基于PI3K/PTEN/AKT信号途径探讨褪黑素对脊髓损伤大鼠突触可塑性的影响
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摘要
目的:探讨褪黑素对脊髓损伤大鼠突触可塑性的影响及磷脂酰肌醇3-激酶/张力蛋白同源基因/蛋白激酶B(PI3K/PTEN/AKT)信号途径在其中的作用。方法:选择4月龄SPF级雄性SD大鼠48只,将其随机分为对照组(CON)、模型组(SCI)、褪黑素组(MT)和褪黑素受体拮抗剂组(LUZ),每组12只大鼠。对照组大鼠背部切口后缝合,余下各组大鼠使用改良的Allen's法建立T9水平的脊髓损伤模型。模型建立后,褪黑素组及褪黑素受体拮抗剂组每天腹腔注射褪黑素及褪黑素抑制剂,剂量为12.5 mg·kg~(-1)·d~(-1),对照组和模型组每天注射同体积的生理盐水。治疗后第3、7、14、21、28天进行BBB评分,实验结束处死大鼠取胸椎8-10节段脊髓组织,分别采用免疫组化方法测尼氏小体数量及Western Blot检测PTEN、Synapsin、PSD-95、Gap-43、Akt蛋白的表达。结果:与SCI模型大鼠相比,MT给药干预14 d后的SCI大鼠BBB评分及痛觉压力值均明显降低(P<0.05),尼氏小体灰度值提高(P <0.05),PTEN、Synapsin、PSD-95、Gap-43、Akt蛋白的表达均显著上调(P <0.05)。结论:MT可能通过激活PI3K/PTEN/Akt信号途径,上调突触可塑性相关蛋白的表达,促进SCI大鼠突触修复。
Objective: To investigate the effects of melatonin on synaptic plasticity in rats with spinal cord injury and the role of PI3 K/PTEN/AKT signaling pathway. Methods: Forty-eight SPF male Sprague-Dawley rats aged 4 months were randomly divided into control group(CON), model group(SCI), melatonin group(MT) and the melaclonin receptor antagonist group(Luz), 12 in each group.Rats in the control group were sutured after the incision, and the remaining groups of rats were used to establish a T9 level spinal cord injury model using the modified Allen's method. After the model was established, the rats in MT and LUZ were intraperitoneally injected with melatonin and melatonin inhibitor at a dose of 12.5 mg·kg~(-1)·d~(-1). Rats in CON and the SCI were injected with the same volume of physiological saline daily. BBB scores were performed on the 3 rd, 7 th, 14 th, 21 st, and 28 th day after treatment. At the end of the experiment, the rats were sacrificed and the spinal cord tissue of 8-10 segments was taken. The number of Nissl bodies and expressio of Synapsin, PSD-95, Gap-43, Akt protein. were detected by immunohistochemistry and Western Blot. Results: After 14 days of MT administration, the BBB score of SCI rats could be significantly reduced, the pain pressure value could be reduced, the gray value of Nissl bodies increased, and the expression of PTEN, Synapsin, PSD-95, Gap-43 and Akt protein could be up-regulated. Conclusion: MT may up-regulate the expression of synaptic plasticity-related proteins by activating the PI3 K/PTEN/Akt signaling pathway and promote synaptic repair in SCI rats.
Objective: To investigate the effects of melatonin on synaptic plasticity in rats with spinal cord injury and the role of PI3 K/PTEN/AKT signaling pathway. Methods: Forty-eight SPF male Sprague-Dawley rats aged 4 months were randomly divided into control group(CON), model group(SCI), melatonin group(MT) and the melaclonin receptor antagonist group(Luz), 12 in each group.Rats in the control group were sutured after the incision, and the remaining groups of rats were used to establish a T9 level spinal cord injury model using the modified Allen's method. After the model was established, the rats in MT and LUZ were intraperitoneally injected with melatonin and melatonin inhibitor at a dose of 12.5 mg·kg~(-1)·d~(-1). Rats in CON and the SCI were injected with the same volume of physiological saline daily. BBB scores were performed on the 3 rd, 7 th, 14 th, 21 st, and 28 th day after treatment. At the end of the experiment, the rats were sacrificed and the spinal cord tissue of 8-10 segments was taken. The number of Nissl bodies and expressio of Synapsin, PSD-95, Gap-43, Akt protein. were detected by immunohistochemistry and Western Blot. Results: After 14 days of MT administration, the BBB score of SCI rats could be significantly reduced, the pain pressure value could be reduced, the gray value of Nissl bodies increased, and the expression of PTEN, Synapsin, PSD-95, Gap-43 and Akt protein could be up-regulated. Conclusion: MT may up-regulate the expression of synaptic plasticity-related proteins by activating the PI3 K/PTEN/Akt signaling pathway and promote synaptic repair in SCI rats.
引文
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[2] Venkataraman P, Selvakumar K, Krishnamoorthy G, et al. Effect of melatonin on PCB(Aroclor 1254)induced neuronal damage and changes in Cu/Zn superoxide dismutase and glutathione peroxidase-4mRNA expression in cerebral cortex, cerebellum and hippocampus of adult rats[J]. Neuroscience Research, 2010, 66(2):189-197
[3]唐蛟,沈达勇,周苏,等.褪黑素对脑梗死的保护作用及研究进展[J].现代生物医学进展, 2017, 17(17):3397-3400
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[5] Slominski AT, Hardeland R, Zmijewski MA, et al. Melatonin:A Cutaneous Perspective on its Production, Metabolism, and Functions[J]. Journal of Investigative Dermatology, 2018, 138(3):490-499
[6]荆瀛黎,刘小野,白帆,等.褪黑素对脊髓损伤大鼠突触可塑性的作用[J].中国康复理论与实践, 2016, 22(7):774-778
[7] Zhao H, Chen S, Gao K, et al. Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway[J]. Neuroscience, 2017, 348:241-251
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[9] Jung SY, Kim DY, Yune TY, et al. Treadmill exercise reduces spinal cord injury-induced apoptosis by activating the PI3K/Akt pathway in rats[J]. Experimental&Therapeutic Medicine, 2014, 7(3):587-593
[10] Zhang P, Zhang L, Zhu L, et al. The change tendency of PI3K/Akt pathway after spinal cord injury[J]. American Journal of Translational Research, 2015, 7(11):2223
[11]刘杨,苗宇船.大鼠脊髓损伤早期PI3K/Akt/mTOR信号转导通路的活性改变及其与后肢运动功能恢复的关系[J].上海交通大学学报(医学版), 2015, 35(6):804-808
[12] Lee Y, Lee S, Lee SR, et al. Beneficial Effects of Melatonin Combined with Exercise on Endogenous Neural Stem/Progenitor Cells Proliferation after Spinal Cord Injury[J]. International Journal of Molecular Sciences, 2014, 15(2):2207-2222
[13]舒海华,李桥波,叶芳,等.环氧化酶-2抑制剂帕瑞昔布对急性芬太尼诱导的大鼠痛觉过敏的抑制作用[J].实用医学杂志, 2015(5):711-714
[14] Schuld C, Franz S, Brüggemann K, et al. International standards for neurological classification of spinal cord injury:impact of the revised worksheet(revision 02/13)on classification performance[J]. Journal of the American Paraplegia Society, 2016, 39(5):504-512
[15] Hutchins GM, Meuli M, Meuli-Simmen C, et al. Acquired spinal cord injury in human fetuses with myelomeningocele[J]. Pediatric Pathology, 2016, 16(5):701-712
[16] Mcdonald JW, Sadowsky C. Spinal-cord injury[J]. Lancet, 2002, 359(9304):417-425
[17]刘杨,高玉亭,苗宇船.丹参注射液对大鼠脊髓损伤后PI3K/Akt/mTOR信号转导通路活性及后肢运动功能的影响[J].中国实验方剂学杂志, 2017(14):152-157
[18] Beattie MS, Farooqui AA, Bresnahan J C. Review of current evidence for apoptosis after spinal cord injury[J]. Journal of Neurotrauma, 2000, 17(10):915
[19] Wang S, Liu Y, Wu C, et al. The Expression of IGFBP6 after Spinal Cord Injury:Implications for Neuronal Apoptosis[J]. Neurochemical Research, 2016, 42(2):1-13
[20]王晓英,张均田.突触可塑性与相关蛋白研究进展[J].中国药理学通报, 2001, 17(4):369-372
[21] Porseva VV, Korzina MB, Smirnova VP, et al. Developmental Changes in NF-200 Neurons in Sensory Ganglia at Different Segmental Levels on Chemical Deafferentation[J]. Neuroscience&Behavioral Physiology, 2013, 43(5):602-606
[22] Kong X, Li X,Cai Z, et al. Melatonin regulates the viability and differentiation of rat midbrain neural stem cells[J]. Cellular&Molecular Neurobiology, 2008, 28(4):569-579
[23] Jenwitheesuk A, Nopparat C, Mukda S, et al. Melatonin regulates aging and neurodegeneration through energy metabolism, epigenetics,autophagy and circadian rhythm pathways[J]. International Journal of Molecular Sciences, 2014, 15(9):16848-16884
[24] Guo Y, Wang F, Li H, et al. Metformin Protects Against Spinal Cord Injury by Regulating Autophagy via the mTOR Signaling Pathway[J].Neurochemical Research, 2018, 43(5):1111-1117
[25] Zhao BB, Long QH, Wang CY, et al. Protective Effects of Liu Wei Di Huang Wan on the Liver, Orbitofrontal Cortex Nissl Bodies, and Neurites in MSG+PH-Induced Liver Regeneration Rat Model[J].2018[Epub ahead of print]
[26] Li J, Wen P Y, Li WW, et al. Upregulation effects of Tanshinone IIA on the expressions of NeuN, Nissl body, and IκB and downregulation effects on the expressions of GFAP and NF-κB in the brain tissues of rat models of Alzheimer's disease[J]. Neuroreport, 2015, 26(13):758-766
[27] Caprini M, Gomis A, Cabedo H, et al. GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity[J]. Embo Journal, 2014, 22(12):3004-3014
[28] Biffo S, Verhaagen J, Schrama LH, et al. B-50/GAP43 Expression Correlates with Process Outgrowth in the Embryonic Mouse Nervous System[J]. European Journal of Neuroscience, 2010, 2(6):487-499
[29] Chi P, Greengard P, Ryan TA. Synapsin dispersion and reclustering during synaptic activity[J]. Nature Neuroscience, 2001, 4(12):1187-1193
[30] Barbieri R, Contestabile A, Ciardo MG, et al. Synapsin I and Synapsin II regulate neurogenesis in the dentate gyrus of adult mice[J]. Oncotarget, 2018, 9(27):18760-18774
[31] Tu Y, Nayak SK, Woodson J, et al. Phosphorylation-regulated inhibition of the Gz GTPase-activating protein activity of RGS proteins by synapsin I[J]. Journal of Biological Chemistry, 2003, 278(52):52273-52281
[2] Venkataraman P, Selvakumar K, Krishnamoorthy G, et al. Effect of melatonin on PCB(Aroclor 1254)induced neuronal damage and changes in Cu/Zn superoxide dismutase and glutathione peroxidase-4mRNA expression in cerebral cortex, cerebellum and hippocampus of adult rats[J]. Neuroscience Research, 2010, 66(2):189-197
[3]唐蛟,沈达勇,周苏,等.褪黑素对脑梗死的保护作用及研究进展[J].现代生物医学进展, 2017, 17(17):3397-3400
[4] He R, Cui M, Lin H, et al. Melatonin resists oxidative stress-induced apoptosis in nucleus pulposus cells[J]. Life Sciences, 2018, 199:122-130
[5] Slominski AT, Hardeland R, Zmijewski MA, et al. Melatonin:A Cutaneous Perspective on its Production, Metabolism, and Functions[J]. Journal of Investigative Dermatology, 2018, 138(3):490-499
[6]荆瀛黎,刘小野,白帆,等.褪黑素对脊髓损伤大鼠突触可塑性的作用[J].中国康复理论与实践, 2016, 22(7):774-778
[7] Zhao H, Chen S, Gao K, et al. Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway[J]. Neuroscience, 2017, 348:241-251
[8] Hemmings BA, Restuccia DF. PI3K-PKB/Akt Pathway[J]. Cold Spring Harbor Perspectives in Biology, 2012, 4(9):a011189
[9] Jung SY, Kim DY, Yune TY, et al. Treadmill exercise reduces spinal cord injury-induced apoptosis by activating the PI3K/Akt pathway in rats[J]. Experimental&Therapeutic Medicine, 2014, 7(3):587-593
[10] Zhang P, Zhang L, Zhu L, et al. The change tendency of PI3K/Akt pathway after spinal cord injury[J]. American Journal of Translational Research, 2015, 7(11):2223
[11]刘杨,苗宇船.大鼠脊髓损伤早期PI3K/Akt/mTOR信号转导通路的活性改变及其与后肢运动功能恢复的关系[J].上海交通大学学报(医学版), 2015, 35(6):804-808
[12] Lee Y, Lee S, Lee SR, et al. Beneficial Effects of Melatonin Combined with Exercise on Endogenous Neural Stem/Progenitor Cells Proliferation after Spinal Cord Injury[J]. International Journal of Molecular Sciences, 2014, 15(2):2207-2222
[13]舒海华,李桥波,叶芳,等.环氧化酶-2抑制剂帕瑞昔布对急性芬太尼诱导的大鼠痛觉过敏的抑制作用[J].实用医学杂志, 2015(5):711-714
[14] Schuld C, Franz S, Brüggemann K, et al. International standards for neurological classification of spinal cord injury:impact of the revised worksheet(revision 02/13)on classification performance[J]. Journal of the American Paraplegia Society, 2016, 39(5):504-512
[15] Hutchins GM, Meuli M, Meuli-Simmen C, et al. Acquired spinal cord injury in human fetuses with myelomeningocele[J]. Pediatric Pathology, 2016, 16(5):701-712
[16] Mcdonald JW, Sadowsky C. Spinal-cord injury[J]. Lancet, 2002, 359(9304):417-425
[17]刘杨,高玉亭,苗宇船.丹参注射液对大鼠脊髓损伤后PI3K/Akt/mTOR信号转导通路活性及后肢运动功能的影响[J].中国实验方剂学杂志, 2017(14):152-157
[18] Beattie MS, Farooqui AA, Bresnahan J C. Review of current evidence for apoptosis after spinal cord injury[J]. Journal of Neurotrauma, 2000, 17(10):915
[19] Wang S, Liu Y, Wu C, et al. The Expression of IGFBP6 after Spinal Cord Injury:Implications for Neuronal Apoptosis[J]. Neurochemical Research, 2016, 42(2):1-13
[20]王晓英,张均田.突触可塑性与相关蛋白研究进展[J].中国药理学通报, 2001, 17(4):369-372
[21] Porseva VV, Korzina MB, Smirnova VP, et al. Developmental Changes in NF-200 Neurons in Sensory Ganglia at Different Segmental Levels on Chemical Deafferentation[J]. Neuroscience&Behavioral Physiology, 2013, 43(5):602-606
[22] Kong X, Li X,Cai Z, et al. Melatonin regulates the viability and differentiation of rat midbrain neural stem cells[J]. Cellular&Molecular Neurobiology, 2008, 28(4):569-579
[23] Jenwitheesuk A, Nopparat C, Mukda S, et al. Melatonin regulates aging and neurodegeneration through energy metabolism, epigenetics,autophagy and circadian rhythm pathways[J]. International Journal of Molecular Sciences, 2014, 15(9):16848-16884
[24] Guo Y, Wang F, Li H, et al. Metformin Protects Against Spinal Cord Injury by Regulating Autophagy via the mTOR Signaling Pathway[J].Neurochemical Research, 2018, 43(5):1111-1117
[25] Zhao BB, Long QH, Wang CY, et al. Protective Effects of Liu Wei Di Huang Wan on the Liver, Orbitofrontal Cortex Nissl Bodies, and Neurites in MSG+PH-Induced Liver Regeneration Rat Model[J].2018[Epub ahead of print]
[26] Li J, Wen P Y, Li WW, et al. Upregulation effects of Tanshinone IIA on the expressions of NeuN, Nissl body, and IκB and downregulation effects on the expressions of GFAP and NF-κB in the brain tissues of rat models of Alzheimer's disease[J]. Neuroreport, 2015, 26(13):758-766
[27] Caprini M, Gomis A, Cabedo H, et al. GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity[J]. Embo Journal, 2014, 22(12):3004-3014
[28] Biffo S, Verhaagen J, Schrama LH, et al. B-50/GAP43 Expression Correlates with Process Outgrowth in the Embryonic Mouse Nervous System[J]. European Journal of Neuroscience, 2010, 2(6):487-499
[29] Chi P, Greengard P, Ryan TA. Synapsin dispersion and reclustering during synaptic activity[J]. Nature Neuroscience, 2001, 4(12):1187-1193
[30] Barbieri R, Contestabile A, Ciardo MG, et al. Synapsin I and Synapsin II regulate neurogenesis in the dentate gyrus of adult mice[J]. Oncotarget, 2018, 9(27):18760-18774
[31] Tu Y, Nayak SK, Woodson J, et al. Phosphorylation-regulated inhibition of the Gz GTPase-activating protein activity of RGS proteins by synapsin I[J]. Journal of Biological Chemistry, 2003, 278(52):52273-52281