内质网应激在医用臭氧导致的大鼠脊髓神经毒性机制中的作用

doi:10.6040/j.issn.1671-7554.0.2018.296

摘要/Abstract

摘要： 目的在体内实验中通过评价臭氧对大鼠脊髓神经细胞中内质网应激相关蛋白表达的影响,探讨内质网应激在臭氧脊髓神经毒性机制中的可能作用。方法将24只健康清洁级SD大鼠随机分为3组:对照组、低浓度组和高浓度组(每组8只),通过大鼠鞘内置管注射臭氧来模拟椎间盘内和硬膜外腔注射治疗中臭氧进入蛛网膜下腔的过程。对照组仅行鞘内穿刺置管不注射医用臭氧,实验组鞘内置管后分别经导管注射30 μg/mL(低浓度组)和60 μg/mL(高浓度组)医用臭氧10 μL。医用臭氧鞘内注射后24 h,每组取3只大鼠,断颈取材,采用Western blotting测定L_2~4节段脊髓组织中GRP78、CHOP、Caspase-12和Sig-1R蛋白表达水平;所有大鼠在注射臭氧前及注射后12 d,每日对大鼠进行步态评分测定及记录。结果与对照组相比,鞘内注射低浓度和高浓度的医用臭氧后,大鼠步态评分升高,差异有统计学意义(P<0.05),且高浓度组大鼠后肢运动功能恢复时间长于低浓度组。Western blotting结果显示,与对照组相比,低浓度和高浓度臭氧作用24 h后脊髓神经细胞中内质网应激相关蛋白GRP78(P<0.05)、Sig-1R(P<0.05)、内质网凋亡相关蛋白CHOP(P<0.05)和Caspase-12(P<0.05)的表达水平均增加(P<0.05),差异有统计学意义。与低浓度组相比,高浓度组臭氧作用24 h后脊髓神经细胞中内质网应激相关蛋白GRP78、Sig-1R、内质网凋亡相关蛋白CHOP和Caspase-12的表达水平增加,差异有统计学意义(P<0.05)。结论在体内实验中,低浓度和高浓度的医用臭氧均可引起脊髓神经元发生内质网应激并最终导致细胞凋亡,导致大鼠运动功能发生不同程度的损伤。其机制可能是低浓度医用臭氧激活CHOP途径的细胞凋亡,而高浓度医用臭氧可以激活CHOP和Caspase-12途径的细胞凋亡。

关键词: 臭氧, 内质网应激, 凋亡, Sigma-1受体, 脊髓神经毒性, 大鼠

Abstract: Objective To investigate the effects of ozone on the expression of endoplasmic reticulum stress associated protein in rats spinal cord neurons in vivo, and to explore the mechanism of endoplasmic reticulum stress in ozone-induced neurotoxicity. Methods A total of 24 healthy and clean SD rats were randomly divided into 3 groups, control group, high concentration group and low concentration group, with 8 rats in each group. The process of ozone entering the subarachnoid space through intervertebral disc and epidural treatment was simulated by injecting ozone into the built-in tube of rats. Rats in the control group were intrathecally catheterized without any medication, while rats in the 60 μg/mL ozone group and 30μg/mL ozone group were intrathecally administered 10 μL 60 μg/mL and 30 μg/mL oxygen-ozone respectively. Twenty-four hours after the injection, 3 rats in each group were sacrificed, and the lumbar spinal cords were collected to detect the protein expressions of GRP78, CHOP, Caspase-12 and Sig-1R with Western blotting. The gait scores of all rats were recorded before and after oxygen-ozone injection once a day for 12 days. Results Compared 山东大学学报 (医学版)57卷3期 -温雪彬,等.内质网应激在医用臭氧导致的大鼠脊髓神经毒性机制中的作用 \=-with the control group, the experimental groups had significantly higher gait scores(P<0.05). The 60 μg/mL group had longer recovery time of hind limb motor function than the 30 μg/mL group. Compared with the control group, the experimental groups had significantly higher expressions of endoplasmic reticulum stress related protein GRP78, CHOP, Caspase-12 and Sig-1R(all P<0.05), and the 60μg/mL group showed higher expressions than the 30 μg/mL group(all P<0.05). Conclusion In vivo, both high and low concentration medical ozone can cause damage on rats’ motor function. The mechanism may be explained that a low concentration of medical ozone activates the apoptosis through CHOP pathway, while high concentration of medical ozone can activate apoptosis through both CHOP and Caspase-12 pathway.

Key words: Ozone, Endoplasmic reticulum stress, Apoptosis, Sigma-1 receptor, Spinal cord neurotoxicity, Rat

中图分类号:

R614.4+2

温雪彬,傅志俭. 内质网应激在医用臭氧导致的大鼠脊髓神经毒性机制中的作用[J]. 山东大学学报 (医学版), 2019, 57(3): 7-12.

WEN Xuebin, FU Zhijian. Role of endoplasmic reticulum stress in ozone-induced neurotoxicity in rats[J]. Journal of Shandong University (Health Sciences), 2019, 57(3): 7-12.

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