膜联蛋白A1激活FPR2/ALX依赖性AMPK/mTOR通路改善创伤性脊髓损伤的机制研究

Annexin A1 protects against traumatic spinal cord injuries by modulating microglia/macrophage polarization via FPR2/ALX-dependent AMPK-mTOR pathway

目的探讨膜联蛋白A1(ANXA1)是否通过激活甲酰受体2/脂蛋白A4受体(FPR2/ALX)依赖的单磷酸腺苷活化蛋白激酶/雷帕霉素靶蛋白(AMPK/mTOR)通路改善创伤性脊髓损伤(SCI)。方法将24只10周龄雄性SD大鼠按随机数表法分为A组(假手术组)、B组(SCI组)、C组[SCI+ANXA1模拟肽(Ac2-26)处理组]、D组(SCI+Ac2-26+WRW4处理组),各6只。建模前3 d,C组大鼠的脊髓T 10段蛛网膜下腔注射1 mg/kg Ac2-26,D组的脊髓T 10段蛛网膜下腔注射1 mg/kg Ac2-26和2.2 mg/kg WRW4,A组和B组注入等量的生理盐水,使用脊髓打击器(参数:高度6 cm,重量10 g)制备成年大鼠SCI模型,通过Basso-Beattie-Bresnahan(BBB)评分检测大鼠的运动功能,活性氧(ROS)试剂盒检测脊髓组织中ROS的含量,ELISA检测脊髓组织TNF-α、IL-1β、IL-6水平,蛋白免疫印迹法检测脊髓组织中单磷酸腺苷活化蛋白激酶(AMPK)、磷酸化单磷酸腺苷活化蛋白激酶(p-AMPK)、雷帕霉素靶蛋白(mTOR)、磷酸化雷帕霉素靶蛋白(p-mTOR)、微管相关轻链蛋白3(LC3)及选择性自噬接头蛋白1(p62)的表达水平。结果与A组比较,B组大鼠BBB评分显著降低(P<0.05);与B组比较,C组大鼠BBB评分[(7.8±1.9)分vs.(16.4±2.5)分]显著升高(P<0.05);与C组相比,D组大鼠BBB评分[(16.4±2.5)分vs.(10.1±1.5)分]升高(P<0.05);与A组比较,B组ROS含量、TNF-α、IL-6、IL-1β水平显著上升(P<0.05);与B组比较,C组ROS含量[(152.33±26.56)%vs.(106.00±22.76)%]、TNF-α[(490.77±13.50)ng/g vs.(268.19±10.94)ng/g]、IL-6[(356.48±13.20)ng/g vs.(194.23±11.60)ng/g]、IL-1β水平[(334.65±9.73)ng/g vs.(153.15±11.61)ng/g]降低(P<0.05)与C组比较,D组ROS含量[(106.00±22.76)%vs.(133.01±28.70)%]、TNF-α[(268.19±10.94)ng/g vs.(394.20±15.24)ng/g、IL-6(194.23±11.60)ng/g vs.(305.77±12.92)ng/g]、IL-1β水平[(153.15±11.61)ng/g vs.(258.74±10.02)ng/g]上升(P<0.05)。与A组比较,B组p-AMPK/AMPK、LC3-Ⅱ/Ⅰ表达水平下调(P<0.05),p-mTOR/mTOR、p62表达水平上调(P<0.05);与B组比较,C组p-AMPK/AMPK(0.32±0.06 vs.0.53±0.09)、LC3-Ⅱ/Ⅰ表达水平(0.21±0.04 vs.0.66±0.08)上调(P<0.05),p-mTOR/mTOR(1.38±0.14 vs.0.76±0.06)、p62表达水平(1.27±0.09 vs.0.79±0.06)下调(P<0.05);与C组比较,D组p-AMPK/AMPK(0.53±0.09 vs.0.43±0.08)、LC3-Ⅱ/Ⅰ表达水平(0.66±0.08 vs.0.57±0.06)下调(P<0.05),p-mTOR/mTOR(0.76±0.06 vs.1.22±0.12)、p62表达水平(0.79±0.06 vs.1.10±0.11)上调(P<0.05)。结论ANXA1具有改善SCI的作用,其机制可能是通过激活FPR2/ALX依赖的AMPK/mTOR通路上调自噬水平、抑制ROS和炎症介质释放介导。

Objective To investigate whether annexin A1(ANXA1)improves traumatic spinal cord injuries(SCIs)by activating the G protein-coupled formyl peptide receptor type 2/lipoxin A4 receptor(FPR2/ALX)-dependent adenosine monophosphate activated protein kinase/mammalian target of rapamycin(AMPK/mTOR)pathway.Methods Twenty-four male SD rats of 10-week old were randomly divided into sham group(group A),SCI group(group B),SCI+Ac2-26 group(treated by mimetic peptide of ANXA1,group C)and SCI+Ac2-26+WRW4 group(antagonism of ANXA1 role,group D),with 6 rats in each group.On the third day before modeling,rats in group C were injected with 1 mg/kg Ac2-26 via the subarachnoid space of T 10 segment,those in group D were injected with 1 mg/kg Ac2-26 and 2.2 mg/kg WRW4 while others in groups A and B with equivalent normal saline via the same segment.The SCI model was established by a spinal cord percussion device(height of 6 cm and weight of 10 g).The motor function of the rats was detected by Basso-Beatie-Bresnahan(BBB)score,and the level of reactive oxygen species(ROS)in the spinal tissue was detected by a ROS kit.The levels of TNF-α,IL-1βand IL-6 in the spinal cord tissue were detected by ELISA,while the expression levels of adenosine monophosphate activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),microtubule-associated light chain protein 3(LC3)and selective autophagy adaptor protein 1(p62)were detected by western blot.Results Compared with group A,the BBB score of group B was significantly decreased(P<0.05),indicating success modeling;compared with group B,the BBB score of group C was significantly increased,indicating a protection role of the ANXA1;compared with group C,the BBB score of rats in group D was significantly increased,indicating an antagonism effect(groups B-D:7.8±1.9 vs.16.4±2.5 vs.10.1±1.5,both P<0.05).Other variables showed a similar trend.Comparison between groups A and B,B and C,C and D showed significantly lower levels in groups A and C(all P<0.05)in terms of ROS(groups B-D,152.33%±26.56%vs.106.00±22.76%vs.133.01%±28.70%),TNF-α(ng/g,groups B-D,490.77±13.50 vs.268.19±10.94 vs.394.20±15.24),IL-6(ng/g,groups B-D,356.48±13.20 vs.194.23±11.60 vs.305.77±12.92)and IL-1β(ng/g,groups B-D,334.65±9.73 vs.153.15±11.61 vs.258.74±10.02).As for the autophagy-related proteins,group C also showed the best results compared to groups B and D respectively(all P<0.05),with significantly up-regulated p-AMPK/AMPK(groups B-D,0.32±0.06 vs.0.53±0.09 vs.0.43±0.08)and LC3-Ⅱ/Ⅰ(groups B-D,0.21±0.04 vs.0.66±0.08 vs.0.57±0.06)and down-regulated p-mTOR/mTOR(groups B-D,1.38±0.14 vs.0.76±0.06 vs.1.22±0.12)and p62(1.27±0.09 vs.0.79±0.06 vs.1.10±0.11).Conclusion ANXA1 can protect against SCIs,which may be mediated by activation of FPR2/ALX-dependent AMPK/mTOR pathways to up-regulate the autophagy level and inhibit ROS and release of inflammatory mediators.