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. 2024 Dec;12(23):e70149.
doi: 10.14814/phy2.70149.

N-butylphthalide (NBP) ameliorated ischemia/reperfusion-induced skeletal muscle injury in male mice via activating Sirt1/Nrf2 signaling pathway

Peng Lu  1   2 , Wei-Peng Li  1 , Ben-Jun Zhou  3 , Wen-Ze Tian  4 , Xiang Lu  1 , Wei Gao  5
Affiliations

N-butylphthalide (NBP) ameliorated ischemia/reperfusion-induced skeletal muscle injury in male mice via activating Sirt1/Nrf2 signaling pathway

Peng Lu et al. Physiol Rep. 2024 Dec.

Abstract

N-butylphthalide (NBP) has been reported to have potential protective effects in ischemic stroke via its antioxidative properties. The present study was aimed to investigate the protective effects of NBP on ischemia/reperfusion (I/R)-induced skeletal muscle injury. Mouse model of I/R-induced skeletal muscle injury and hypoxia/reoxygenation (H/R)-induced C2C12 myotube injury model were constructed to test the protective effects of NBP both in vivo and in vitro. Our results showed that I/R resulted in skeletal muscle injury, as evidenced by elevated levels of LDH, CK, ROS, 3-NT, MDA, and 4-HNE as well as decreased activities of SOD, GSH-Px, and decreased expression of Myog and MyoD in gastrocnemius muscle, which was ameliorated by NBP treatment. Mechanistically, NBP treatment increased the expression of Sirt1 and Nrf2 in the injured skeletal muscle. Notably, the protective effects of NBP on I/R-induced skeletal muscle injury was diminished by the treatment of Sirt1 inhibitor. Further studies in H/R-induced C2C12 myotubes injury model also showed that NBP activated the Sirt1/Nrf2 pathway. NBP treatment upregulated the expression of myog and MyoD in H/R-stimulated C2C12 myotubes, which was eliminated by silencing of Sirt1. Taken together, our results suggest that NBP may alleviated I/R-induced skeletal muscle injury by activating Sirt1/Nrf2 signaling pathway.

Keywords: Nrf2; N‐butylphthalide; Sirt1; ischemia/reperfusion; skeletal muscle.

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Conflict of interest statement

None of the authors has any conflict of interest to disclose.

Figures

FIGURE 1
FIGURE 1
NBP alleviates I/R‐induced skeletal muscle injury. (a–c) H&E staining and immunofluorescence staining of the gastrocnemius muscle indicate the extent of muscle fiber injury and changes in muscle fiber cross‐sectional area across different groups. n = 8 per group. Scale bar = 100 μm. A microscope with a 40× objective was used to capture the images. (d) Western blot analysis in gastrocnemius muscle indicates the effects of NBP on Myog, MyoD after I/R. Data are presented as relative fold change to the control group. n = 6 per group. (e) Western blot analysis in gastrocnemius muscle indicates the effects of NBP on 4‐HNE expression. Data are presented as relative fold change to the control group (set to 1). n = 6 per group.
FIGURE 2
FIGURE 2
NBP activates Sirt1/Nrf2 signaling pathway in I/R‐induced skeletal muscle injury. (a) Western blot analysis in gastrocnemius muscle indicates the effects of NBP on Sirt1, Nrf2 expression. Data are presented as relative fold change to the control group (set to 1). n = 6 per group. (b–d) H&E staining and immunofluorescence staining of the gastrocnemius muscle indicate the extent of muscle fiber injury and changes in muscle fiber cross‐sectional area across different groups after EX527 treatment. n = 8 per group. Scale bar = 100 μm. A microscope with a 40× objective was used to capture the images. (e–g) Western blot analysis in gastrocnemius muscle indicates the effects of 4‐HNE, Sirt1, Nrf2, Myog and MyoD expressions after EX527 treatment. Data are presented as relative fold change to the I/R group. n = 6 per group.
FIGURE 3
FIGURE 3
NBP alleviates H/R induced C2C12 myotube injury. (a) C2C12 myotubes were treated with different concentrations of NBP for 24 h and changes in myotubes viability were assessed. n = 5 per group. (b) C2C12 myotubes were pretreated with NBP at different concentrations for 24 h followed by H/R and changes in myotubes viability were assessed. n = 5 per group. (c) Myog and MyoD expressions were detected by Western blot in each group of C2C12 myotubes. Data are presented as relative fold change to the control group. n = 6 per group. (d, e) Representative immunocytochemistry staining of myotubes showed the changes in myotubes diameter in different groups. n = 8 per group. Scale bar = 50 μm. A microscope with a 40× objective was used to capture the images. (f) Western blot analysis in each group of C2C12 myotubes indicates the effects of NBP on 4‐HNE expression. Data are presented as relative fold change to the control group (set to 1). n = 6 per group.
FIGURE 4
FIGURE 4
NBP activates Sirt1/Nrf2 pathway in H/R‐induced injury in C2C12 myotubes. (a) Sirt1 and Nrf2 expressions were detected by Western blot in each group of C2C12 myotubes. Data are presented as relative fold change to the control group. n = 6 per group. (b) The efficiency of siRNA targeting Sirt1. Data are presented as relative fold change to the control group. (c–e) The effects of NBP on the expression of 4‐HNE, Sirt1, Nrf2, Myog and MyoD after silencing of Sirt1. Data are presented as relative fold change to the H/R group. n = 6 per group.
FIGURE 5
FIGURE 5
Schematic of the mechanism by which NBP attenuates I/R‐induced skeletal muscle injury. NBP may alleviate I/R‐induced skeletal muscle injury by activating the Sirt1/Nrf2 signaling pathway and promoting the expression of Myog and MyoD, thereby exerting a protective effect during the progression of I/R‐induced skeletal muscle damage.
See this image and copyright information in PMC

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