摘要
目的探讨丙泊酚介导细胞自噬及雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路对脊髓损伤(spinal cord injury,SCI)小鼠的保护机制。方法将60只健康C57BL/6雌性小鼠随机分为假手术组(S组)、模型组(I组)、甲泼尼龙组(M组)和丙泊酚组(P组),每组15只。S组仅切除T9全椎板、不损伤脊髓,其余各组均采用Allen’s打击方法制备SCI模型。S组和I组均于术后30 min内腹腔注射等容量的0.9%生理盐水,M组和P组分别于造模成功后30 min内腹腔注射甲泼尼龙(30 mg/kg)和丙泊酚(50 mg/kg)。给药即刻、3 h、6 h时,采用改良Tarlov评分评估运动功能。给药6 h后,处死各组小鼠,取新鲜脊髓组织,H-E染色观察脊髓组织病理变化,透射电镜观察脊髓组织超微结构变化,Western blot法检测自噬相关蛋白P62、LC3Ⅱ及mTOR信号通路相关因子mTOR、p-mTOR蛋白的表达。结果随着给药时间的延长,M组和P组的Tarlov评分均升高,差异有统计学意义(P<0.05);给药即刻、3 h、6 h,I组、M组、P组的Tarlov评分均低于S组,差异有统计学意义(P<0.05);给药3 h、6 h,M组和P组的Tarlov评分均高于I组,差异有统计学意义(P<0.05);而给药3 h、6 h,M组和P组的Tarlov评分比较,差异无统计学意义(P>0.05)。S组小鼠脊髓结构完整,神经元形态正常且分布均匀,脊髓组织无出血、水肿,细胞呈现正常的超微结构;I组小鼠脊髓细胞肿胀、变形,正常神经元大量减少,坏死神经元增多,脊髓组织出血、水肿严重,细胞明显变性;M组和P组小鼠脊髓细胞肿胀、变形减轻,神经元坏死数目减少,正常神经元增多,脊髓组织内仅少部分区域可见间质水肿和出血,胶质细胞结构基本正常。与S组比较,I组P62、mTOR、p-mTOR蛋白表达明显上调,LC3Ⅱ蛋白表达明显下调,差异均有统计学意义(P<0.05);与I组比较,M组和P组P62、mTOR、p-mTOR蛋白表达下调,LC3Ⅱ蛋白表达上调,差异均有统计学意义(P<0.05);M组和P组P62、LC3Ⅱ、mTOR、p-mTOR蛋白表达差异无统计学意义(P>0.05)。结论丙泊酚对于SCI小鼠的神经保护作用可能是通过抑制mTOR信号通路、诱导细胞自噬来实现的。
Objective To explore the protective mechanisms of propofol-mediated cellular autophagy and mammalian target of rapamycin(mTOR)signalling pathway in mice with spinal cord injury(SCI).Methods Sixty healthy C57BL/6 female mice were randomly divided into the sham operation group(S group),model group(I group),methylprednisolone group(M group),and propofol group(P group),with 15 mice in each group.In the S group,only the whole T9 vertebral plate was resected without damaging the spinal cord,and SCI models were prepared by using Allen’s percussion method for the rest of the groups.Groups S and I were injected intraperitoneally with an equal volume of 0.9%saline within 30 min,and groups M and P were injected intraperitoneally with methylprednisolone(30 mg/kg)and propofol(50 mg/kg)within 30 min after successful modelling,respectively.The modified Tarlov score was used to evaluate the motor function at the moment of drug administration,3 h and 6 h.The spinal cord tissue of each group was killed after 6 h.The pathological changes of the spinal cord tissue were observed by HE staining.Fresh spinal cord tissues were taken from each group of mice after 6 h of drug administration,and the pathological changes of spinal cord tissues were observed by HE staining,ultrastructural changes of spinal cord tissues were observed by transmission electron microscopy,and the expression of autophagy-related proteins P62,LC3II,and mTOR and p-mTOR,which were related to the mTOR signalling pathway,were detected by Western blot.Results With the prolongation of the administration time,the Tarlov scores of both groups M and P increased,and the difference was statistically significant(P<0.05);at the moment of administration,3 h and 6 h,the Tarlov scores of groups I,M and P were lower than those of group S,and the difference was statistically significant(P<0.05);at the administration of 3 h and 6 h,the Tarlov scores of groups M and P were higher than those of group I,and the difference was statistically significant(P<0.05);while the Tarlov scores of groups M and P were compared at 3 h and 6 h of drug administration,the difference was not statistically significant(P>0.05).In group S mice,the spinal cord structure was intact,the neuron morphology was normal and uniformly distributed,there was no haemorrhage and oedema in the spinal cord tissue,and the cells showed normal ultrastructure;in group I mice,the spinal cord cells were swollen and deformed,the normal neurons were reduced in large number,the necrotic neurons were increased,the haemorrhage and oedema were severe in the spinal cord tissue,and the cells were obviously denatured;in mice in group M and group P,the spinal cord cells were swollen and deformed to reduce the swelling and deformity of neuron cells,the necrosis of neurons was reduced in number,the number of normal neurons was increased,the interstitial oedema and haemorrhage could be seen in a small portion of the spinal cord tissue,and the structure of glial cells was basically normal.Compared with group S,group I showed significant up-regulation of P62,mTOR,and p-mTOR protein expression,and significant down-regulation of LC3 II protein expression,and the differences were statistically significant(P<0.05);compared with group I,group M and group P showed down-regulation of P62,mTOR,and p-mTOR protein expression,and up-regulation of LC3 II protein expression,and the differences were statistically significant(P<0.05);there were no significant differences in the expression of P62,LC3 II,mTOR,and p-mTOR protein expression between group M and group P(P>0.05).Conclusion The neuroprotective effect of propofol in SCI mice may be achieved by inhibiting the mTOR signalling pathway and inducing cellular autophagy.
作者
吴驰
曹殿青
傅彩娟
WU Chi;CAO Dian-qing;FU Cai-juan(Affiliated Hospital of Guangdong Medical University,Zhanjiang,Guangdong 524022,China;Second Affiliated Hospital of Guangdong Medical University,Zhanjiang,Guangdong 524000,China)
出处
《颈腰痛杂志》
2024年第2期199-204,共6页
The Journal of Cervicodynia and Lumbodynia
基金
国家自然科学基金面上项目(编号:81771161)。
关键词
脊髓损伤
丙泊酚
细胞自噬
雷帕霉素靶蛋白信号通路
spinal cord injury
propofol
cellular autophagy
rapamycin target protein signalling pathway
作者简介
吴驰(1985-),男,广东籍,主治医师;通信作者:曹殿青。
