Effect of Enolase inhibitor on autophagy and motor function after spinal cord injury in rats / 中华创伤杂志

ABSTRACT

Objective:To investigate the effect of Enolase inhibition (ENOblock) on autophagy- related protein expression and motor function promotion after spinal cord injury in rats.Methods:A total of 160 female SD rats were divided into sham-operation group, 3-methyladenine (3-MA) autophagy inhibitor treatment group (3-MA group), spinal cord injury group and ENOblock treatment group (ENOblock group) according to the random number table, with 40 rats per group. Back laminectomy without injury to the spinal cord was performed in sham-operation group. Spinal cord injury at T 8 was induced by using a modified Allen weight-drop apparatus to establish a spinal cord injury model in the rest three groups. 3-MA and ENOblock groups were injected 3-MA (2.5 mg/kg) and ENOblock (100 μg/kg) into the caudal vein immediately after injury, respectively. Sham-operation and spinal cord injury groups were injected same dose of isotonic sodium chloride solution into the caudal vein. At 1, 3, 7, 14 and 21 days after injury, BBB score was used to evaluate lower limb motor function. At day 3 after injury, the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I and protein expressions of autophagy effector protein (Beclin-1) and polyubiq-uitinbinding protein (p62) were detected by Western blotting. At day 7 after injury, LC3-Ⅱ and Beclin-1 positive cells in the injured area of the spinal cord were determined by immunofluorescence staining. At day 3 after injury, the mRNA expressions of Beclin-1 and Enolase in the injured area of the spinal cord were detected by RT-PCR. Results:At 1, 3, 7, 14 and 21 days after injury, BBB score was lowered in 3-MA group [(1.4±1.1)points, (2.4±0.9)points, (3.8±1.8)points, (7.6±1.1)points, (9.0±2.1)points], spinal cord injury group [(0.8±0.5)points, (1.8±0.9)points, (3.6±0.9)points, (6.2±1.3)points, (8.0±0.7)points] and ENOblock group [(2.0±0.9)points, (2.2±0.8)points, (4.8±1.1)points, (10.6±1.5)points, (13.2±0.8)points] compared to sham-operation group [(21.0±0.0)points at all time points] (all P<0.05). Moreover, the score in ENOblock group was significantly higher than that in spinal cord injury group at 14, 21 days after injury, and the score in 3-MA group was significantly higher than that in spinal cord injury group at day 21 after injury (all P<0.05). At day 3 after injury, Western blotting showed that the ratio of LC3-II to LC3-I and protein expressions of Beclin-1 and p62 were 0.46±0.10, 0.41±0.03, 0.81±0.03 in sham-operation group, 0.66±0.06, 0.69±0.02, 0.59±0.05 in 3-MA group, 0.85±0.06, 1.07±0.03, 0.41±0.02 in spinal cord injury group and 0.68±0.06, 0.66±0.08, 0.55±0.02 in ENOblock group. By comparison, spinal cord injury group showed significantly higher ratio of LC3-II to LC3-I and protein expression of Beclin-1 and significantly lower protein expression of p62 than sham-operation group (all P<0.05); 3-MA and ENOblock groups showed significantly lower ratio of LC3-II to LC3-I and protein expression of Beclin-1 and significantly higher protein expression of p62 than spinal cord injury group (all P<0.05); there was no significant difference in the ratio of LC3-II to LC3-I and protein expressions of Beclin-1 and p62 between 3-MA and ENOblock groups (all P>0.05). At day 7 after injury, immunofluorescence staining showed that LC3-II and Beclin-1 positive cells in 3-MA and ENOblock groups were less than those in spinal cord injury group. At day 3 after injury, RT-PCR showed that mRNA expressions of Beclin-1 and Enolase in spinal cord injury group (1.08±0.16, 0.98±0.17) were higher than those in sham-operation group (0.25±0.06, 0.29±0.03). Moreover, mRNA expressions of Beclin-1 and Enolase in 3-MA group (0.77±0.11, 0.72±0.04) and ENOblock group (0.81±0.10, 0.64±0.09) were lower than those in spinal cord injury group (all P<0.05). There was no significant difference in mRNA expressions of Beclin-1 and Enolase between 3-MA and ENOblock groups (all P>0.05). Conclusions:Autophagy activity is significantly up-regulated after spinal cord injury in rats. ENOblock can inhibit autophagy and promote motor function recovery in rats by regulating the expression of autophagy-related proteins.