Dexmedetomidine attenuates heat stress-induced oncosis in human skeletal muscle cells by activating the Nrf2/Ho-1 pathway

doi:10.12122/j.issn.1673-4254.2025.03.18

Abstract

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

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.

Figures/Tables 10

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

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).

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).

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).

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).

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).

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.

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).

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).

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).

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