6‐Shogaol Attenuates Doxorubicin‐Induced Cardiac and Skeletal Muscle Atrophy by Inhibiting E3 Ubiquitin Ligases and Necroptosis

Figures

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  FIGURE 1. 6‐SH alleviated the loss of body weight, muscle weight, heart weight, grip strength and food intake in healthy mice after DOX treatment. (A) The chemical structure of 6‐SH. (B) Schematic representation of the experimental protocol. C57BL/6 mice were treated with DOX (5 mg/kg/3d, 4 doses, i.p.) and given low doses of 6‐SH (2.5 mg/kg, qd, i.p.) or high doses of 6‐SH (10 mg/kg, qd, i.p.) at the same time for 14 days. (C) Body weight was measured every day. (D–I). The gastrocnemius, tibialis anterior, epididymal adipose tissue and heart were weighed, and the heart weight‐to‐tibial length ratio and grip strength were measured. (J) The cumulative food intake of each group, n = 6/group. The data are shown as the means ± SEM, n = 6/group, **p < 0.01, ***p < 0.001 versus the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the DOX group.

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  FIGURE 2. 6‐SH prevented DOX‐induced cachexia symptoms and heart atrophy in LLC tumor‐bearing mice and enhanced the antitumor effect of DOX. (A) Schematic representation of the experimental protocol. C57BL/6 mice were implanted with LLC cells in the right flank, treated with DOX (5 mg/kg/3d, 4 doses, i.p.) and given low doses of 6‐SH (2.5 mg/kg, qd, i.p.) or high doses of 6‐SH (10 mg/kg, qd, i.p.) at the same time for 14 days. (B) Body weight was measured every 2 days. (C) The tumor size was assessed every 2 days after the start of drug administration. (D) After the mice were sacrificed, body weights without tumors were measured. (E–K) The gastrocnemius, tibialis anterior, epididymal adipose tissue and heart were weighed, and the heart weight‐to‐tibial length ratio and grip strength were measured. The data are shown as the means ± SEM, n = 6/group, *p < 0.05, **p < 0.01, ***p < 0.001 versus the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the DOX group.

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  FIGURE 3. 6‐SH alleviated the reduction in CSA of skeletal muscle fibers and epididymal fat induced by DOX. (A) H&E‐stained sections of mouse gastrocnemius. (B) The CSA was measured via ImageJ software, and the gastrocnemius muscle fibers CSA distribution was shown (n = 300). (C) Statistical analysis of gastrocnemius muscle fibers CSA (n = 300). (D) H&E‐stained sections of epididymal fat. (E) The epididymal fat CSA distribution was shown (n = 300). (F) Statistical analysis of epididymal fat CSA (n = 300). ***p < 0.001 versus the CON group; ###p < 0.001 versus the DOX group.

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  FIGURE 4. Effects of 6‐SH on SDH activity and the distribution of muscle fiber types in the gastrocnemius after DOX treatment. (A) SDH‐stained sections of mouse gastrocnemius tissue. (B) Sections of mouse gastrocnemius tissue were stained with MyHC I and MyHC II and observed under a fluorescence microscope. (C) The SDH intensity was quantitatively analyzed via ImageJ software. (D) Muscle fiber type distribution in the gastrocnemius. (E) Quantitative analysis of fluorescence intensity in fast‐twitch fibers and slow‐twitch fibers via ImageJ software. **p < 0.01, ***p < 0.001 versus the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the DOX group.

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  FIGURE 5. 6‐SH alleviated cardiac injury, myocardial fibrosis and cardiac atrophy induced by DOX. (A) H&E‐stained sections of mouse hearts. (B) Masson‐stained sections of mouse hearts. (C) WGA‐stained sections of mouse hearts. (D) Statistical analysis of the CSA of the mouse heart (n = 300). (E) Distribution of the CSA of the mouse heart (n = 300). ***p < 0.001 versus the CON group; ###p < 0.001 versus the DOX group.

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  FIGURE 6. 6‐SH alleviated the decrease in cardiac systolic and diastolic function induced by DOX. (A) Representative echocardiography images of mouse hearts. (B–M) Echocardiography parameters. (B) Left ventricle ejection fraction (LVEF). (C) Left ventricle fractional shortening (LVFS). (D) Left ventricle diastolic internal dimension (LVIDd). (E) Left ventricle systolic internal dimension (LVIDs). (F) Left ventricle diastolic volume (LVvol;d). (G) Left ventricle systolic volume (LVvol;s). (H) Left ventricle diastolic posterior wall thickness (LVPWd). (I) Left ventricle systolic posterior wall thickness (LVPWs). (J) Left ventricle diastolic interventricular septum thickness (LVS;d). (K) Left ventricle systolic interventricular septum thickness (LVS;s). (L) E/A ratio. (M) Left ventricle mass. The data are shown as the means ± SEM, n = 6/group, *p < 0.05, **p < 0.01, ***p < 0.01 versus the CON group; #p < 0.05, ##p < 0.01 versus the DOX group.

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  FIGURE 7. 6‐SH attenuated DOX‐induced oxidative stress, cardiac injury, and inflammation. (A) The levels of MDA, SOD, GSH, and CAT in the gastrocnemius. (B) The levels of MDA, SOD, GSH, and CAT in the heart. (C) Serum cTnT levels. (D) Serum BNP levels. (E) Serum TNF‐α levels. (F) Serum IL‐6 levels. The data are shown as the means ± SEM, n = 6/group, *p < 0.05, ***p < 0.001 versus the CON group; ##p < 0.01, ###p < 0.001 versus the DOX group.

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  FIGURE 8. Effects of 6‐SH on E3 ubiquitin ligases and myogenic factors in the gastrocnemius after DOX treatment. (A) The protein expression of Atrogin1, MuRF1, MyHC, MyoD, and MyoG was evaluated via western blotting. GAPDH was used as an internal control. (B) The relative expression levels of the proteins were quantified via ImageJ software, normalized to the level of GAPDH, and normalized to the levels in the CON group (n = 3/group). (C) The mRNA expression of Atrogin1, MuRF1, Myh2, Myh4, Myh7, MyoD, and MyoG in gastrocnemius was assessed via qRT–PCR, and RPS18 was used as an internal control (n = 5/group). The data are presented as the means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the DOX group.

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  FIGURE 9. Effects of 6‐SH on E3 ubiquitin ligases and myogenic factors in the heart after DOX treatment. (A) The protein levels of Atrogin1, MuRF1, MyHC, MYH6, MYH7, MyoD and MyoG were evaluated via western blotting. GAPDH was used as an internal control. (B) The relative expression levels of the proteins were quantified via ImageJ software, normalized to the levels of GAPDH, and normalized to the levels in the CON group (n = 3/group). (C) The mRNA expression of Atrogin1, MuRF1, Myh6, Myh7, MyoD and MyoG in the heart was assessed via qRT–PCR, and RPS18 was used as an internal control (n = 5/group). The data are presented as the means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus the CON group; #p < 0.05, ##p < 0.01, ###: p < 0.001 versus the DOX group.

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  FIGURE 10. 6‐SH inhibited DOX‐induced necroptosis in skeletal and cardiac muscle. (A) Representative blot bands of p‐RIPK1, RIPK1, p‐RIPK3, RIPK3, p‐MLKL and MLKL in the gastrocnemius. (B) Relative expression levels of necroptosis‐related proteins in the gastrocnemius. (n = 3/group). (C) Representative blot bands of p‐RIPK1, RIPK1, p‐RIPK3, RIPK3, p‐MLKL and MLKL in the heart. (D) Relative expression levels of necroptosis‐related proteins in the heart. (n = 3/group). The data are presented as the means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the DOX group.