胞液型磷脂酶A2抑制剂上调自噬通量改善脊髓损伤后神经功能的作用及机制研究

Neuroprotective effect and mechanism of cPLA2 inhibitor increases autophagic flux on spinal cord injury

目的:探讨胞液型磷脂酶A2(cytosolic phospholipase A 2,cPLA 2)抑制剂改善脊髓损伤(spinal cord injury,SCI)后神经功能的机制。方法:36只雌性SD大鼠,体质量(280±20)g,3月龄,分为3组(n=12):假模型组、SCI组、SCI+花生四烯酸三氟甲基酮(arachidonyl trifluoromethyl ketone,AACOCF3)组。3组均建立球囊压迫SCI模型。假模型组置入球囊后不做加压处理,余两组球囊加压持续48 h造脊髓压迫模型。造模成功后,对SCI+AACOCF3组大鼠腹腔注射cPLA2的特异性抑制剂AACOCF3。余两组大鼠腹腔注射生理盐水。分别于造模后7、14 d分批处死动物,于受损脊髓组织取材,行组织形态学观察,检测cPLA2和多种自噬通量相关分子的表达,测试运动功能的恢复情况。结果:脊髓组织形态检测显示:假模型组脊髓组织结构完整,神经元和胶质细胞数量和形态正常。在SCI组可见脊髓组织断裂,有大而突出的脊髓空洞。在SCI+AACOCF3组,脊髓组织比SCI组更完整,可见脊髓空洞融合,有更多存活的神经元,胶质细胞增生较少。Western blot检测显示:同SCI组和SCI+AACOCF3组相比,假模型组的LC3-Ⅱ、Beclin 1、p62和cPLA2的蛋白表达量最低(P<0.05),LC3-Ⅰ的蛋白表达最高(P<0.05)。SCI组的p62和cPLA2表达高于SCI+AACOCF3组(P<0.05)。行为学观察显示:SCI组和SCI+AACOCF3组BBB法(basso,beattie,bresnahan)运动评分对应时间均明显低于假模型组(P<0.05);SCI+AACOCF3组在加压后3、7、14 d的运动评分均高于SCI组(P<0.05)。结论:cPLA2抑制剂可通过改善溶酶体膜通透性,调节自噬通量,减轻SCI继发性损伤对神经元损害,促进神经功能恢复,改善运动功能。

Objective To investigate the mechanism of cytosolic phospholipase A2(cPLA2)inhibitor to improve neurological function after spinal cord injury(SCI).Methods Thirty-six 3 months old female SD rats,with body mass(280±20)g,were divided into three groups(n=12):sham group,SCI group,and SCI+arachidonyl trifluoromethyl ketone(AACOCF3)group.Balloon compression SCI model was established in all three groups.In the sham model group,the spinal cord compression model was created after the balloon was placed without pressure treatment,and the remaining two groups were pressurized with the balloon for 48 h.After successful modeling,rats in the SCI+AACOCF3 group were injected intraperitoneally with AACOCF3,a specific inhibitor of cPLA2.The remaining two groups of rats were injected intraperitoneally with saline.The animals were sacrificed in batches on 7 and 14 days after modeling,respectively.And the damaged spinal cord tissues were sampled for pathomorphological observation,to detect the expression of cPLA2 and various autophagic fluxPrelated molecules and test the recovery of motor function.Results Spinal cord histomorphometry examination showed that the spinal cord tissue in the sham group was structurally intact,with normal numbers and morphology of neurons and glial cells.In the SCI group,spinal cord tissue fractures with large and prominent spinal cord cavities were seen.In the SCI+AACOCF3 group,the spinal cord tissue wasmore intact than in the SCI group,with more fused spinal cord cavities,more surviving neurons,and less glial cell hyperplasia.Western blot showed that the sham group had the lowest protein expression of LC3-Ⅱ,Beclin 1,p62,and cPLA2 compared with the SCI and SCI+AACOCF3 groups(P<0.05)and the highest protein expression of LC3-Ⅰ(P<0.05).P62 and cPLA2 expression in the SCI group were higher than in the SCI+AACOCF3 group(P<0.05).Behavioral observations showed that the time corresponding to BBB exercise scores was significantly lower in both the SCI and SCI+AACOCF3 groups than in the sham group(P<0.05).Scores at 3,7,and 14 days after pressurization were higher in the SCI+AACOCF3 group than in the SCI group(P<0.05).Conclusion cPLA2 inhibitors can reduce neuronal damage secondary to SCI,promote neurological recovery and improve motor function by improving lysosomal membrane permeability and regulating autophagic flux.