右美托咪定通过激活Nrf2/HO-1通路减轻热应激诱导的人骨骼肌细胞胀亡

doi:10.12122/j.issn.1673-4254.2025.03.18

摘要/Abstract

摘要：

目的研究右美托咪定（DEX）对于热打击诱导人骨骼肌细胞（HSKMC）胀亡的保护作用及可能机制。方法体外培养HSKMC，热打击组（HS组）将细胞置于43 ℃的水浴锅中热打击4 h，构建细胞胀亡模型，设置对照组、HS组、30 μmol/L DEX+HS组、ML385+30 μmol/L DEX+HS组、si-Nrf2+HS组、si-Nrf2+30 μmol/L DEX+HS组。通过CCK-8法检测细胞活力，透射电子显微镜观察细胞超微结构，Annexin V-FITC/PI免疫荧光流式细胞术检测细胞胀亡和细胞凋亡，使用谷胱甘肽（GSH）和GSH-px试剂盒检测细胞中GSH及GSH-px含量，TBA法检测丙二醛（MDA），比色法检测乳酸脱氢酶（LDH）、超氧歧化酶（SOD）、三磷酸腺苷（ATP）表达水平，ELISA法检测肿瘤坏死因子（TNF）-α、白介素（IL）-6、IL-1β水平，qRT-PCR及Western blotting法检测porimin、caspase-3、Nrf2、p-Nrf2、HO-1、NQO1等蛋白表达水平变化，荧光探针法检测细胞内活性氧（ROS），JC-1荧光染色法检测线粒体膜电位损伤。结果与对照组相比，HS组细胞和细胞器肿胀、胞质内空泡化明显，符合胀亡表现，细胞活力下降，细胞双阳性细胞率（Annexin-V+/PI+）、porimin蛋白表达量增加（P<0.05），caspase-3蛋白在各组间的差异无统计学意义（P>0.05），ROS、MDA、LDH、TNF-α、IL-6和IL-1β的含量增多（P<0.05），ATP、线粒体膜电位、GSH、GSH- px和SOD水平降低（P<0.05）。与HS组相比，30 μmol/L DEX+HS组细胞损伤减轻，细胞活力升高，细胞双阳性细胞率下降（P<0.05），ROS、MDA、LDH、TNF-α、IL-6和IL-1β的含量下降（P<0.05），ATP、线粒体膜电位、GSH、GSH- px和SOD含量升高（P<0.05），Nrf2、p-Nrf2、HO-1和NQO1蛋白表达升高（P<0.05）。与30 μmol/L DEX+HS组相比，经ML385或si-Nrf2干预后，DEX对HSKMC细胞的保护作用减弱（P<0.05）。结论热打击诱导人骨骼肌细胞发生胀亡，DEX抑制热打击诱导的胀亡，可能机制是激活Nrf2/HO-1信号通路减轻HSKMC的氧化损伤以及抑制炎症因子分泌。

关键词: 右美托咪定, 横纹肌溶解症, 胀亡, 人骨骼肌细胞, Nrf2/HO-1通路

Abstract:

Objective To investigate the protective effects of dexmedetomidine (DEX) against heat stress (HS)-induced oncosis in human skeletal muscle cells (HSKMCs) and its underlying mechanisms. Methods A HSKMC model of HS-induced oncosis were established by 43 ℃ water bath for 4 h, and the effects of treatments with 30 μmol/L DEX, ML385 (a Nrf2 inhibitor) +DEX, si-Nrf2+HS, and si-Nrf2+DEX prior to modeling on cell viability was assessed using CCK-8 assay. Oncosis characteristics were evaluated using transmission electron microscopy and Annexin V-FITC/PI flow cytometry. The oxidative stress markers (GSH, GSH-Px, MDA, SOD and ROS), mitochondrial membrane potential, energy metabolism, and inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the cells were quantified using standard kits, and the expressions of porimin, caspase-3 and Nrf2 pathway proteins were analyzed using Western blotting and qRT-PCR. Results HS induced typical oncotic features in HSKMCs including organelle swelling and cytoplasmic vacuolization. DEX pretreatment significantly attenuated these changes, reduced Annexin V⁺/PI⁺ cell ratio and cellular porimin expression, and lowered the levels of ROS and MDA while restoring GSH and SOD levels. DEX pretreatment also significantly increased the mitochondrial membrane potential and ATP level, upregulated the expressions of Nrf2, p-Nrf2, HO-1 and NQO1, and suppressed the expressions of TNF-α, IL-6 and IL-1β. The protective effects of DEX were obviously attenuated by interventions with ML385 or si-Nrf2. Conclusion DEX mitigates HS-induced HSKMC oncosis by activating the Nrf2/HO-1 pathway to relieve oxidative stress, mitochondrial dysfunction, and inflammatory responses.

Key words: dexmedetomidine, rhabdomyolysis, oncosis, human skeletal muscle cells, Nrf2/HO-1 pathway

刘洋, 贾亿卿, 李程程, 毛汉丁, 刘树元, 单毅. 右美托咪定通过激活Nrf2/HO-1通路减轻热应激诱导的人骨骼肌细胞胀亡[J]. 南方医科大学学报, 2025, 45(3): 603-613.

Yang LIU, Yiqing JIA, Chengcheng LI, Handing MAO, Shuyuan LIU, Yi SHAN. Dexmedetomidine attenuates heat stress-induced oncosis in human skeletal muscle cells by activating the Nrf2/Ho-1 pathway[J]. Journal of Southern Medical University, 2025, 45(3): 603-613.

图/表 10

表1 引物序列

Tab.1 The sequences of primers

Target name	Primer sequences
Actin	F	TCCTCCTGAGCGCAAGTACTCC
Actin	R	CATACTCCTGCTTGCTGATCCAC
Porimin	F	CTCGGAACAATGGGACTCGG
Porimin	R	TATGTTTGCAGATGCCGCC
Caspase-3	F	ATGACATCTCGGTCTGGTA
Caspase-3	R	CTTTAGAAACATCACGCATC
Nrf2	F	TCAGCGACGGAAAGAGTATGA
Nrf2	R	CCACTGGTTTCTGACTGGATGT
HO-1	F	CCTTCCCCAACATTGCCAGT
HO-1	R	CTTGGCCTCTTCTATCACCCTC
NQO1	F	GCTGGTTTGAGCGAGTGTTC
NQO1	R	CTGCCTTCTTACTCCGGAAGG

图1 热打击后分别复温0、12、24、48 h的细胞活力

Fig.1 Cell viability at 0, 12, 24, and 48 h after heat stress (HS). *P<0.05 vs NC group (Mean±SD, n=6).

图2 DEX用药浓度筛选

Fig.2 Screening of dexmedetomidine (DEX) dosing concentrations. A: Effects of different concentrations of DEX on HSKMC viability. *P<0.05 vs NC group (n=6). B: Effect of DEX (1, 30 and 60 μmol/L) on viability of HSKMC cells after heat stress. *P<0.05 vs NC group; ^P<0.05 vs HS group (n=6).

图3 DEX对各组HSKMC细胞活力的影响

Fig.3 Effect of DEX on viability of HSKMC cells in each group. *P<0.05 vs NC group; ^P<0.05 vs HS group; +P<0.05 vs 30 μmol/L DEX+HS group (n=6).

图4 透射电子显微镜观察HSKMC结构变化

Fig.4 Observation of structural changes of HSKMCs under transmission electron microscope. Black arrows indicated mitochondrial swelling after heat stress. A: NC group (Original magnification: ×2000). B: HS group (×2000), C: 30 μmol/L DEX+HS group (×2000). D: NC group (×10 000). E: HS group (×10 000). F: 30 μmol/L DEX+HS group (×10 000). G: ML385+30 μmol/L DEX+HS group (×2000) . H: si-Nrf2+HS group (×2000). I: si-Nrf2+30 μmol/L DEX+HS group (×2000). J: ML385+30 μmol/L DEX+HS group (×10 000). K: si-Nrf2+HS group (×10 000) . L: si-Nrf2+30 μmol/L DEX+HS group (×10 000).

图5 各组细胞双阳性细胞率(Annexin-V+/PI+)比较

Fig.5 Comparison of Annexin-V+/PI+ double-positive cell rate in each group. *P<0.05 vs NC group; ^P<0.05 vs HS group; +P<0.05 vs 30 μmol/L DEX+HS group (n=3).

图6 各组HSKMC中porimin(A)、caspase-3(B)、Nrf2(C)、p-Nrf2(D)、HO-1(E)和NQO1(F)蛋白表达量

Fig.6 Protein expression levels of porimin (A), caspase-3 (B), Nrf2 (C), p-Nrf2 (D), HO-1 (E), and NQO1 (F) in HSKMCs in each group. *P<0.05 vs NC group; ^P<0.05 vs HS group; +P<0.05 vs 30 μmol/L DEX+HS group (Mean±SD, n=3). a: NC group; b: HS group; c: 30 μmol/L DEX+HS group; d: ML385+30 μmol/L DEX+HS group; e: si-Nrf2+HS group; f: si-Nrf2+DEX +HS group.

图7 各组HSKMC中GSH(A)、GSH- px(B)、SOD(C)、MDA(D)、LDH(E)、ATP(F)、TNF-α(G)、IL-6(H)和IL-1β(I)水平

Fig.7 GSH (A), GSH-px (B), SOD (C), MDA (D), LDH (E), ATP (F), TNF-α (G), IL-6 (H), and IL-1β (I) levels in HSKMC in each group. *P<0.05 vs NC group; ^P<0.05 vs HS group; +P<0.05 vs 30 μmol/L DEX+HS group (Mean±SD, n=3).

图8 各组HSKMC中ROS荧光检测及荧光强度检测

Fig.8 ROS fluorescence detection and fluorescence intensity in HSKMCs in each group (×100). *P<0.05 vs NC group; ^P<0.05 vs HS group (Mean±SD, n=3).

图9 各组HSKMC线粒体荧光及膜电位检测

Fig.9 HSKMC mitochondrial fluorescence and membrane potential assay in each group (×100). *P<0.05 vs NC group; ^P<0.05 vs HS group (Mean±SD, n=3).

参考文献 35

1	Torres PA, Helmstetter JA, Kaye AM, et al. Rhabdomyolysis: pathogenesis, diagnosis, and treatment[J]. Ochsner J, 2015, 15(1): 58-69.
2	Zutt R, van der Kooi AJ, Linthorst GE, et al. Rhabdomyolysis: review of the literature[J]. Neuromuscul Disord, 2014, 24(8): 651-9.
3	Cabral BMI, Edding SN, Portocarrero JP, et al. Rhabdomyolysis[J]. Dis Mon, 2020, 66(8): 101015.
4	Peters AA, Jamaludin SN, Yapa KS, et al. Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells[J]. Oncogene, 2017, 36(46): 6490-500.
5	Zheng JY, Tan HL, Matsudaira PT, et al. Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis[J]. Cell Death Differ, 2017, 24(3): 546-58.
6	Sun YX, Yu J, Liu XR, et al. Oncosis-like cell death is induced by berberine through ERK1/2-mediated impairment of mitochondrial aerobic respiration in gliomas[J]. Biomed Pharmacother, 2018, 102: 699-710.
7	Liu XM, Chen QH, Hu Q, et al. Dexmedetomidine protects intestinal ischemia-reperfusion injury via inhibiting p38 MAPK cascades[J]. Exp Mol Pathol, 2020, 115: 104444.
8	Weerink MAS, Struys MMRF, Hannivoort LN, et al. Clinical pharmacokinetics and pharmacodynamics of dexmedetomidine[J]. Clin Pharmacokinet, 2017, 56(8): 893-913.
9	Tian L, Liu Q, Wang X, et al. Fighting ferroptosis: Protective effects of dexmedetomidine on vital organ injuries[J]. Life Sci, 2024, 354: 122949.
10	Bao NR, Tang B. Organ-protective effects and the underlying mechanism of dexmedetomidine[J]. Mediators Inflamm, 2020, 2020: 6136105.
11	李程程, 刘洋, 毛汉丁, 等. 右美托咪定通过肾上腺素能α2受体调控NLRP3/IL-1β通路缓解劳力性热射病相关性横纹肌溶解[J/OL]. 解放军医学杂志, 2024: 1-14.
12	Qi YJ, Fu S, Pei DG, et al. Luteolin attenuated cisplatin-induced cardiac dysfunction and oxidative stress via modulation of Keap1/Nrf2 signaling pathway[J]. Free Radic Res, 2022, 56(2): 209-21.
13	Ulasov AV, Rosenkranz AA, Georgiev GP, et al. Nrf2/Keap1/ARE signaling: towards specific regulation[J]. Life Sci, 2022, 291: 120111.
14	D’Arcy MS. Cell death: a review of the major forms of apoptosis, necrosis and autophagy[J]. Cell Biol Int, 2019, 43(6): 582-92.
15	Lecoeur H, Prévost MC, Gougeon ML. Oncosis is associated with exposure of phosphatidylserine residues on the outside layer of the plasma membrane: a reconsideration of the specificity of the annexin V/propidium iodide assay[J]. Cytometry, 2001, 44(1): 65-72.
16	Zhou X, Sun WJ, Wang WM, et al. Artesunate inhibits the growth of gastric cancer cells through the mechanism of promoting oncosis both in vitro and in vivo [J]. Anticancer Drugs, 2013, 24(9): 920-7.
17	Duanghathaipornsuk S, Farrell EJ, Alba-Rubio AC, et al. Detection technologies for reactive oxygen species: fluorescence and electrochemical methods and their applications[J]. Biosensors, 2021, 11(2): 30.
18	豆春丽. ROS介导线粒体功能障碍在热应激骨骼肌细胞胀亡中机制研究[D]. 广州: 广州中医药大学, 2020.
19	Peiris AN, Jaroudi S, Noor R. Heat stroke[J]. JAMA, 2017, 318(24): 2503.
20	Kodadek L, Carmichael Ii SP, Seshadri A, et al. Rhabdomyolysis: an American association for the surgery of trauma critical care committee clinical consensus document[J]. Trauma Surg Acute Care Open, 2022, 7(1): e000836.
21	Yue RC, Hu HX, Yiu KH, et al. Lycopene protects against hypoxia/reoxygenation-induced apoptosis by preventing mitochondrial dysfunction in primary neonatal mouse cardiomyocytes[J]. PLoS One, 2012, 7(11): e50778.
22	Chi XJ, Zhang R, Shen N, et al. Sulforaphane reduces apoptosis and oncosis along with protecting liver injury-induced ischemic reperfusion by activating the Nrf2/ARE pathway[J]. Hepatol Int, 2015, 9(2): 321-9.
23	Weerasinghe P, Maximilian Buja L. Oncosis: an important non-apoptotic mode of cell death[J]. Exp Mol Pathol, 2012, 93(3): 302-8.
24	Trump BF, Berezesky IK, Chang SH, et al. The pathways of cell death: oncosis, apoptosis, and necrosis[J]. Toxicol Pathol, 1997, 25(1): 82-8.
25	Wang YP, Gao WQ, Shi XY, et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin[J]. Nature, 2017, 547(7661): 99-103.
26	Ge Y, Cai YM, Bonneau L, et al. Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis [J]. Cell Death Differ, 2016, 23(9): 1493-501.
27	豆春丽, 袁芳芳, 刘斌, 等. 热打击诱导骨骼肌细胞胀亡而非凋亡[J]. 解放军医学杂志, 2020, 45(10): 1047-51.
28	Wu C, Chen RL, Wang Y, et al. Acacetin alleviates myocardial ischaemia/reperfusion injury by inhibiting oxidative stress and apoptosis via the Nrf-2/HO-1 pathway[J]. Pharm Biol, 2022, 60(1): 553-61.
29	Park WH. Antiapoptotic effects of caspase inhibitors on H₂O₂-treated lung cancer cells concerning oxidative stress and GSH[J]. Mol Cell Biochem, 2018, 441(1/2): 125-34.
30	Zhang Q, Dong XW, Xia JY, et al. Obestatin plays beneficial role in cardiomyocyte injury induced by ischemia-reperfusion in vivo and in vitro [J]. Med Sci Monit, 2017, 23: 2127-36.
31	Yilmaz Y, Tumkaya L. Effects of hyperbaric oxygen and iloprost on intestinal ischemia-reperfusion induced acute lung injury[J]. Ann Surg Treat Res, 2019, 96(1): 34-40.
32	Yang L, Xing GL, Wang L, et al. Acute kidney injury in China: a cross-sectional survey[J]. Lancet, 2015, 386(10002): 1465-71.
33	Lankadeva YR, Ma S, Iguchi N, et al. Dexmedetomidine reduces norepinephrine requirements and preserves renal oxygenation and function in ovine septic acute kidney injury[J]. Kidney Int, 2019, 96(5): 1150-61.
34	Yao H, Chi XJ, Jin Y, et al. Dexmedetomidine inhibits TLR4/NF-κB activation and reduces acute kidney injury after orthotopic autologous liver transplantation in rats[J]. Sci Rep, 2015, 5: 16849.
35	Kopacz A, Kloska D, Forman HJ, et al. Beyond repression of Nrf2: an update on Keap1[J]. Free Radic Biol Med, 2020, 157: 63-74.

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