P-Coumaric Acid Improves Skeletal Muscle Atrophy in Chronic Kidney Disease by Modulating TLR4/MyD88/NF-κB-Mediated Inflammation and Oxidative Stress

Abstract

Skeletal muscle atrophy is a prevalent complication in chronic kidney disease (CKD), and its pathogenesis is closely related to inflammation and oxidative stress. P-Coumaric acid (PCA) is a phenolic acid with anti-inflammatory and antioxidant pharmacological actions. This research aims to investigate the effect of PCA on CKD-induced muscle atrophy and its underlying mechanism. In our study, in vivo and in vitro models were established by using 5/6 nephrectomized rats and LPS-induced C2C12 myoblasts. The experimental results showed that PCA ameliorated kidney injury in CKD rats and increased skeletal muscle weight and the cross-sectional area of muscle fibres. In both CKD rats and LPS-induced C2C12 myoblasts, PCA also exhibited anti-inflammatory and antioxidant effects, reduced the levels of pro-inflammatory cytokines and enhanced the activity of antioxidant enzymes. Network pharmacology studies have identified 165 common targets between PCA and skeletal muscle atrophy. Furthermore, the experimental results also demonstrated that PCA decreased the expression of TLR4, MyD88, NF-κB p65, MurF1 and MAFbx at both the protein and mRNA levels. Additionally, in vitro experiments showed that the use of TLR4 agonists could reverse the muscle-protective effect of PCA. In summary, this study illustrated that PCA ameliorated skeletal muscle atrophy in CKD rats by inhibiting the TLR4/MyD88/NF-κB pathway.

Keywords: P‐Coumaric acid; TLR4/MyD88/NF‐κB pathway; inflammation; oxidative stress; skeletal muscle atrophy.

FIGURE 1

PCA alleviated renal function and protein‐energy wasting in CKD rats. (A) Animal experiment design and treatment timeline. (B–E) Scr (B), BUN (C), 24‐h urinary protein (D) and urine output (E) Levels in each group. (F) Typical photographs of H&E staining of renal tissues in each group (scale bars: 100 μm). The kidney anatomy in CKD model rats was disordered, with an extensive number of inflammatory cells infiltrating the renal interstitium (black arrow), glomeruli being significantly atrophic and cystic cavities expanding (green arrow). (G) Weekly weight fluctuations among rats in all groups. (H) Final body weight of rats on the day of sampling. (I) Weight of wet EAT in each group. (J, K) Serum Alb (J) and leptin (K) levels in each group. (L, M) Results of the IPGTT. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, vs. the sham group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^####p < 0.0001, vs. the model group (mean ± SD, n = 6).

FIGURE 2

PCA alleviated skeletal muscle atrophy in CKD rats. (A–C) TA (A), GA (B) and soleus (C) Weights in each group (mean ± SD, n = 5–6). (D) Representative H&E‐stained images of GA (scale bars: 100 μm). (E) Muscle fibres CSA of each group (570 ~ 885 myofibres were measured in each group, mean ± SD, n = 6). (F) Representative electron micrographs. Mitochondria in CKD model rats were decreased, swollen, arranged disorderly and cristae were loose, accompanied by the appearance of membrane rupture or large vacuoles. The black arrows indicate mitochondria (scale bars: 2 μm and 500 nm). **p < 0.01, ***p < 0.001, ****p < 0.0001, vs. the sham group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, vs. the model group.

FIGURE 3

PCA mitigated inflammation and oxidative stress in CKD rats. (A–G) Levels of Tnf, Il1b, Il6, Il17a, Il10, Vcam1 and Icam1 inflammatory cytokines in rat muscle (mean ± SD, n = 4–6). (H–J) Expression of the inflammatory cytokines IL‐6, TNF‐α and CRP in serum (mean ± SD, n = 4–5). (K–R) Contents of SOD, CAT, MDA and GSH‐Px in serum and muscles of each group (mean ± SD, n = 4–6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, vs. the sham group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. the model group.

FIGURE 4

Network pharmacology forecasted the probable mechanism of PCA ameliorating skeletal muscle atrophy. (A) Molecular structure diagram of PCA. (B) Venn analysis of the intersection genes between PCA and skeletal muscle atrophy. (C) PPI network of overlapping genes. (D) PPI network of top 30 target genes. (E) KEGG enrichment analysis of overlapping genes. (F) GO analysis of overlapping genes (from left to right in turn for molecular functions, biological processes and cellular components).

FIGURE 5

Molecular docking of PCA with target proteins. Docking situation of PCA and TLR4 (A), MyD88 (B), IKKβ (C), NF‐κB p65 (D) and MurF1 (E).

FIGURE 6

PCA inhibited the TLR4/MyD88/NF‐κB pathway in the muscle tissues of CKD rats. (A) The heatmap analysis of important genes in the TLR4/MyD88/NF‐κB signalling pathway. (B–F) Expression levels of Tlr4 (B), Myd88 (C), Ikbkb (D), Nfkbia (E) and Rela (F) in muscles of rats (mean ± SD, n = 6). (G) Western blot analysis of the representative bands of TLR4, MyD88, p‐p65, p65 and GAPDH proteins. (H–K) Statistical results of Western blot expression of TLR4, MyD88, p‐p65 and p65 proteins, with GAPDH as a control (mean ± SD, n = 3–4). (L, M) The levels of Murf1 and Mafbx mRNA expression in rat muscle (mean ± SD, n = 6). (N, O) Representative Western blot bands of MurF1, MAFbx and GAPDH protein. (P, Q) Statistical results of Western blot expression of MurF1 and MAFbx proteins, with GAPDH as a control (mean ± SD, n = 4). *p < 0.05, **p < 0.01, vs. the sham group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001, vs. the model group; ns, no significant differences.

FIGURE 7

PCA inhibited the TLR4/MyD88/NF‐κB signalling pathway in C2C12 myoblasts. The MTT results showed that 40 μM PCA emerged as the most effective concentration. (A) Viability of C2C12 myoblasts after 24‐h exposure to PCA (mean ± SD, n = 5). (B) Viability of C2C12 myoblasts after 24‐h exposure to LPS (mean ± SD, n = 5). (C) LPS‐treated C2C12 myoblasts viability after treatment with PCA for 24 h (mean ± SD, n = 4). (D) Viability of C2C12 myoblasts after 24‐h exposure to PA (mean ± SD, n = 5). (E) Western blot analysis of the representative bands of TLR4, MyD88, p‐p65, p65, MAFbx, MurF1 and GAPDH proteins. (F–H) Statistical results of Western blot expression of TLR4, MyD88, p‐p65 and p65 proteins, with GAPDH as a control (mean ± SD, n = 5). (I, J) Statistical results of Western blot expression of MAFbx and MurF1 proteins, with GAPDH as a control (mean ± SD, n = 4). *p < 0.05, **p < 0.01, ****p < 0.0001, vs. the control group; ^# p < 0.05, ^## p < 0.01, vs. the LPS group; ^&p < 0.05, ^&&p < 0.01, vs. the LPS + PCA group.

FIGURE 8

PCA suppressed the inflammation and oxidative stress induced by LPS in C2C12 myoblasts. (A–E) Expression of the inflammatory cytokines Tnf, Il1b, Crp, Il6 and Il10 in C2C12 myoblasts. (F) Representative pictures of ROS fluorescence in C2C12 myoblasts. (G) Figures of the statistical results of ROS fluorescence. (H–K) SOD, CAT, MDA and GSH‐Px contents in C2C12 myoblasts of different groups. *p < 0.05, **p < 0.01, vs. the control group; ^# p < 0.05, ^## p < 0.01, vs. the LPS group; ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001, vs. the LPS + PCA group; ns, no significant difference (mean ± SD, n = 4).

FIGURE 9

A brief overview of PCA's mechanism for alleviating skeletal muscle atrophy in CKD (by Figdraw).