| Objective: Spinal cord ischemia/reperfusion injury(SCII)can lead to a series of complications after spinal or thoracic abdominal aneurysm surgery,resulting in paraplegia or even threatening life.The mechanism of spinal cord ischemia/reperfusion injury is very complex,including the release of excitatory amino acids,inflammatory response and neuronal apoptosis.However,we can’t get comprehensive understanding of the mechanism from current research,resulting in limited clinical effective treatment and protection measures.AP-1 family is composed of Jun(c-Jun,Jun B and Jun D)and Fos(c-Fos,Fra B,Fra-1 and Fra-2).Fra-1 and c-Fos are the members of Fos family,and the increased expression of c-Fos is considered as a marker of neuronal activation after SCII.AP-1 family is related to regulating the expression of cytokines and apoptosis.S100A8 protein is the ligand of Toll-like receptor 4(TLR4)and is known as an important therapeutic target for myocardial ischemia.However,the roles and effects of Fra-1 and S100A8 after SCII remain unclear.In addition,The TLR4 receptor inhibitor,TAK-242,can inhibit the downstream TLR4 pathways,but whether it inhibits the activation of S100A8/TLR4 pathway and reduces apoptosis and inflammatory response after SCII requires further research.Micro RNAs(miRNAs)play a certain role in spinal cord ischemia-reperfusion injury,but the relationship between miRNAs and Fra-1 has not been studied.Therefore,by studying the Fra-1,S100A8,TLR4 pathways and miRNAs,we can understand the mechanism of SCII and provide new ideas for clinical treatment.Methods:1.The spinal ischemia reperfusion model was established by clipping aortic arch of SD rats for 14 minutes,and they were randomly divided into sham group(sham group)and ischemia reperfusion group(IR group).The spinal cord of rats from L4-L6 was obtained at 6h,12 h,24h,36 h,48h and 72 h after SCII.Fra-1 m RNA and protein levels were detected by Quantitative real-time polymerase chain reaction(q RT-PCR)and Western blotting at different time points.The cleaved caspase-3 and S100A8 protein levels were detected by Western blotting at different time points.The cell localization of Fra-1 was quantitatively detected by immunofluorescence staining,and the expression of Fra-1 in sham group and I/R group were analyzed.The Neun(neuron marker),Iba-1(microglia marker)and GFAP(astrocyte marker)were used to label different cell types.2.The oxygen-glucose deprivation/reperfusion(OGD/R)model was established and randomly divided into control group and OGD/R group.Absorbance value was detected by CCK-8 reagent.The cell viability of cells in OGD/R group were established at 0.5h,1h,2h,4h,6h and 8h after oxygen-glucose deprivation,and at 12 h,24h and 36 h after OGD/R.The expression of Fra-1 is changed by means of small interfering RNA(si RNA)and vector-mediated overexpression(OE).The transfection effect was detected by western blot,and the cells were divided into the following groups: control group,oxygen-glucose deprivation/reperfusion(OGD/R group),tansfection of si RNA targeting Fra-1 group(si-Fra-1 group),tansfection of overexpression targeting Fra-1 group(OEFra-1 group),negative control group(NC-Fra-1 group),and WB was used to detect protein expression levels of cleaved caspase-3,Bax,and Bcl-2 in different groups to detect cell apoptosis.Immunofluorescence staining was used to quantitatively detect protein expression levels of cleaved caspase-3 and Fra-1 in the control group,OGD/R group,OGD/R+NC group,and OGD/R+ si-Fra-1 group.Chromatin Immunoprecipitation(Ch IP)was used to analyze the relationship between Fra-1 and S100A8.3.SCII model was established by clipping aortic arch of SD rats for 14 minutes.The Fra-1 and S100A8 genes were targeted with short hairpin RNA(sh RNA)mediated by adeno-associated virus(AAV),followed by intrathecal injection in rats.The rats were divided into sham operation group(sham group),ischemia/reperfusion injury group(I/R group),adeno-associated virus-mediated sh-Fra-1 group(IR+sh-Fra-1 group),adenoassociated virus-mediated sh-S100A8 group(IR+sh-S100A8)and corresponding negative control group(NC group).Hematoxylin-eosin staining(HE)was used to analyze the number of intact neurons.Toe footprint analysis was used to evaluate neuromotor function in different groups of rats.The expression of S100A8 was upregulated by recombinant protein S100A8(r S100A8),and the activation of downstream TLR4 pathways was inhibited by TLR4 receptor inhibitor(TAK-242).Intrathecal injection of r S100A8 and intraperitoneal injection of TAK-242 were performed in rats,and the rats were divided into sham group,sham +r S100A8 group,I/R group,and I/R+r S100A8+TAK-242 group.Cleaved caspase-3,Bax,Bcl-2,TLR4,nuclear transcription factor-κB(NF-κB),phosphorylated NF-κB(p-NF-κB),extracellular regulated protein kinases1/2(ERK1/2),phosphorylated ERK1/2(p-ERK1/2),and interleukin-1β(IL-1β)were detected by western blot.Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL)was used to detect apoptosis response in different groups.4.SCII rats were established by clipping aortic arch for 14 minutes.Rats were injected intrathecally with miR-29a-mimic,miR-29a-inhibitor and corresponding NC plasmids.The rats were divided into sham group(sham group),spinal cord ischemiareperfusion injury group(I/R group),miR-29 a mimic group(I/R+mimic group),miR-29 a inhibitor group(I/R+inhibitor group)and corresponding negative control group(I/R+mimic-NC group and I/R+ inhibitor-NC group).The targeted binding relationship and binding sites of Fra-1 and miR-29 a were analyzed by dual-luciferase reporter assay.Tarlov score analysis was used to evaluate neuromotor function in different groups of rats.PCR was used to detect the expression of Fra-1 and miR-29 a between different groups.The protein expression of Fra-1 in different groups was detected by WB.Results:1.After spinal cord ischemia-reperfusion injury in rats,the mRNA and protein levels of Fra-1 increased significantly,and peaking 48 h after SCII induction.Western blotting results showed that cleaved caspase-3 and S100A8 increased significantly after SCII,and S100A8 was correlated with Fra-1(r=0.78,P<0.0001).At 48 h after reperfusion,immunofluorescence showed Fra-1 expression was increased in neurons and microglia,especially in neurons,where Fra-1 expression was highest.After intrathecal injection of AAV-sh-Fra-1,western blotting results showed that compared with I/R group,in I/R+sh-Fra-1 group the expression levels of Fra-1,cleaved caspase-3 and Bax were significantly decreased,while the expression of Bcl-2 was significantly increased.HE staining results showed that the number of intact neurons in I/R group was significantly reduced compared with sham group.But compared with I/R group,the number of intact neurons in I/R+ sh-Fra-1 group was significantly increased.The results of toe footprint analysis showed that compared with sham group,rats in I/R group were unable to stand on hind limbs,walked mainly on forelimbs,and the step length and the Tarlov score were significantly reduced.Compared with I/R group,hind limb motor function of rats in I/R+sh-Fra-1 group was improved and step length and the Tarlov score were significantly increased.2.After oxygen-glucose deprivation/reperfusion in VSC4.1 cells,western blotting results showed that cleaved caspase-3 and Bax protein expression were increased and Bcl-2protein expression was decreased in the OGD/R group compared with the control group.After transfection with si-fra-1,Fra-1 expression was knocked down.Compared with the OGD/R group,in the OGD/R+ si-Fra-1 group,the expression of Fra-1,cleaved caspase-3,and Bax protein was decreased,and the expression of Bcl-2 was increased.Overexpression of Fra-1 using OE-Fra-1 significantly increased expression levels of Fra-1,cleaved caspase-3,and Bax and significantly decreased Bcl-2 expression in the OGD/R+ OE-Fra-1 group compared to the OGD/R group.Immunofluorescence staining results showed that compared with the control group,the expression of Fra-1 and cleaved caspase-3 was significantly increased in the OGD/R group;Fra-1 and cleaved caspase-3expression levels were significantly reduced in the OGD/R+ si-Fra-1 group compared to the OGD/R group.Ch IP-qPCR results showed that after inducing OGD/R,Fra-1 was found to bind to the promoter region of S100A8.After sh-Fra-1 was intrathecally injected into rats,q RT-PCR results showed that the m RNA level of S100A8 in I/R+ shFra-1 group was significantly lower than that in I/R group.Western blotting results showed that the protein level of S100A8 in I/R+ sh-Fra-1 group was significantly lower than that in I/R group.Tarlov score results showed that the Tarlov score in I/R+ shS100A8 group was significantly higher than that in I/R group.Therefore,it is further suggested that Fra-1 binds to the S100A8 promoter to promote the transcriptional expression of S100A8 after SCII.3.Western blotting results showed that protein levels of cleaved caspase-3,TLR4,pERK1/2,p-NF-κB,IL-1β and Fra-1 were significantly increased in sham+r S100A8 group compared with sham group.Compared with I/R group,the protein expression levels of cleaved caspase-3,TLR4,p-ERK1/2,p-NF-κB,IL-1β were significantly decreased in I/R+ sh-Fra-1 group.The protein levels of cleaved caspase-3,TLR4,p-ERK1/2/ERK1/2,p-NF-κB/NF-κB,and IL-1β were significantly increased in the I/R+sh-Fra-1+r S100A8 group compared with the I/R+ sh-Fra-1 group.The western blotting results showed that the expression levels of p-ERK1/2/ERK1/2,p-NF–κB/NF-κB,IL-1β and Fra-1 in I/R+r S100A8+TAK-242 group were significantly decreased compared with I/R group.TUNEL results showed that compared with sham group,cell apoptosis rate was increased in sham +r S100A8 group.Compared with I/R group,cell apoptosis rate was decreased in I/R+r S100A8+TAK-242 group and the Tarlov score in I/R+r S100A8+TAK-242 group was increased.4.PCR results showed that after SCII,the expression of miR-29 a was significantly decreased at 12,24 and 48 h after reperfusion compared with sham group.After intrathecal injection of miR-29a-mimic,the expression of miR-29a-3p in I/R+mimic group was significantly increased compared with I/R group,while the m RNA and protein expression of Fra-1 and S100A8 were significantly decreased.Tarlov score showed that compared with I/R group,the score was significantly increased in I/R+ mimic group.Conclusions: Mitigated I/R injury in rats with downregulated Fra-1 expression was associated with improved motor function,as determined by a reduction in the apoptosis rate and inflammation in the spinal cord.We found that S100A8 was a direct target of Fra-1.After SCII,increased S100A8 upregulated expression of TLR4,p-NF-κB and pERK,and increased production of IL-1β.These S100A8-induced changes were inhibited by intraperitoneal injection of TAK-242.TAK-242 also inhibited the activation of ERK and NF-κB pathways,thereby decreasing Fra-1 expression.miR-29 a can target to bind Fra-1 and regulate Fra-1 and S100A8 expression.Together,the Fra-1-targeted S100A8 also appears upstream of Fra-1 and Fra-1 /S100A8 interacts to form a feedback loop in the signaling pathway activated by SCII. |
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