大鼠坐骨神经损伤后局部自噬激活的动态变化
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摘要
目的观察大鼠坐骨神经压迫(CCI)模型中,自噬在坐骨神经中的动态变化。方法将36只雄性SD大鼠分为假手术组(Sham组)和坐骨神经结扎组(CCI组)各18只。以大鼠机械性缩足反射阈值评价疼痛,于术前1 d测定足底基础痛阈值,术后1、4、7 d再次评定。术后4、7 d,取术侧坐骨神经,采用蛋白免疫印迹法检测微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、自噬底物P62表达水平,用免疫荧光标记LC3,于透射电镜下观察自噬体的形成。结果 CCI模型中大鼠疼痛阈值降低,具有明显病理性疼痛行为学改变。与Sham组比较,CCI组术后4 d LC3-Ⅱ蛋白水平增高,P62蛋白表达减少(P均<0.01);至术后7 d,LC3-Ⅱ蛋白水平进一步上升(P<0.001),P62蛋白表达水平也较术后4d高(P<0.05)。电镜下可见,CCI组中的术后4 d组及术后7 d组的施万细胞及神经元轴突均出现自噬体。结论在大鼠CCI模型中,坐骨神经轴突及施万细胞的自噬被激活,且大鼠坐骨神经损伤后局部自噬激活发生了动态变化。
Objective To observe the dynamic changes of autophagy in the sciatic nerve of the rat models with chronic constriction injury( CCI) of the sciatic nerve. Methods Thirty-six male Sprague-Dawley rats were randomly divided into the sham-operation group( n = 18) and the CCI group( n = 18). The degree of pain was assessed by the paw mechanical withdrawal threshold in rats. The baseline pain threshold was measured 1 day before surgery. The pain was assessed again at 1,4 and 7 days after surgery. The operated sciatic nerve was obtained at postoperative 4 and 7 days. Expression of microtubule associated protein light chain 3-Ⅱ( LC3-Ⅱ) and P62 were detected by Western blot. Immunofluorescence was utilized for LC3 labeling. The formation of autophagosome was observed under transmission electron microscopy. Results In the CCI rat models,the pain threshold was decreased and evident neuropathic pain behavioral changes were observed. Compared with the sham-operation group,the expression of LC3-Ⅱ was significantly up-regulated,whereas that of P62 was considerably down-regulated in the CCI group at postoperative 4 days( both P < 0. 01). At postoperative 7 days,the expression of LC3-Ⅱ was further up-regulated( P < 0. 001),whereas that of P62 was further down-regulated( P < 0. 05). Under the electron microscopy,autophagosome was observed in the Schwann cells and neuron axons of sciactic nerve in the CCI group at postoperative 4 and 7 days. Conclusions In the CCI rat models,the autophagy of the sciatic nerve axon and Schwann cells is activated. Dynamic changes occur in the topical activation of autophagy after sciatic nerve injury.
Objective To observe the dynamic changes of autophagy in the sciatic nerve of the rat models with chronic constriction injury( CCI) of the sciatic nerve. Methods Thirty-six male Sprague-Dawley rats were randomly divided into the sham-operation group( n = 18) and the CCI group( n = 18). The degree of pain was assessed by the paw mechanical withdrawal threshold in rats. The baseline pain threshold was measured 1 day before surgery. The pain was assessed again at 1,4 and 7 days after surgery. The operated sciatic nerve was obtained at postoperative 4 and 7 days. Expression of microtubule associated protein light chain 3-Ⅱ( LC3-Ⅱ) and P62 were detected by Western blot. Immunofluorescence was utilized for LC3 labeling. The formation of autophagosome was observed under transmission electron microscopy. Results In the CCI rat models,the pain threshold was decreased and evident neuropathic pain behavioral changes were observed. Compared with the sham-operation group,the expression of LC3-Ⅱ was significantly up-regulated,whereas that of P62 was considerably down-regulated in the CCI group at postoperative 4 days( both P < 0. 01). At postoperative 7 days,the expression of LC3-Ⅱ was further up-regulated( P < 0. 001),whereas that of P62 was further down-regulated( P < 0. 05). Under the electron microscopy,autophagosome was observed in the Schwann cells and neuron axons of sciactic nerve in the CCI group at postoperative 4 and 7 days. Conclusions In the CCI rat models,the autophagy of the sciatic nerve axon and Schwann cells is activated. Dynamic changes occur in the topical activation of autophagy after sciatic nerve injury.
引文
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[2]van Hecke O,Austin SK,Khan RA,Smith BH,Torrance N.Neuropathic pain in the general population:a systematic review of epidemiological studies.Pain,2014.155(4):654-662.
[3]Blyth FM.Global burden of neuropathic pain.Pain,2018,159(3):614-617.
[4]Ke PY.Horning cell self-digestion:autophagy wins the 2016 Nobel prize in physiology or medicine.Biomed J,2017,40(1):5-8.
[5]李文,胡喆,林智君,杨玥,陈煜森,钟望涛,林春霞,赵斌,冯杜.miR-137对Parkin诱导的线粒体自噬的影响.新医学,2015,46(5):283-288.
[6]Nixon RA.The role of autophagy in neurodegenerative disease.Nat Med,2013,19(8):983-997.
[7]Askanas V,Engel WK,Nogalska A.Sporadic inclusion-body myositis:a degenerative muscle disease associated with aging,impaired muscle protein homeostasis and abnormal mitophagy.Biochim Biophys Acta,2015,1852(4):633-643.
[8]Chen L,Zhang B,Toborek M.Autophagy is involved in nanoalumina-induced cerebrovascular toxicity.Nanomedicine,2013,9(2):212-221.
[9]刘永达,韩光,王志彬,赵平.两种不同神经病理性疼痛模型中自噬的改变.中国疼痛医学杂志,2017,23(7):489-493.
[10]潘勤,柏晓勇.脊髓神经元自噬与大鼠糖尿病神经病理性疼痛关系的研究.中国糖尿病杂志,2016,24(11):1016-1020.
[11]Berliocchi L,Maiaru M,Varano GP,Russo R,Corasaniti MT,Bagetta G,Tassorelli C.Spinal autophagy is differently modulated in distinct mouse models of neuropathic pain.Mol Pain,2015.11:3.
[12]陈红光,陈亚军,谢克亮,于泳浩.自噬在神经病理性痛大鼠脊髓星形胶质细胞活化中的作用.中华麻醉学杂志,2015,35(11):1307-1309.
[13]Bennett GJ,Xie YK.A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.Pain,1988,33(1):87-107.
[14]Chaplan SR,Bach FW,Pogrel JW,Chung JM,Yaksh TL.Quantitative assessment of tactile allodynia in the rat paw.J Neurosci Methods,1994,53(1):55-63.
[15]Shibutani ST,Yoshimori T.A current perspective of autophagosome biogenesis.Cell Res,2014,24(1):58-68.
[16]Piao ZX,Wang WS,Xu X,Wang QW,Huo X,Han MH,Piao YJ.Autophagy of neuron axon during regeneration of rat sciatic nerves.第一军医大学学报,2004,24(4):361-364.
[17]Lee YK,Lee JA.Role of the mammalian ATG8/LC3 family in autophagy:differential and compensatory roles in the spatiotemporal regulation of autophagy.BMB Rep,2016,49(8):424-430.
[18]Lippai M,Low P.The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy.Biomed Res Int,2014,2014:832704.
[19]Marinelli S,Nazio F,Tinari A,Ciarlo L,D'Amelio M,Pieroni L,Vacca V,Urbani A,Cecconi F,Malorni W,Pavone F.Schwann cell autophagy counteracts the onset and chronification of neuropathic pain.Pain,2014,155(1):93-107.
[20]Gautron M,Jazat F,Ratinahirana H,Hauw JJ,Guilbaud G.Alterations in myelinated fibres in the sciatic nerve of rats after constriction:possible relationships between the presence of abnormal small myelinated fibres and pain-related behaviour.Neurosci Lett,1990,111(1-2):28-33.