of 7d in overexpression group increased.Highly significant;double immunofluoresc ence results showed that: the number of GFAP-positive astrocytes in SCI + LV-sponge group was lower than that in injury group,and there was st atistical difference;GF-miRNA-136-5p group GFAP positive staining star glue The number of cytoplasmic cells was higher than that of the injured group,and there was statistical difference.The levels of inflammatory fa ctors in the experimental group were increased,compared with the control group(P < 0.05),and the overexpression group was 7 days after modeli ng.The increase of inflammatory factors was the most obvious;it decreased in the order of overexpression group,model group and inhibition grou p.The above results suggest that miRNA-136-5p in rat astrocytes can pro mote acute spinal cord injury through target IKK? regulation of NF-?B si gnaling pathway.Inflammatory response in rats.?Conclusion?In vivo experiments: Basso scoring(Basso,Beattie,Bresnahan Scale)scores 1 day after model establishment,SCI + LV-miRNA-NC group,SC I + LV-sponge group,LV-miRNA-136-5p group compared with sham grou p Significant decrease(p<0.05),indicating that the SCI animal model was successfully established.The expression of IKK? and p-NF-?B protein in the injured spinal cord segment was detected by Western Blot.The mod el group,overexpression group and inhibition group were higher than the normal control group(P < 0.05),with different time after modeling(1st,3d).And 7d)gradually increased,and the expression level of the 7d day in the overexpression group was the most significant;the double immunof luorescence results showed that the number of GFAP positive staining astr ocytes in the SCI + LV-sponge group was lower than that in the injury g roup,and there were statistics.The difference of GFAP positive staining a strocytes in LV-miRNA-136-5p group was higher than that in injury group,and there was statistical difference;compared with normal control group,the levels of other three groups of inflammatory factors were increased(P < 0.05),the level of various inflammatory factors in the overexpressio n group was more obvious on the 7th day after model establishment;the levels of inflammatory factors in each group were gradually decreased in the order of overexpression group,model group and inhibition group;the above results suggest that rat star In glial cells,miRNA-136-5p regulates NF-?B signaling pathway through target IKK? to promote inflammatory re sponse in rats with acute spinal cord injury. |
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