| Spinal cord ischemia/reperfusion injury(SCII)is a special type of spinal cord injury.It refers to the symptoms of nerve damage resulted from the recanalization of the spinal cord after ischemia and compression,such as numbness of the skin,weakness of the limbs and even paraplegia,the incidence of paraplegia is 2.9%-40%.Characterized by unpredictability and refractoriness,SCII becomes a serious type of spinal cord injury.SCII brings not only worse quality of personal life to patients,but also huge burdens to the family and society.Therefore,How to treat SCII is difficult and significant for medical staffs.Synthesized by mammalian cells,Lipoxin A4(LXA4)is a member of lipoxin family which has strong anti-inflammation effect.LXA4 has been observed that it can involve in the treatment of respiratory infection and gastrointestinal inflammation by inhibiting inflammatory response and it takes neuroprotective effect on the center nevous system injury.Therefore,in our study,LXA4 is considered to be applied to the rat SCII animal model in order to obtain better therapeutic effects.AimIn our study,we set up the model of SCII in rat successfully and applied LXA4 to the animal model.Firstly,The neuroprotective effect of LXA4 on SCII rat model by inhibiting an inflammatory response and apoptosis was investigated.Secondly,the protective mechanism of LXA4 for the SCII was studied in our study.Finally,the effect of autophagy on SCII and the effect of LXA4 on autophagy were explored.Method 1.The protective effect of LXA4 on SCII in rats by inhibiting inflammatory response.Rat spinal cord ischemia-reperfusion injury models were established by double clipping abdominal aorta method and randomly divided into experimental group(LXA4 group),ischemia-reperfusion group(SCII group)and sham group.LXA4 group rats were administered to get intrathecal injection of 10μl LXA4(300 pmol)30 min after modeling and the SCII group rats were given sodium chloride injection in the same way.The rats of the three groups were score-d by Basso-Beattie-Bresnahan(BBB)score and measured for the activity of my-eloperoxidase(MPO)and IL-1β、TNF-α at 6、12、24 and 48 h after reperfusion.Then,the morphological changes of spinal cord were observed by HE staining 24 h after reperfusion.2.The protective effect of LXA4 on SCII in rats by inhibiting apoptosis.The rat SCII models were constructed and grouped in the way of the first part.The rats of the three groups were sacrificed by cervical dislocation at 24 hours after reperfusion.And then,the apoptotic cells were observed by Tunel method,the level of Bcl-2 and Bax by Elisa method and the content of Cleaved-Caspase-3 by Western blot.3.Mechanism of protective effect of LXA4 on SCII model in rats.The rat SCII models were constructed and grouped in the way of the first part.The rats of the three groups were sacrificed by cervical dislocation at 24 hours after reperfusion.Then,the expression of FPR2/ALX,p-IKKβ and p-NF-κB in lumbar spinal cord tissue were analyzed by Western blot.4.The role of autophagy in SCII and the effect of lipoxin A4 on it.The rat SCII models were constructed and grouped in the same way of the first part.The rats of three groups were sacrificed by cervical dislocation at 24 hours after reperfusion and the lumbar spinal cord was removed after sacrificed.The apoptosis-positive cells of the three groups were stained and counted by LC3 B double-labeled fluorescent staining and the expression of LC3-II/LC3-I,GABARAP and mTOR protein were detected by Western blot.Result 1.The protective effect of LXA4 on SCII of rats by inhibiting inflammatory responseCompared with the sham group,the BBB scores of LXA4 group and SCII group were significantly decreased and the MPO activity,IL-1β and TNF-α levels were increased remarkably(P<0.05).Some differences between LXA4 group and SCII group were shown as follow:(1)The BBB score of LXA4 group was increased excellentlly at 12,24 and 48 h after reperfusion(P<0.05).(2)The MPO activity of LXA4 group was significantly decreased at each time point(P<0.05).(3)At 12 h and 24 h after reperfusion,the IL-1β and TNF-α levels in LXA4 group were significantly lower than those in SCII group(P<0.05).(4)A large number of degenerated neurons emerged in the gray matter of spinal cord through HE stain in SCII group with inflammatory cells infiltrated and the nucleus morphology of spinal cord returned to near-normal form in LXA4 group with less inflammatory cells infiltrated.2.The protective effect of LXA4 on SCII of rats by inhibiting apoptosis(1)Apoptotic cells in spinal cord tissue of sham-operated rats were rare and a large number of apoptotic cells were observed in SCII group and LXA4 group.The number of apoptotic cells in LXA4 group was obviously higher than SCII group(P<0.05).(2)The content of Bcl-2 and Bax in SCII group and LXA4 group was remarkably higher than that in sham group.The content of Bcl-2 was significantly increased and Bax decreased in LXA4 compared with SCII group with statistical significance(P<0.05).(3)The expression of Cleaved-Caspase-3 in SCII group and LXA4 group was significantly higher than that in sham group(P<0.05).After treatment with LXA4,The expression of Cleaved-Caspase-3 in spinal cord tissue decreased significantly(P<0.05).3.The mechanism of protective effect of LXA4 on SCII in rats(1)The content of FPR2/ALX of spinal cord tissue in SCII group and LXA4 group was remarkably increased compared with sham group(P<0.05),The FPR2/ALX content in LXA4 group increased significantly compared with SCII group with no statistically significance.(2)The content of p-IKKβ was high in the sham group.The expression of p-IKKβ protein in SCII group and the LXA4 group was remarkably lower than that in the sham group(P<0.05)and it was notably higher in LXA4 group than that in the SCII group(P<0.05).(3)The expression of p-NF-κB was higher in the sham group.The expression of p-NF-κB in SCII group and LXA4 group was significantly lower than that in the sham group(P<0.05)and it was significantly higher in LXA4 group than that in SCII group(P<0.05).4.Autophagy involved in the protective effect of LXA4 on SCII in rats(1)There were fewer LC3 B positive cells in the sham group.Compared to that in the sham group,the number of LC3 B positive cells in SCII group and LXA4 group was significantly increased(P<0.05)and there were significantly lower in LXA4 group than those in SCII group(P<0.05).(2)The ratio of LC3-II/LC3-I in SCII group and LXA4 group was significantly higher than that in sham group(P<0.05).After treatment with LXA4,the ratio of LC3-II/LC3-I in spinal cord tissue had a significantly decline compared with SCII group(P<0.05).(3)The expression of GABARAP in SCII group and LXA4 group was significantly higher than that in sham group(P<0.05).After treatment with LXA4,the expression of GABARAP in LXA4 group had a certain decline compared with SCII group with no statistical difference(P>0.05).(4)The content of mTOR was shown no significant difference among the three groups,while the content of p-mTOR was shown significant difference.Compared with the sham group,the expression of p-mTOR protein in SCII group was significantly lower(P<0.05).When LXA4 given,the expression of p-mTOR was significantly higher than that in the sham group and SCII group(P<0.05).Conclusion 1.LXA4 has a protective effect on SCII in rats which is achieved by inhibiting the inflammatory response and apoptosis of SCII.2.The mechanism by which LXA4 inhibits inflammatory response and apoptosis is shown as follow: LXA4 activates IKKβ in cytoplasm to increase the phosphorylation of IKKβ by binding to FPR2/ALX and the phosphorylation of IKKβ decreases the activation of NF-κB which inhibits the inflammatory response and apoptosis.3.The conclusion that autophagy is activated when SCII occurring indicates that the autophagy plays an important role in the process of SCII.LXA4 can be used to treat SCII by the inhibition of autophagy which is regulated by the activation of m-TOR. |
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