转位分子蛋白经由Keap1/Nrf2/HO-1通路激活自噬缓解大鼠糖尿病神经病理性疼痛

doi:10.3969/j.issn.1674-8115.2023.08.006

摘要/Abstract

摘要：

目的·探讨转位分子蛋白（translocator protein，TSPO）激动剂Ro5-4864对糖尿病神经病理性疼痛（diabetic neuropathic pain，DNP）大鼠坐骨神经自噬和Kelch样ECH关联蛋白（Kelch-like ECH-associated protein 1，Keap1）/核因子E2 相关因子（nuclear factor erythroid-derived-2-like 2，Nrf2）/血红素加氧酶-1（heme oxygenase-1，HO-1）通路的影响。方法·通过高脂饮食和链脲佐菌素构建2型糖尿病模型，经行为学筛选获得DNP大鼠。24只大鼠根据数字表随机分配到假手术组（Sham组）、DNP模型组（DNP组）、TSPO激动剂Ro5-4864处理组（Ro组）和TSPO激动剂Ro5-4864合用Nrf2抑制剂ML385处理组（Ro+ML385组）。采用up-down法测量大鼠50%机械缩足反射阈值（paw 50% mechanical withdrawal threshold，50% PMWT），时间点选定为处理前（baseline），处理后第3（D3）、7（D7）、14（D14）、21（D21）和28（D28）天。在最后1天收集坐骨神经，使用免疫荧光和Western blotting分析鞘内注射Ro5-4864对DNP大鼠坐骨神经中自噬相关蛋白和Keap1/Nrf2/HO-1通路相关蛋白的影响。结果·与Sham组相比，DNP组大鼠的50% PMWT自第3天开始大幅下降，全程无改善（各时间点均P=0.000）；另外，DNP大鼠坐骨神经中Bcl-2相互作用蛋白（Bcl-2 interacting coiled-coil protein 1，Beclin-1）、微管相关蛋白轻链3-Ⅱ（microtubule-associated protein light chain 3-Ⅱ，LC3-Ⅱ）、HO-1和核Nrf2蛋白的含量显著减少（均P=0.000），p62则显著增加（P=0.000）。鞘内注射Ro5-4864改善了以上损伤，大鼠50% PMWT与DNP组相比逐渐上升（D14 P=0.039，D21和D28均P=0.000），自噬和Keap1/Nrf2/HO-1通路的损伤也被修复，表现为Beclin-1、LC3-Ⅱ、HO-1和核Nrf2蛋白含量的升高（均P=0.000）以及p62含量的降低（P=0.001）。但是，Ro5-4864的效应被ML385完全抑制。结论·TSPO激动剂Ro5-4864缓解了DNP，其作用机制可能与通过对Keap1/Nrf2/HO-1通路的正向调节激活了DNP大鼠坐骨神经中的自噬相关。这可能为治疗DNP提供了一个新的策略。

关键词: 转位分子蛋白, 神经病理性疼痛, 糖尿病, 自噬, Keap1/Nrf2/HO-1通路

Abstract:

Objective ·To study the effects of translocator protein (TSPO) agonist Ro5-4864 on autophagy and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid-derived-2-like 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling in diabetic neuropathic pain (DNP) rats. Methods ·Type 2 diabetic rats were established by high-fat diet and streptozotocin (STZ), and DNP rats were filtered by behavioral assessment. Twenty-four rats were randomly assigned to the Sham group, DNP group, TSPO agonist Ro5-4864 group (Ro group), and TSPO agonist Ro5-4864 combined with Nrf2 inhibitor ML385 group (Ro+ML385 group). Up-Down method was used to measure paw 50% mechanical withdrawal threshold (50% PMWT) of the rats before high-fat diet (baseline), and on Day 3, 7, 14, 21 and 28 after STZ. Sciatic nerves were collected on the last day to analyze the effects of Ro5-4864 on autophagy related proteins and Keap1/Nrf2/HO-1 signaling related proteins of DNP rats by using immunofluorescence and Western blotting. Results ·The 50% PMWT in the DNP group decreased from D3 to D28 (P=0.000 at all timing), and the expression of Bcl-2 interacting coiled-coil protein 1 (Beclin-1), microtubule-associated protein light chain 3-Ⅱ (LC3-Ⅱ), HO-1, and nuclear Nrf2 (P=0.000) were significantly reduced in the sciatic nerves of DNP rats (all P=0.000), compared with those in the sham group, but p62 was significantly increased (P=0.000). Administration of Ro5-4864 attenuated these changes in the rats of the Ro group. There was a gradual increase in the 50% PMWT, compared with that of the rats in the DNP group (D14 P=0.039, both D21 and D28 P=0.000), and the impairment of autophagy and the Keap1/Nrf2/HO-1 signaling was repaired, which was demonstrated by increases of Beclin-1, LC3-Ⅱ, HO-1, and nuclear Nrf2 protein contents (all P=0.000) and a decrease in p62 content (P=0.001). However, the beneficial effects of Ro5-4864 were totally abrogated by ML385 in rats of the Ro+ML385 group. Conclusion ·TSPO alleviates DNP in rats, of which the mechanism involves activation of autophagy via upregulation of the Keap1/Nrf2/HO-1 signaling in sciatic nerves. This study provides a new strategy for the treatment of DNP.

Key words: translocator protein (TSPO), neuropathic pain (NP), diabetes, autophagy, Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid-derived-2-like 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling (Keap1/Nrf2/HO-1 signaling)

中图分类号:

R614

高楠, 郝璨, 马冰洁, 靳天, 马柯, 刘晓明. 转位分子蛋白经由Keap1/Nrf2/HO-1通路激活自噬缓解大鼠糖尿病神经病理性疼痛[J]. 上海交通大学学报（医学版）, 2023, 43(8): 988-996.

GAO Nan, HAO Gem, MA Bingjie, JIN Tian, MA Ke, LIU Xiaoming. Translocator protein activates autophagy in diabetic neuropathic pain rats via regulation of the Keap1/Nrf2/HO-1 signaling[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(8): 988-996.

图/表 7

图1 2型糖尿病大鼠并发神经病理性疼痛 (n=6)Note：A/B. The levels of HOMA-IR (A) and ISI (B) of the Sham group and the DNP group 3 d after STZ injection. C. 50% PMWT of the Sham group and the DNP group 3 d after STZ injection. ①P=0.000, compared with the Sham group.

Fig 1 T2DM rats showed pain hypersensitivity (n=6)

图2 鞘内注射TSPO激动剂Ro5-4864对DNP大鼠50% PMWT的影响 (n=6)Note：①P=0.000, compared with the Sham group; ②P=0.039, ③P=0.000, compared with the DNP group.

Fig 2 Effects of TSPO agonist Ro5-4864 on the 50% PMWT in DNP rats (n=6)

图3 鞘内注射TSPO激动剂Ro5-4864对DNP大鼠坐骨神经中LC3表达变化的影响 (n=3)Note： A. Representative immunofluorescent pictures of single staining of LC3 in the sciatic nerves of the rats. B. Quantitative analysis of LC3 signal intensity. ①P=0.000, compared with the Sham group; ②P=0.000, compared with the DNP group.

Fig 3 Effects of TSPO agonist Ro5-4864 on the change of LC3 expression in the sciatic nerves of DNP rats (n=3)

图4 鞘内注射TSPO激动剂Ro5-4864改善DNP大鼠坐骨神经中自噬相关蛋白表达的变化 (n=3)Note：A. Representative immunoblots of p62, Beclin-1 and LC3-II in the sciatic nerves of the rats. B?D. Illustration of the relative expression of p62 (B), Beclin-1 (C), and LC3-Ⅱ (D). ①P=0.000, compared with the Sham group; ②P=0.001, ③P=0.000, compared with the DNP group.

Fig 4 TSPO agonist Ro5-4864 improved the changes in the expression of autophagy-related proteins in the sciatic nerves of DNP rats (n=3)

图5 鞘内注射TSPO激动剂Ro5-4864改善DNP大鼠坐骨神经Nrf2表达的降低 (n=3)Note：A. Representative immunofluorescent pictures of single staining of Nrf2 in the sciatic nerves of the rats. B. Quantitative analysis of Nrf2 signal intensity. ①P=0.000, compared with the Sham group; ②P=0.000 compared with the DNP group.

Fig 5 TSPO agonist Ro5-4864 blocked the decrease in the expression of Nrf2 in the sciatic nerves of DNP rats (n=3)

图6 鞘内注射TSPO激动剂Ro5-4864对DNP大鼠坐骨神经中Keap1/Nrf2/HO-1通路相关蛋白表达的影响 (n=3)Note：A/B. Representative immunoblots of nuclear Nrf2 (A) and HO-1 (B). C/D. Illustration of the relative expression of nuclear Nrf2 (C) and HO-1 (D). ①P=0.000, compared with the Sham group; ②P=0.000, compared with the DNP group.

Fig 6 Effects of TSPO agonist Ro5-4864 on the expression of Keap1/Nrf2/HO-1 signaling-related proteins in the sciatic nerves of DNP rats (n=3)

图7 Nrf2抑制剂ML385对TSPO激动剂Ro5-4864作用的影响Note：A. Effects of Nrf2 inhibitor ML385 on the 50% PMWT of the rats (n=6). B. Representative immunoblots of nuclear Nrf2 and HO-1 in the sciatic nerves (n=3). C. Representative immunoblots of p62, Beclin-1 and LC3-Ⅱ (n=3). D/E. Illustration of the relative expression of nuclear Nrf2 (D) and HO-1 (E). F?H. Illustration of the relative expression of p62 (F), Beclin-1 (G), LC3-Ⅱ (H). ①P=0.000, ②P=0.006, ③P=0.003, compared with the Ro group.

Fig 7 Effects of Nrf2 inhibitor ML385 on the changes induced by TSPO agonist Ro5-4864 in DNP rats

参考文献 23

1	RHEE E J, KIM J H, MOON S J, et al. Encountering COVID-19 as endocrinologists[J]. Endocrinol Metab (Seoul), 2020, 35(2): 197-205.
2	FELDMAN E L, CALLAGHAN B C, POP-BUSUI R, et al. Diabetic neuropathy[J]. Nat Rev Dis Primers, 2019, 5: 41.
3	GALLUZZI L, GREEN D R. Autophagy-independent functions of the autophagy machinery[J]. Cell, 2019, 177(7): 1682-1699.
4	MARTANO M, ALTAMURA G, POWER K, et al. Beclin 1, LC3 and P62 expression in equine sarcoids[J]. Animals, 2021, 12(1): 20.
5	CAO W Y, LI J H, YANG K P, et al. An overview of autophagy: mechanism, regulation and research progress[J]. Bull Cancer, 2021, 108(3): 304-322.
6	KLIONSKY D J, PETRONI G, AMARAVADI R K, et al. Autophagy in major human diseases[J]. EMBO J, 2021, 40(19): e108863.
7	CHUNG Y C, LIM J H, OH H M, et al. Calcimimetic restores diabetic peripheral neuropathy by ameliorating apoptosis and improving autophagy[J]. Cell Death Dis, 2018, 9(12): 1163.
8	YU C, XIAO J H. The Keap1-Nrf2 system: a mediator between oxidative stress and aging[J]. Oxid Med Cell Longev, 2021, 2021: 6635460.
9	BAIRD L, YAMAMOTO M. The molecular mechanisms regulating the KEAP1-NRF2 pathway[J]. Mol Cell Biol, 2020, 40(13): e00099-e00020.
10	LI S, YANG C, FANG X, et al. Role of Keap1-Nrf2 signaling in anhedonia symptoms in a rat model of chronic neuropathic pain: improvement with sulforaphane[J]. Front Pharmacol, 2018, 9: 887.
11	FRIAS D P, GOMES R L N, YOSHIZAKI K, et al. Nrf2 positively regulates autophagy antioxidant response in human bronchial epithelial cells exposed to diesel exhaust particles[J]. Sci Rep, 2020, 10(1): 3704.
12	RYTER S W. Heme oxygenase-1/carbon monoxide as modulators of autophagy and inflammation[J]. Arch Biochem Biophys, 2019, 678: 108186.
13	RUPPRECHT R, WETZEL C H, DOROSTKAR M, et al. Translocator protein (18kDa) TSPO: a new diagnostic or therapeutic target for stress-related disorders?[J]. Mol Psychiatry, 2022, 27(7): 2918-2926.
14	HAO C, MA B J, GAO N, et al. Translocator protein (TSPO) alleviates neuropathic pain by activating spinal autophagy and nuclear SIRT1/PGC-1α signaling in a rat L5 SNL model[J]. J Pain Res, 2022, 15: 767-778.
15	RASHID K, VERHOYEN M, TAIWO M, et al. Translocator protein (18 kDa) (TSPO) ligands activate Nrf2 signaling and attenuate inflammatory responses and oxidative stress in human retinal pigment epithelial cells[J]. Biochem Biophys Res Commun, 2020, 528(2): 261-268.
16	GHEIBI S, KASHFI K, GHASEMI A. A practical guide for induction of type-2 diabetes in rat: incorporating a high-fat diet and streptozotocin[J]. Biomedecine Pharmacother, 2017, 95: 605-613.
17	CHAPLAN S R, BACH F W, POGREL J W, et al. Quantitative assessment of tactile allodynia in the rat paw[J]. J Neurosci Methods, 1994, 53(1): 55-63.
18	BARRESI E, ROBELLO M, COSTA B, et al. An update into the medicinal chemistry of translocator protein (TSPO) ligands[J]. Eur J Med Chem, 2021, 209: 112924.
19	LIU X M, LIU H J, XU S S, et al. Spinal translocator protein alleviates chronic neuropathic pain behavior and modulates spinal astrocyte-neuronal function in rats with L5 spinal nerve ligation model[J]. Pain, 2016, 157(1): 103-116.
20	BLOMS-FUNKE P, SCHUMACHER M, LIU S, et al. A novel dual mode-of-action anti-hyperalgesic compound in rats which is neuroprotective and promotes neuroregeneration[J]. Eur J Pharmacol, 2022, 923: 174935.
21	MARINELLI S, NAZIO F, TINARI A, et al. Schwann cell autophagy counteracts the onset and chronification of neuropathic pain[J]. Pain, 2014, 155(1): 93-107.
22	ARRURI V, KOMIRISHETTY P, ARETI A, et al. Nrf2 and NF-κB modulation by Plumbagin attenuates functional, behavioural and biochemical deficits in rat model of neuropathic pain[J]. Pharmacol Rep, 2017, 69(4): 625-632.
23	DI W, SHI X L, LV H, et al. Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats[J]. J Headache Pain, 2016, 17(1): 99.

[1]	吴凌恒, 陈建雄, 张梦娇, 沙蕾, 曹萌萌, 沈崔琴, 杜联芳, 李朝军. 血糖控制不理想对2型糖尿病患者亚临床心肌收缩功能的影响研究[J]. 上海交通大学学报（医学版）, 2023, 43(8): 1024-1031.
[2]	孟祥雨, 闫丹丹, 陈香慧, 赖思宇, 徐云, 耿瑞娜, 张红, 张蓉, 胡承, 严婧. 家系全外显子组测序鉴定Wolfram综合征致病突变及其临床性状分析[J]. 上海交通大学学报（医学版）, 2023, 43(7): 898-905.
[3]	吴淇琦, 汪豪, 林砺, 晏博, 张舒林. miR-185-5p通过抑制巨噬细胞自噬促进胞内分枝杆菌生长[J]. 上海交通大学学报（医学版）, 2023, 43(6): 699-708.
[4]	金芳全, 樊成虎, 唐晓栋, 陈彦同, 齐兵献. 线粒体功能障碍与骨质疏松症相关性研究进展[J]. 上海交通大学学报（医学版）, 2023, 43(6): 761-767.
[5]	刘芊若, 方子晨, 吴宇涵, 钟羡欣, 国沐禾, 贾洁. 肠道菌群及其代谢产物与妊娠期糖尿病相关性的研究进展[J]. 上海交通大学学报（医学版）, 2023, 43(5): 641-647.
[6]	张越, 瞿蕾, 谷沁, 朱亦清, 马莉莹, 孙文广. 妊娠期糖尿病孕妇尿酮体持续阳性对母婴临床结局的影响[J]. 上海交通大学学报（医学版）, 2023, 43(3): 314-319.
[7]	陈奕馨, 程丽珍, 林祎嘉, 苗雅. 2型糖尿病脑病小鼠海马中转录因子EB活性与自噬功能的变化[J]. 上海交通大学学报（医学版）, 2023, 43(2): 162-170.
[8]	吴佳晋, 钟晨, 李大伟, 陈若洋, 瞿俊文, 张明. 甲基转移酶3调控pri-miR-21甲基化修饰在糖尿病肾病肾脏纤维化中的作用[J]. 上海交通大学学报（医学版）, 2023, 43(1): 1-7.
[9]	王雪敏, 王亚楠, 牛爱琴, 叶英, 李飞. 微RNA-30b-5p通过靶向Atg5抑制多囊卵巢综合征大鼠卵巢颗粒细胞自噬[J]. 上海交通大学学报（医学版）, 2023, 43(1): 20-28.
[10]	贾伟平. 血糖监测技术的进步与展望[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1171-1175.
[11]	李雯, 李苑, 叶海昀, 张笑笑, 乔彤, 李嫔. 1型糖尿病儿童眼底黄斑区早期微血管变化的观察[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1311-1314.
[12]	王婕, 吴慧, 卢凌鹏, 杨科峰, 祝捷, 周恒益, 姚蝶, 高雅, 冯宇婷, 刘玉红, 贾洁. 妊娠期糖尿病女性肠道菌群的变化特征及其与血糖、血脂和膳食的相关性[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1336-1346.
[13]	高菲, 卢宇, 董书琴, 杨柳, 吴邵花, 韦静. 孕早期成纤维生长因子19亚族对妊娠期糖尿病的预测价值[J]. 上海交通大学学报（医学版）, 2022, 42(7): 898-903.
[14]	吴侠霏, 方婕, 漆洪波, 余昕烊. 妊娠期糖尿病对C57BL/6J子代成年鼠神经精神功能的影响[J]. 上海交通大学学报（医学版）, 2022, 42(4): 422-432.
[15]	吴亚, 殷峻. 生酮饮食调控肠道菌群在疾病治疗中的研究进展[J]. 上海交通大学学报（医学版）, 2022, 42(4): 545-550.