| Objective: Spinal cord ischemia-reperfusion injury(SCIRI)is one of the most serious complications of large vessel related surgery such as thoracic and abdominal aorta.It can lead to sensory and motor dysfunction below the level of spinal cord nerve injury.The occurrence of this situation will bring huge psychological and economic burden to the patients themselves,family members and society.Existing studies have shown that SCIRI can adversely affect neural function through a variety of mechanisms,but the current research believes that the mechanism of SCIRI is complex and has not been fully understood.Studying the pathological mechanism of SCIRI can contribute to promoting the recovery of neurological function after SCIRI.Apoptosis and inflammatory response play a very important role in SCIRI,which is the main mechanism of SCIRI,and the death of neurons is irreversible.The injury of neurons in the spinal cord is the cause of lower limb motor dysfunction and sensory impairment.The challenge is the nerve repair and functional recovery of the injured area after SCI.Noncoding RNA is a kind of RNA without coding effect.Previous studies have proved that noncoding RNA is involved in the pathophysiological functions of multiple organs,including the regulation of ischemiareperfusion injury.In recent years,there is increasing evidence that noncoding RNA plays an increasingly important role in central nervous system diseases.Some microarray results suggest that there are changes in the levels of lncrna and micro RNA after SCIRI.Combined with the existing research results,it can be confirmed that lnc RNA and micro RNA are involved in the regulation of SCIRI related mechanisms.lnc RNA TUG1 is one of the earliest discovered lnc RNAs related to human diseases.It actively participates in a variety of physiological and pathological processes,including nervous system diseases.A recent study showed that TUG1 gene knockout can prevent neuronal death and reduce acute spinal cord injury.mi R-9a-5p has been shown to be involved in the regulation of a variety of disease processes,including nervous system diseases.DIAPH1 is a member of formin family.It can bind to the cytoplasmic domain of advanced glycation end product receptor(RAGE)and mediate the intracellular signal transduction of RAGE receptor.DIAPH1 plays a very important role in the regulation of related cellular functions and intracellular signal transduction by rage ligand.In this study,we explored the effect of lnc RNA TUG1 on SCIRI and its targeted regulation of DIAPH1 through competitive endogenous RNA mechanism combined with mi R-9a-5p,thereby affecting neuronal apoptosis.Methods:1.The SCIRI model was established.The aortic arch was clamped at the proximal end of the left subclavian artery for 14 minutes.The expression levels of mi R-9a-5p and DIAPH1 were detected by q RT-PCR and western blot at 12 h,24 h,48 h and 72 h after SCIRI.Based on the results of the above experiments,we choose the research time point after SCIRI.The expression of mi R-9a-5p was up-regulated and inhibited by intrathecal injection of mi R-9a-5p mimic for three consecutive days before operation.Tarlov score was used to measure lower limb motor function scores after SCIRI and after mi R-9a-5p mimic and inhibitor intrasheath injection.Structural changes and counting of motor neurons in the anterior horn of spinal cord were observed by HE staining.The relative expression of mi R-9a-5p was detected by q RT-PCR.The targeted binding relationship between mi R-9a-5p and DIAPH1 was verified by dual luciferase reporter assay.Tarlov scoring system and TDT mediated d UTP nick end labeling(TUNEL)experiment were used to evaluate neural function and neuronal apoptosis.Apoptosis factors Bax,Bcl-2,cleaved caspase-3 and pathway protein NF-κB p65 were detected by western blot and its phosphorylated form p-NF-κB p65 protein expression level.The colocalization of DIAPH1 and neuronal cells was detected by immunofluorescence staining,and the fluorescence intensity of DIAPH1 was observed.2.q RT-PCR was used to detect the relative expression levels of lnc RNA TUG1 and mi R-9a-5p at different time points.Based on the above results,we selected the most serious time point of lnc RNA TUG1 and mi R-9a-5p damage after SCIRI.Compared with IR group,the Tarlov score was increased after intrasheath injection of Si-Tug1,and the number of neurons with intact morphology was increased by HE staining.Dual luciferase reporter assay verified the direct binding relationship between lnc RNA TUG1 and mi R-9a-5p.lnc RNA TUG1 was silenced by intrathecal injection of small interfering RNA(si RNA)for three consecutive days before operation,and then the expression level of lnc RNA TUG1 was detected by q RT-PCR to verify the silencing effect of si-TUG1,and mi R-9a-5p was detected to verify the effect of silencing lnc RNA TUG1 on the expression level of mi R-9a-5p in spinal cord.3.lnc RNA TUG1 was silenced by intrathecal injection of si-TUG1 for three consecutive days before operation.DIAPH1,apoptosis factor Bax,Bcl-2,cleaved caspase-3 and pathway protein NF-κB in sham group,SCIRI group,NC group and si-TUG1 group were detected by q RT-PCR and western blotting and its phosphorylated form p-NF-κB p65 protein expression level.In sham group,SCIRI group,NC group and si-TUG1 group,the colocalization of DIAPH1 and neuronal cells was detected by immunofluorescence staining,and the fluorescence intensity of DIAPH1 was observed.Tarlov scoring system and TUNEL experiment were used to evaluate neural function and neuronal apoptosis.Result:1.Mi R-9a-5p decreased significantly at 24 h and 48 h after SCIRI,and DIAPH1 increased at 48 h after SCIRI.The results of dual luciferase reporter assay showed that mi R-9a-5p could directly bind to the predicted site of 3’UTR region of DIAPH1.After intrathecal injection of mi R-9a-5p mimic for 3 consecutive days before operation,compared with IR group,the relative expression of mi R-9a-5p increased significantly,Tarlov score showed that the motor function of lower limbs was improved,and the number of neurons with complete morphology and structure was increased by HE staining.The content of DIAPH1 protein decreased significantly.At the same time,the levels of apoptosis factors Bax and cleaved caspase-3 decreased significantly,the level of Bcl-2increased and phosphorylated NF-κB p65 relative expression decreased.The results of immunofluorescence double labeling showed that DIAPH1 could be colocated with neuronal marker Neu N.Compared with sham group,the relative fluorescence value of DIAPH1 in IR group and NC group was significantly higher,and the relative fluorescence value of mi R-9a-5p mimic group was lower compared with IR group.The percentage of TUNEL positive cells in IR group was significantly higher than that in sham group.Compared with IR group,the percentage of TUNEL positive cells in mi R-9a-5p mimic group decreased.2.The RNA expression level of lnc RNA TUG1 increased 48 hours after spinal cord ischemia-reperfusion injury.The RNA expression level of mi R-9a-5p decreased significantly 48 hours after spinal cord ischemia-reperfusion injury.There is a correlation between the two RNA relative expression level.After intrathecal injection of si-TUG1,the content of lnc RNA TUG1 in spinal cord decreased significantly.The results of dual luciferase reporter assay showed that lnc RNA TUG1 could directly bind to mi R-9a-5p.After intrathecal injection of si-TUG1,the content of lnc RNA TUG1 in spinal cord decreased.At the same time,silencing lnc RNA TUG1 could regulate the increase of mi R-9a-5p level.3.After silencing lnc RNA TUG1,the m RNA expression level of DIAPH1 increased significantly.At the same time,WB results also showed that after silencing lnc RNA TUG1,the protein expression level of DIAPH1 increased,the levels of apoptosis factors Bax and cleaved caspase-3 decreased significantly,the level of Bcl-2 increased and phosphorylated NF-κB p65 The relative level expression of decreased.Immunofluorescence results showed that compared with sham group,the relative fluorescence value of DIAPH1 in IR group and NC group increased significantly,and the relative fluorescence value of siTUG1 group decreased compared with IR group.In TUNEL experiment,compared with sham group,the number of apoptotic cells in IR group increased significantly,while the ratio of apoptotic cells in si-TUG1 group decreased significantly compared with IR group.Conclusion: In the process of SCIRI,mi R-9a-5p is directly combined with DIAPH1.Intrathecal injection of mi R-9a-5p mimic can inhibit the level of DIAPH1,alleviate cell apoptosis and play a neuroprotective role.lnc RNA TUG1 has a competitive relationship with mi R-9a-5p.Silencing lnc RNA TUG1 can inhibit DIAPH1 by up regulating the level of mi R-9a-5p,through the pathway of NF-κB,reduce the neuroapoptotic response after SCIRI,and alleviate the motor dysfunction of lower limbs in rats. |
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