| Background:Spinal cord injury(SCI)is one of the ten major disability diseases in China,and there is still a lack of effective treatment.Acetyl-L-carnitine(ALC)is a strong antioxidant that can enter mitochondria and participate in the tricarboxylic acid cycle.It is also an important substrate for mitochondria-oxidation and can cross the blood-brain barrier to reach the nerve center for material exchange.Silence information regulator3(SIRT3)is a mitochondrial deacetylase that plays an important role in energy metabolism,reactive oxygen species production,apoptosis and proliferation.In addition,SIRT3 can also induce and regulate the occurrence and process of mitophagy.Mitochondria are involved in nerve injury in multiple links.Secondary spinal cord injury can lead to cell energy metabolism disorder and a large amount of reactive oxygen species.The main role of ALC is to participate in energy metabolism and inhibit oxidative stress,which is closely related to the function of mitochondria.Objective:To observe the neuroprotective effect of ALC on SCI in rats,and to explore the protective effect and mechanism of ALC on mitochondria in SCI rats,so as to provide new ideas for clinical application of ALC in the treatment of SCI.Methods:1.The acute SCI model of rats was established by HI-0400 spinal cord striker,which caused T10 injury in rats.The rats were randomly divided into Sham group,SCI group,SCI + ALC group and SCI + ALC + 3-MA group.BBB score was used to evaluate the recovery of motor function.SOD activity and GSH content in plasma were determined by enzyme-linked immunosorbent assay.HE staining was used to observe the histomorphological changes of spinal cord.The expression of neuronal marker NeuN was detected by Western Blot and immunofluorescence staining.2.Western Blot and immunofluorescence staining were used to detect the expression of mitochondrial oxidative stress-related proteins GRP75,SOD2,SIRT3,apoptotic protein Cleaved Caspase 3,and autophagy pathway proteins PINK1 and Parkin;the activity of oxidative stress related factors SOD and CAT,the content of lipid peroxidation product MDA and the content of ATP were detected by enzyme labeling method.TUNEL staining was used to detect neuronal apoptosis.3.SCI rats were randomly divided into AAV-NC group,AAV-NC + ALC group and AAV-sh SIRT3 + ALC group.Western Blot was used to verify the interference efficiency of SIRT3 and the expression of PINK1,Parkin,LC3 Ⅱ/Ⅰ,p62 and NeuN in spinal cord of SCI rats.The m RNA levels of SIRT3,PINK1 and Parkin were detected by qRT-PCR.The expression of SIRT3,PINK1,Parkin,LC3,Caspase 3 and NeuN were detected by immunofluorescence.Results:1.BBB score results showed that the score of SCI + ALC group was significantly higher than that of SCI group and SCI + ALC + 3-MA group.The score of SCI + ALC +3-MA group was higher than that of SCI group.The results of enzyme labeling showed that SOD activity and GSH content in serum of rats with spinal cord injury increased significantly after ALC intervention.Compared with the SCI + ALC group,the activity of SOD and the content of GSH in the serum of rats decreased after the intervention of 3-MA and ALC.After ALC intervention,the spinal nerve fibers of rats were arranged relatively neatly,the nucleolus was slightly larger,and the junction of gray matter and white matter was clearer;the recovery results of SCI + ALC + 3-MA group were between SCI group and SCI + ALC group.NeuN protein expression increased.2.Western Blot and immunofluorescence staining showed that mitophagy increased and mitochondrial oxidative stress and apoptosis decreased in spinal cord tissue of SCI rats after ALC intervention.The results of enzyme labeling showed that the activity of antioxidant enzymes in the spinal cord of rats increased after ALC intervention,the content of lipid peroxidation products decreased significantly,and the content of ATP in serum increased significantly.TUNEL staining showed that neuronal apoptosis was significantly reduced after ALC treatment.The recovery results of SCI + ALC + 3-MA group were between SCI group and SCI + ALC group.3.Western Blot results showed that the expression of SIRT3 in the spinal cord was interfered by adeno-associated virus infection,and the expression level could be reduced by more than 60 %.Immunofluorescence staining,qRT-PCR and Western Blot results showed that the protective effect of ALC on spinal cord mitochondria was weakened and the expression of NeuN was decreased after SIRT3 knockdown.Conclusion:1.ALC has neuroprotective effect on spinal cord injury in rats.2.ALC protects mitochondria from damaged spinal cord by promoting mitophagy,inhibiting oxidative stress and inhibiting neuronal apoptosis.3.ALC activates the PINK1 / Parkin pathway through SIRT3 to promote nerve repair in rats after spinal cord injury. |
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