| Objective:The incidence of thoracic and abdominal aortic aneurysms and aortic dissections is increasing.With the advancement of diagnostic and surgical techniques,temporary or permanent spinal cord ischemia resulting in paraplegia or paresis during surgeries for thoracic and abdominal aortic aneurysms and aortic dissections,whether performed through traditional open surgery or endovascular repair techniques,remains an unpredictable and severe complication.Therefore,there is an urgent need to explore the mechanisms of spinal cord ischemia/reperfusion injury(SCIRI)and identify effective protective strategies.This study aims to investigate the impact of overexpressed long noncoding RNA(lnc RNA)HOXD-AS1 on SCIRI in rats and its possible molecular mechanisms.The study will explore whether lnc RNA HOXD-AS1can target and regulate downstream mi RNA-130a-3p and mi RNA-330-5p genes in a rat model.Additionally,changes in the expression of lnc RNA HOXD-AS1 and downstream mi RNAs in spinal cord tissue will be assessed after inducing SCIRI modeling.The study aims to investigate whether modulation of lnc RNA HOXD-AS1expression in the rat spinal cord can mitigate spinal cord ischemia/reperfusion injury caused by thoracic aortic occlusion.Methods:1.Bioinformatics techniques and dual-luciferase reporter assays will be used to predict and confirm the mi RNAs that can be targeted and regulated by LncRNA HOXD-AS1.2.Lentivirus vectors(LV)will be constructed to overexpress LncRNA HOXD-AS1,mi RNA-130a-3p,and mi RNA-330-5p.Rat spinal cord neurons will be transfected with these vectors via intrathecal injection to establish the intervention model.3.The spinal cord ischemia/reperfusion(IR)model will be established using an arterial occlusion method,involving occlusion of the descending aorta and subclavian artery for 15 minutes.4.A total of 150 rats will be randomly divided into 10 groups for two parts of the experiment.The first part,the validation experiment,consists of four groups:(1)Sham group:rats undergoing thoracotomy without arterial occlusion(n=15);(2)Control group:rats receiving intrathecal injection of PBS(10μL)7 days before ischemia and arterial occlusion for 15 minutes(n=15);(3)Vector group:rats receiving intrathecal injection of empty lentivirus vector(1×10~7TU,10μL)7 days before ischemia and arterial occlusion for 15 minutes(n=15);(4)LncRNA HOXD-AS1 group:rats receiving intrathecal injection of LV-LncRNA HOXD-AS1(1×10~7TU,10μL)7 days before ischemia and arterial occlusion for 15 minutes(n=15).The second part,the recovery experiment,includes six groups:(5)Control vector group:rats receiving intrathecal injection of control vector(1×10~7TU,10μL)5 days before ischemia and arterial occlusion for 15 minutes(n=15);(6)mi RNA-130a group:rats receiving intrathecal injection of LV-pre-mi RNA-130a(1×10~7TU,10μL)5 days before ischemia and arterial occlusion for 15 minutes(n=15);(7)mi RNA-330 group:rats receiving intrathecal injection of LV-pre-mi RNA-330(1×10~7TU,10μL)5 days before ischemia and arterial occlusion for 15 minutes(n=15);(8)Control vector+LncRNA HOXD-AS1 group:rats receiving intrathecal injection of LV-LncRNA HOXD-AS1(1×10~7TU,10μL)7 days before ischemia and intrathecal injection of control vector(1×10~7TU,10μL)5 days before arterial occlusion for 15 minutes(n=15);(9)LncRNA HOXD-AS1+mi RNA-130a group:rats receiving intrathecal injection of LV-LncRNA HOXD-AS1(1×10~7TU,10μL)7 days before ischemia and intrathecal injection of LV-pre-mi RNA-130a(1×10~7TU,10μL)5 days before arterial occlusion for 15 minutes(n=15);(10)LncRNA HOXD-AS1+mi RNA-330 group:rats receiving intrathecal injection of LV-LncRNA HOXD-AS1(1×10~7TU,10μL)7 days before ischemia and intrathecal injection of LV-pre-mi RNA-330(1×10~7TU,10μL)5 days before arterial occlusion for15 minutes(n=15).5.Assessment of motor deficit index(MDI)in the hind limbs of each group of rats(n=5/time point)at 24 hours,48 hours,7 days,14 days,and 21days after SCIRI.6.Sacrifice of rats from each group at 24 hours after SCIRI,collection of spinal cord tissue samples,and evaluation of the expression levels of LncRNA HOXD-AS1,mi RNA-130a-3p,and mi RNA-330-5p using RT-q PCR.Assessment of the expression levels of Homeobox A5,Aquaporins 4 M1,Me CP2,E2F8,E2F1,Occludin,and phospho-Akt in spinal cord tissue using Western blot.Nissl staining and TUNEL assay will be performed on spinal cord tissue sections for apoptosis detection,and fluorescent staining will be used to observe the formation of microvascular gaps in spinal cord tissue.Results:1.There were no significant differences in body weight and rectal temperature among the 10 groups of rats(P>0.05).Compared to the Sham group,the average arterial pressure at the proximal and distal ends significantly decreased in the other 9 groups after arterial occlusion(P<0.05).2.The dual-luciferase assay confirmed the binding sites between LncRNA HOXD-AS1 and mi RNA-130a,mi RNA-330,indicating a negative regulatory relationship.3.Motor deficit index(MDI)evaluation showed that compared to the Sham group,the IR group had increased MDI scores at all 5 observation points after surgery(P<0.008).The MDI scores were higher in the intrathecal injection groups of mi RNA-130a and mi RNA-330 compared to the non-injection group(P<0.003).The MDI scores were lower in the intrathecal injection group of LncRNA HOXD-AS1 compared to the non-injection group(P<0.003).4.RT-q PCR results showed that compared to the Sham group,the expression of mi RNA-130a-3p and mi RNA-330-5p increased in the IR group after surgery.The intrathecal injection groups of mi RNA-130a and mi RNA-330 had higher expression of mi RNA-130a-3p and mi RNA-330-5p compared to the non-injection group,while the intrathecal injection group of LncRNA HOXD-AS1 had lower expression of mi RNA-130a-3p and mi RNA-330-5p and higher expression of LncRNA HOXD-AS1(P<0.05).5.Western blot results showed that overexpression of LncRNA HOXD-AS1 increased the expression levels of Me CP2,Homeobox A5,Occludin,Aquaporins 4 M1,E2F8,E2F1,and p-Akt,while overexpression of mi RNA-130a and mi RNA-330 had the opposite effect(P<0.05).6.Nissl staining results showed that the overexpression of LncRNA HOXD-AS1 group had a higher number of normal neurons in the spinal cord tissue compared to the Control group,while the overexpression of mi RNA-130a and mi RNA-330 groups had a decreased number of normal neurons(P<0.05).7.TUNEL staining results showed that the group with overexpression of LncRNA HOXD-AS1 had significantly reduced apoptotic cells compared to the Control group,while the groups with overexpression of mi RNA-130a and mi RNA-330 had increased apoptotic cells(P<0.05).8.Fluorescent staining results showed that compared to the Control group,the group with overexpression of LncRNA HOXD-AS1 had increased fluorescence intensity of Occludin and decreased fluorescence intensity of CD31,while the groups with overexpression of mi RNA-130a and mi RNA-330 had decreased fluorescence intensity of Occludin and increased fluorescence intensity of CD31(P<0.05).Conclusion:Overexpression of LncRNA HOXD-AS1 negatively regulates mi RNA-130a and mi RNA-330,thereby alleviating spinal cord ischemia-reperfusion injury by promoting neurite outgrowth,enhancing neuronal survival,inhibiting neural tissue edema,and suppressing cell apoptosis.It improves motor function after spinal cord ischemia-reperfusion injury. |
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