Inhibition Of MicroRNA-204 Attenuates The Ischemia-reperfusion Injury In Early Phase Of Spinal Cords In Rats Via The Promotion Of Neuron Autophagy And Regulation Of Apoptosis

Objective:Spinal ischemia reperfusion injury is always the difficult promble in clinic.However,the effective prevention measures and therapist method are still scarce to protect the spinal cord neuron when it is occure.Further studies are still necessary to find the new method to promote the tolerability of the spine to ischemia.Autophagy is a hot topic on modern research,and play an important role in cell metabolism and repairde.microRNAs are found in eukaryotes,a highly conserved noncoding single-stranded RNA molecules,which are important regulatory molecules for translation involved in regulating many life processes and disease,including autophagy.microRNA-204,which is close to autophagy and change significantiy in the early phase of spinal injury.In our research,we build a model of spinal cord ischemia reperfusion injury,evaluate the neuroprotective effects by inhibitionthe expression of microRNA-204 regulate autophagy and apoptosis against ischemia-reperfusion injury of spinal cords,and the molecular mechanisms were also explored.Aimed to find the new method to prevent and cure the spinal cord injury.Methods:This study randomly selected 50 male SD rats,weight about 250 g,and randomly divided into 5 groups(n=10).sham group,6h group,12h group,24h group and 48h group.Exceptfor the sham group,the other groups were used to establish the rat spinal cord ischemia-reperfusion injury model by occlusion of the descending aorta just distal to the subclavian artery for 14 min.After reperfusion duration in 6 hours,12hours,24 hours,48 hours,we observed the changesin motor function of rats with MDI movement function score,histopathological changes(HE staining)and caculating motor neurons;Observing the ultrastructure of neuron by electron microscope,evaluati-On the activation of autophagy by detecting specific autophagy protein LC3 and the autophagy regulate protein beclin 1 by western-blot.according to 4 different reperfusion durations.At the meantime,we observed the expression of microRNA-204 and its target protein bcl–2,and evaluated their correlationship with the autophagy.Another Male SD rats were divided into 6 groups randomly(n=10).Sham group:rats underwent thoractomy without aortic occlusion;Control group:rats underwent spinal cord ischemia for 14 min;vector group:1×10~7 TU empty lentivirus vectors in a total volume of 10?l were intrathecally injected 5 days before spinal cords ischemia;antagomiRNA-204 group:1×10~7 TU lentivirus vectors containing antagomiRNA-204in a total volume of 10?l were intrathecally injected 5 days before spinal cords ischemia.3-MA group:100?g 3-MA in 2?l saline was intrathecally injected before a14-minute of spinal cord ischemia;antagomiRNA-204+3-MA group:1×10~7 TU lentivirus vectors containing antagomiRNA-204 in a total volume of 10?l were intrathecally injected 5 days before spinal cords ischemia and 100?g 3-MA in 2?l saline was intrathecally injected just before ischemia.Hind-limb motor function was assessed using the Motor Deficit Index(MDI)at 48h after reperfusion.Then the animals were sacrificed and lumbar spinalcords were harvested for HE and TUNEL staining.The intact motor neurons and apoptotic cells were calculated in the anterior horn.Detecting the expression of apoptotic protein caspase-3 by western-blot method.In a parallel series of experiments,spinal cords were additionally collected 6 hours after reperfusion from the 6 groups to evaluate expressions of microRNA-204 by qRT-PCR method.The expressions of beclin-1,LC3 II and bcl-2 protein by western blot method.Result:1.The model of spinal cord ischemia-reperfusion injury in rats is exact effect.Compare of sham group,thehind legs of rats MID score of the 4 point time group was increase significantly(P<0.05).There was also corresponding pathological changes such as neurons swelling,necrosis,vague change of Nissl body of different degree in spinal cord tissue,consistent with the neural function changes2.Autophy is activated in ischemia reperfusion injury in rats.We can detect extensive cytoplasm edema,swelling and vacuolation of mitochondria,decompaction and vacuolation of rough endoplasmic reticulum,and double membrane packed autophagosome with organelles fragments in spinal cord anterior horn motor neurons under transmission electron microscopy(TEM),24hours after spinal cord ischemia reperfusion injury.Immunohistochemical staining analysis also show the expression of LC3II was significantly increase.The ratio of LC3-II/LC3-I in spinal cords markedly increased from 12h after the transient ischemia(vs the sham group,P<0.05)and peaked at 24h after reperfusion(vs the sham group,P<0.05).Although the ratios of LC3-II/LC3-I at both 6h and 48h after reperfusion were higher then that of the sham group,the differences did not reach a statistical significance(vs the sham group,P=0.206 and 0.854,all P>0.05).The expression of becline-1 increased slightly after reperfusion,but no significant differences were detected at the 6,12,24 and 48h after reperfusion 4 time points compared with the sham group(vs the sham group at,P=1.000,0.952,1.000 and 1.000 respectively,all P>0.05).3.In the early phase after reperfusion,the expression of microRNA-204 was significantly increase,and its target protein bcl-2 wasdecreased.Compared with the sham animals,expressions of microRNA-204 were significantly enhanced at 6,12 and24h after reperfusion(P<0.05,respectively).Then the expression of microRNA-204decreased to a level nearly to the sham group at 48h after reperfusion(P>0.05).Compared with the sham animals,the expression of BCL-2 in spinal cords was significantly inhibited at 6,12 and 24h after reperfusion(P<0.05,respectively).At 48h after reperfusion,the expression of BCL-2 was restored(vs sham group P>0.05).4.Intrathecal injection of antagomiRNA-204,but not thelentivirus vector,can dramatically inhibited the expression of microRNA-204 in the spinal cord.Compared with the control group,the expression of microRNA-204 was markly decrease in antagomiRNA-204 group and antagomiRNA-204+3-MA group(P<0.05,respectively)6h after reperfusion.Administration of 3-MA and lentivirus vector did not affect the expression of microRNA-204 in spinal cords after reperfusion.Compared with the control group,no significant difference in microRNA-204 expression was detected between the vector group and the 3-MA group(P>0.05,respectively).5.Intrathecal injection of the lentivirus vector of antagomiRNA-204 remarkably enhanced the motor function of hind limbs after spinal cord ischemia,but administration of 3-MA abolish the neuroprotectionin of antagomiRNA-204 completely.Equivalent MDI scores were observed in the control group and the control vector group,at the 4 observation time points after reperfusion(all P>0.05).By contrast,intrathecal injection of lentivirus of antagomiR-204 remarkably enhanced the motor function of hind limbs after spinal cord ischemia,as indicated by the significantly lower MDI scores of the antagomiR-204 group at the 4 observation time points after reperfusion(all P<0.05 vs the control group,at the 4 time points after reperfusion).Administration of 3-MA abolished the neuroprotective effects of antagomiR-204 completely.There was no significant difference of MDI scores between the antagomiR-204+3-MA group and the control group at any observation time point after reperfusion(P>0.05,respectively).Compared with the antagomiR-204 group,MDI scores of the antagomiR-204+3-MA group were even much higher at 12 and 24h after reperfusion(P<0.05,respectively).6.Intrathecal injection of the lentivirus vector of antagomiRNA-204 remarkably enhanced the number of intact motor neurons and decreased the number of apoptotic neurons.Severe neurologic damage of spinal cords characterized by vacuolization,frank necrosis,and loss of motor neurons was detected in both the control group and the control vector group.Slighter histologic changes were found in lumbar spinal cords of animals in the antagomiR-204 group,and the number of intact motor neurons in the antagomiR-204 group was much greater than that in the control group(P<0.05).Compared with the the antagomiR-204 group,intact motor neurons in the antagomiR-204+3-MA group was significantly fewer(P<0.05).No significant difference of the number of intact neurons was detected between the control group and the antagomiR-204+3-MA group(P>0.05).The number of apoptotic neurons in the antagomiR-204 group was still much smaller than that in the control group(P<0.05).Compared with the the antagomiR-204 group,the number of apoptoticneurons in the antagomiR-204+3-MA group was significantly increase(P<0.05),but not diffreent with control group(P>0.05).7.Intrathecal injection of antagmiR-204 induced a significant increase of the expression of Beclin 1and the ratio of LC3-II/LC3-I in spinal cords after reperfusion 6hour.Such effection was inhibited completely by administration of 3-MA.Intrathecal injection of antagmiR-204 induced a significant increase of the ratio of LC3-II/LC3-I in spinal cords after reperfusion(the antagomiR-204 group vs the control group,P<0.05).Such an increase of the ratio of LC3-II/LC3-I was inhibited completely by administration of 3-MA in the antagomiR-204+3-MA group(theantagomiR-204+3-M Agroup vs the antagomiR-204 group,P<0.05).Becline-1expressionin spinal cords was also dramatically enhanced by pretreatment with antagomiR-204 compared with the control group(the antagomiR-204 group vs the control group,P<0.05).Compared with the antagomiR-204 group,becline-1 expression in the antagomiR-204+3-MA group was markedly reduced(the antagomiR-204+3-MA group vs the antagomiR-204group,P<0.05).8.Intrathecal injection of antagomiR-204 dramatically increased the expression of BCL-2 after reperfusion.6h.The BCL-2 expression in the antagomiR-204 group was much higher than that in the control group(the antagomiR-204 group vs the control group,P<0.05).Injection of 3-MA did not affect the effect of antagmiR-204 on the expression of BCL-2.BCL-2 expression in the antagomiR-204+3-MA group was equivalent to that in the antagomiR-204 group(the antagomiR-204+3-MA group vs the antagomiR-204 group,P>0.05).9.Intrathecal injection of antagomiR-204 dramatically decreased the expression of caspase-3after reperfusion 48 hour.The expression of caspase-3in spinal cords was increased significantly in the control and vector groups(the control group and the vector group vs the sham group,P<0.05 respectively).Administration of antagomiR-204 reduced the caspase-3 expression markedly in the antagomiR-204group(the antagomiR-204 group vs the control group,P<0.05).3-MA did not affect the caspase-3 expression in spinal cords.Caspase-3 expression in the 3-MA group was equivalent to that in the control group(the 3-MA group vs the control group,P>0.05).Consistently,no statistical difference of caspase-3 expression was detected between the antagomiR-204 group and the antagomiR-204+3-MA group(antagomiR-204+3-MA group vs the antagomiR-204group,P>0.05).Conclusion:Inhibition of microRNA-204 attenuates the ischemia-reperfusion injury in early phase of spinal cords in rats via the promotion of neuron autophagy and Inhibition of apoptosis.