Capsaicin alleviates cisplatin-induced muscle loss and atrophy in vitro and in vivo

Abstract

Background: Cisplatin (CP) is a widely used chemotherapeutic drug with subsequent adverse effects on different organs and tissues including skeletal muscle loss and atrophy as the most common clinical symptoms. The molecular mechanism of cisplatin-induced muscle atrophy is not clearly understood. However, recent significant advances indicate that it is related to an imbalance in both the protein status and apoptosis. Capsaicin (CAP) is one of the major ingredients in chilli peppers. It is a valuable pharmacological agent with several therapeutic applications in controlling pain and inflammation with particular therapeutic potential in muscle atrophy. However, the mechanisms underlying its protective effects against cisplatin-induced muscle loss and atrophy remain largely unknown. This study aims to investigate capsaicin's beneficial effects on cisplatin-induced muscle loss and atrophy in vitro and in vivo.

Methods: The anti-muscle-atrophic effect of capsaicin on cisplatin-induced muscle loss was investigated using in vivo and in vitro studies. By using the pretreatment model, pretreated capsaicin for 24 h and treated with cisplatin for 48 h, we utilized a C₂ C₁₂ myotube formation model where cell viability analysis, immunofluorescence, and protein expression were measured to investigate the effect of capsaicin in hampering cisplatin-induced muscle atrophy. C57BL/6 mice were administered capsaicin (10, 40 mg/kg BW) as a pretreatment for 5 weeks and cisplatin (3 mg/kg BW) for seven consecutively days to assess muscle atrophy in an animal model for protein and oxidative stress examination, and the grip strength was tested to evaluate the muscle strength.

Results: Our study results indicated that cisplatin caused lower cell viability and showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in the myotube diameter, repression of Akt, and mTOR protein expression. However, pretreatment with capsaicin could ameliorate cisplatin-induced muscle atrophy by up-regulating the protein synthesis in skeletal muscle as well as down-regulating the markers of protein degradation. Additionally, capsaicin was able to downregulate the protein expression of apoptosis-related markers, activated TRPV1 and autophagy progress modulation and the recovery of lysosome function. In vivo, capsaicin could relieve oxidative stress and cytokine secretion while modulating autophagy-related lysosome fusion, improving grip strength, and alleviating cisplatin-induced body weight loss and gastrocnemius atrophy.

Conclusions: These findings suggest that capsaicin can restore cisplatin-induced imbalance between protein synthesis and protein degradation pathways and it may have protective effects against cisplatin-induced muscle atrophy.

Keywords: Capsaicin; Cisplatin; Muscle atrophy; Myotube.

Figure 1

Effect of capsaicin and cisplatin on C₂C₁₂ cell viability and morphology change. C₂C₁₂ were cultured in 2% horse serum DMEM to differentiate over 6 days. After differentiation, cells were treated with various concentrations of (A) capsaicin or (B) cisplatin for 24 and 48 h. Cells were pretreated with various capsaicin concentrations for 24 h, then treated with cisplatin for 48 h. (C) Cell viability was analysed by MTT assay and the (D) morphology was captured by microscopy: Scale bar 10 μm, ×100. (E) Trypan blue assay was used to determine the cell number change. (F) Cell staining was measured by crystal violet. Data represent the means ± SD. **P < 0.01; ***P < 0.001 compared with control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with the cisplatin group. C, control; CP, cisplatin (μM); CAP, capsaicin (μM); T, testosterone (1 μM).

Figure 2

Effect of capsaicin on C₂C₁₂ cell myotube diameter and muscle atrophy‐related protein expression. C₂C₁₂ cells were pretreated with various capsaicin concentrations for 24 h and then treated with cisplatin for 48 h. (A) Cells were observed under a fluorescent microscope for MyH expression by ICC (green). Scale bar, 300 μm, ×100. (B) The cell myotube diameter was measured by ImageJ software according to MyH expression by ICC (green). (C) The fluorescence intensity was measured by ImageJ software. Western blot was used to explore the muscle‐atrophy‐related protein expression of (D) MaFbx, (E) MuRF‐1, and (F) myostatin. Data are represented as mean ± SD. **P < 0.01; ***P < 0.001 compared with control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with the cisplatin alone group. C, control; CP, cisplatin (μM); CAP, capsaicin (μM); T, testosterone (1 μM).

Figure 3

Capsaicin administration recovered protein synthesis and apoptosis‐related protein expression. C₂C₁₂ cells were pretreated with various capsaicin concentrations for 24 h, then treated with cisplatin for 48 h. Western blot was used to assess the expression of protein‐synthesis‐related markers (A) p‐mTOR and mTOR, (B) p‐Akt/Akt, and apoptosis‐related markers (C) Bax/Bcl‐2, (D) Caspase3 and PARP protein expression. (E) The potential modulated pathway in protein synthesis and apoptosis was assessed. Data were represented as the mean ± SD. **P < 0.01; ***P < 0.001 compared with the control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with the cisplatin group.

Figure 4

Capsaicin recovered cisplatin‐induced autophagy dysfunction. C₂C₁₂ cells were pretreated with various capsaicin concentrations for 24 h, then treated with cisplatin for 48 h. Western blot was used to assess the expression of autophagy‐related markers (A) p62, (B) LC3B, and (C) LAMP1. To evaluate the effect of lysosome fusion, C₂C₁₂ cells were pretreated with BafA1 (200 nM) for 3 h and treated with capsaicin 50 μM for 24 h, then treated with cisplatin for 40 μM for 48 h. Western blot was used to assess the expression of apoptosis and autophagy‐related marker (D) PARP (E) LC3B. Data were represented as the mean ± SD. **P < 0.01; ***P < 0.001 compared with control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with the cisplatin group. †††P < 0.001 compared with capsaicin‐treated group. ^P < 0.05 compared with cisplatin combined BafA1 group.

Figure 5

The role of TRPV1 channel in cisplatin‐induced muscle atrophy and the recovery effect of capsaicin. C₂C₁₂ cells were pretreated with various capsaicin concentrations for 24 h then treated with cisplatin for 48 h. Western blot was used to assess the expression of (A) TRPV1 protein expression. To evaluate TRPV1's role, C₂C₁₂ cells were co‐treated with SB366791 (10 μM) and capsaicin 50 μM for 24 h, then treated with cisplatin for 48 h. Western blot was used to assess the expression of apoptosis and autophagy related marker (B) PARP (C) LC3B. Data are represented as the mean ± SD. **P < 0.01; ***P < 0.001 compared with control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with the cisplatin group.

Figure 6

The effect of capsaicin on cisplatin‐induced muscle atrophy in vivo. (A) Flowchart of the in vivo experiment using cisplatin‐induced muscle atrophy animal model. (B) Body weight loss during the experiment and after cisplatin treatment for one week. (C) Gastrocnemius figure in each treatment group and the representative images of a haematoxylin and eosin (H&E)‐stained gastrocnemius muscle. Scale bar, 200 μm, ×200. Used ImageJ software quantify the fibre cross‐sectional area of the gastrocnemius muscle. Quantification of the fibre cross‐sectional area of the gastrocnemius muscle were calculated 30–50 muscle fibre/fields for more than 10 replicates per animal (D) the forelimb hanging before cisplatin injection. After injection for 7 days the grip strength was assessed using a grip strength meter and was expressed in absolute values (gram) and normalized with body weight. (E) Serum TNF‐α levels in mice. (F) The oxidative stress of serum and tissue were evaluated by MDA concentration. Data are expressed as mean ± SEM (n = 4–6). *P < 0.05; **P < 0.01; ***P < 0.001 compared with control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with the cisplatin group. C, control; CP, cisplatin; CAP, capsaicin; T, testosterone.

Figure 7

Capsaicin treatment recovers cisplatin‐induced muscle atrophy and autophagy‐related protein expression. After sacrifice, we used Western blot to analyse the expression of muscle‐degradation‐related proteins (A) myostatin, (B) MuRF‐1 and MaFbx expression, and autophagy‐related markers (C) p62 and LC3B and lysosome marker (D) Cathepsin B protein expression and activity, showing that the (E) autophagy dysfunction‐related modulation was involved in muscle atrophy. Data were expressed as mean ± SEM (n = 4–6). *P < 0.05; **P < 0.01; ***P < 0.001 compared with control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with the cisplatin group. C, control; CP, cisplatin; CAP, capsaicin; T, testosterone.

Figure 8

Schematic representation of the effect of capsaicin on cisplatin‐induced muscle atrophy. Capsaicin could stimulate protein synthesis in skeletal muscle, and down‐regulate protein degradation‐related protein expression and apoptosis‐related markers, recovering the muscle atrophy‐related protein expression while restoring the autophagy‐related lysosome dysfunction through TRPV1 modulation.