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跑台运动通过调节大鼠前扣带回线粒体自噬改善神经病理性疼痛

李翠^1,2,3, 王晓歌^1,3, 杨帅^1,3, 吕怡杭^1,3, 高晓娟¹, 曹靖^2,3, 臧卫东^2,3,*

¹郑州大学体育学院(校本部)，郑州 450001；²郑州大学基础医学院，郑州 450001；³郑州大学神经科学研究所，郑州 450001

摘要

本研究旨在考察跑台运动对神经病理性疼痛的影响，并探讨大鼠前扣带回皮质(anterior cingulate cortex, ACC)线粒体自噬在运动改善神经病理性疼痛中的潜在作用。为了探讨神经病理性疼痛对线粒体自噬的影响，采用慢性坐骨神经压迫损伤(chronic constriction injury of the sciatic nerve, CCI)方法建立神经病理性疼痛Sprague-Dawley (SD)大鼠模型。Von-Frey丝检测大鼠的机械性缩足反射阈值(paw withdrawal threshold, PWT)，热辐射仪检测大鼠的热缩足潜伏期(paw withdrawal latency, PWL)，qPCR检测ACC组织中线粒体自噬相关Pink1、Parkin、Fundc1、Bnip3 mRNA的表达，Western blot检测PINK1、PARKIN蛋白水平。为了探讨线粒体自噬诱导剂羰基氰化物间氯苯腙(carbonyl cyanide m-chlorophenylhydrazone, CCCP)激活线粒体自噬对CCI大鼠痛行为的影响，将24只SD大鼠随机分为CCI对照组(CCI+Veh)、CCI+CCCP低剂量组(CCI+CCCP0.25)、CCI+CCCP中剂量组(CCI+CCCP2.5)和CCI+CCCP高剂量组(CCI+CCCP5)，在第0、1、3、5、7天检测疼痛行为。为了探究跑台运动是否通过调节线粒体自噬来调控疼痛，将24只SD大鼠分为假手术组(Sham)、CCI组、CCI+运动(CCI+Exe)组，CCI+Exe组在造模一周后进行4周的中低强度跑台训练，在0、7、14、21、35天检测各组大鼠机械痛和热痛行为。Western blot检测ACC线粒体自噬蛋白PINK1、PARKIN、LC3 II/LC3 I、P62含量的变化；透射电镜观察ACC线粒体的超微结构。结果显示：(1)与Sham组相比，CCI组术侧PWT与PWL均显著下降(P < 0.001)；ACC中Pink1 mRNA和蛋白表达显著上升，而Parkin mRNA和蛋白表达显著下降(P < 0.05)。(2)与CCI+Veh组相比，CCCP各剂量组疼痛阈值均有上升，ACC中PINK1、LC3 II/LC3 I蛋白表达显著上升(P < 0.05, P < 0.01)。(3)与CCI组相比，CCI+Exe组PWT和PWL均显著上升(P < 0.001, P < 0.01)，ACC中PINK1、P62蛋白表达显著下降(P < 0.001, P < 0.01)，PARKIN、LC3 II/LC3 I蛋白表达显著上升(P < 0.01, P < 0.05)，且CCI+Exe组ACC出现杆状线粒体形态，未见明显线粒体破碎、肿胀或空泡。以上结果提示，神经病理性疼痛模型大鼠ACC线粒体PINK1/PARKIN自噬通路受阻，跑台运动可通过调节PINK1/PARKIN通路恢复线粒体稳态，缓解神经病理性疼痛。

关键词： 跑台运动; 神经病理性疼痛; 前扣带回皮质; 线粒体自噬

Treadmill exercise alleviates neuropathic pain by regulating mitophagy of the anterior cingulate cortex in rats

LI Cui^1,2,3, WANG Xiao-Ge^1,3, YANG Shuai^1,3, LYU Yi-Hang^1,3, GAO Xiao-Juan¹, CAO Jing^2,3, ZANG Wei-Dong^2,3,*

¹School of Physical Education (Main Campus), Zhengzhou University, Zhengzhou 450001, China；²School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China；³Institute of Neuroscience, Zhengzhou University, Zhengzhou 450001, China

Abstract

This study aimed to investigate the effect of treadmill exercise on neuropathic pain and to determine whether mitophagy of the anterior cingulate cortex (ACC) contributes to exercise-mediated amelioration of neuropathic pain. Chronic constriction injury of the sciatic nerve (CCI) was used to establish a neuropathic pain model in Sprague-Dawley (SD) rats. Von-Frey filaments were used to assess the mechanical paw withdrawal threshold (PWT), and a thermal radiation meter was used to assess the thermal paw withdrawal latency (PWL) in rats. qPCR was used to evaluate the mRNA levels of Pink1, Parkin, Fundc1, and Bnip3. Western blot was used to evaluate the protein levels of PINK1 and PARKIN. To determine the impact of the mitophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP) on pain behaviors in CCI rats, 24 SD rats were randomly divided into CCI drug control group (CCI+Veh group), CCI+CCCP low-dose group (CCI+CCCP0.25), CCI+CCCP medium-dose group (CCI+CCCP2.5), and CCI+CCCP high-dose group (CCI+CCCP5). Pain behaviors were assessed on 0, 1, 3, 5, and 7 days after modeling. To explore whether exercise regulates pain through mitophagy, 24 SD rats were divided into sham, CCI, and CCI+Exercise (CCI+Exe) groups. The rats in the CCI+Exe group underwent 4-week low-moderate treadmill training one week after modeling. The mechanical pain and thermal pain behaviors of the rats in each group were assessed on 0, 7, 14, 21, and 35 days after modeling. Western blot was used to detect the levels of the mitophagy-related proteins PINK1, PARKIN, LC3 II/LC3 I, and P62 in ACC tissues. Transmission electron microscopy was used to observe the ultrastructure of mitochondrial morphology in the ACC. The results showed that: (1) Compared with the sham group, the pain thresholds of the ipsilateral side of the CCI group decreased significantly (P < 0.001). Meanwhile, the mRNA and protein levels of Pink1 were significantly higher, and those of Parkin were lower in the CCI group (P < 0.05). (2) Compared with the CCI+Veh group, each CCCP-dose group showed higher mechanical and thermal pain thresholds, and the levels of PINK1 and LC3 II/LC3 I were elevated significantly (P < 0.05, P < 0.01). (3) The pain thresholds of the CCI+Exe group increased significantly compared with those of the CCI group after treadmill intervention (P < 0.001, P < 0.01). Compared with the CCI group, the protein levels of PINK1 and P62 were decreased (P < 0.001, P < 0.01), and the protein levels of PARKIN and LC3 II/LC3 I were increased in the CCI+Exe group (P < 0.01, P < 0.05). Rod-shaped mitochondria were observed in the ACC of CCI+Exe group, and there were little mitochondrial fragmentation, swelling, or vacuoles. The results suggest that the mitochondrial PINK1/PARKIN autophagy pathway is blocked in the ACC of neuropathic pain model rats. Treadmill exercise could restore mitochondrial homeostasis and relieve neuropathic pain via the PINK1/PARKIN pathway. 

Key words: treadmill exercise; neuropathic pain; anterior cingulate cortex; mitophagy

收稿日期：　　录用日期：

通讯作者：臧卫东　　E-mail: zwd@zzu.edu.cn

DOI: 10.13294/j.aps.2022.0070

引用本文：

李翠, 王晓歌, 杨帅, 吕怡杭, 高晓娟, 曹靖, 臧卫东. 跑台运动通过调节大鼠前扣带回线粒体自噬改善神经病理性疼痛[J]. 生理学报 2023; 75 (2): 160-170.

LI Cui, WANG Xiao-Ge, YANG Shuai, LYU Yi-Hang, GAO Xiao-Juan, CAO Jing, ZANG Wei-Dong. Treadmill exercise alleviates neuropathic pain by regulating mitophagy of the anterior cingulate cortex in rats. Acta Physiol Sin 2023; 75 (2): 160-170 (in Chinese with English abstract).