Ficus carica L. Attenuates Denervated Skeletal Muscle Atrophy via PPARα/NF-κB Pathway

Abstract

Treatment options for denervated skeletal muscle atrophy are limited, in part because the underlying molecular mechanisms are not well understood. Unlike previous transcriptomics studies conducted in rodent models of peripheral nerve injury, in the present study, we performed high-throughput sequencing with denervated atrophic biceps muscle and normal (non-denervated) sternocleidomastoid muscle samples obtained from four brachial plexus injury (BPI) patients. We also investigated whether Ficus carica L. (FCL.) extract can suppress denervated muscle atrophy in a mouse model, along with the mechanism of action. We identified 1471 genes that were differentially expressed between clinical specimens of atrophic and normal muscle, including 771 that were downregulated and 700 that were upregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the differentially expressed genes were mainly enriched in the GO terms "structural constituent of muscle," "Z disc," "M band," and "striated muscle contraction," as well as "Cell adhesion molecules," "Glycolysis/Gluconeogenesis," "Peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway," and "P53 signaling pathway." In experiments using mice, the reduction in wet weight and myofiber diameter in denervated muscle was improved by FCL. extract compared to saline administration, which was accompanied by downregulation of the proinflammatory cytokines interleukin (IL)-1β and IL-6. Moreover, although both denervated groups showed increased nuclear factor (NF)-κB activation and PPARα expression, the degree of NF-κB activation was lower while PPARα and inhibitor of NF-κB IκBα expression was higher in FCL. extract-treated mice. Thus, FCL. extract suppresses denervation-induced inflammation and attenuates muscle atrophy by enhancing PPARα expression and inhibiting NF-κB activation. These findings suggest that FCL. extract has therapeutic potential for preventing denervation-induced muscle atrophy caused by peripheral nerve injury or disease.

Keywords: Ficus carica; NF-κß; PPAR; denervated muscle atrophy; muscle atrophy; peripheral nerve injury.

FIGURE 1

HE and Masson’s trichrome staining showing atrophy of human sternocleidomastoid muscle (left figures) and biceps muscle (right figures). (A,B) HE staining showing the irregular arrangement of myocytes in denervated muscle and a smaller stained area compared to normal (non-denervated) sternocleidomastoid muscle. (C,D) Masson’s trichrome staining showing the intercellular matrix of denervated muscle covered with blue collagen fiber. Scale bar, 200 μm.

FIGURE 2

DEGs identified by RNA-seq and associated GO terms. (A,B) Heatmap and volcano plot of DEGs. (C) Top 30 GO terms.

FIGURE 3

KEGG pathways of DEGs. (A) Top 20 KEGG pathways of DEGs. (B) Top four pathways gene network.

FIGURE 4

FCL. extract attenuates skeletal muscle atrophy and inhibits IL-1β and IL-6 production in denervated muscle (n = 4 mice in each group). (A,B) The wet weight ratio of gastrocnemius at 21 days post-denervation. (C,D) HE staining of muscle tissue and means ± SEM of fibro-diameter showing muscle atrophy that was reduced by FCL. extract treatment. (E,F) Compared to control group muscle, IL-1β and IL-6 positive-expression was increased in the denervated muscle of Den-saline group mice; the levels of both cytokines were lower in the Den-FCL. mice. Scale bar, 50 μm. **p < 0.01 versus Control. ##p < 0.01 versus Den-Saline.

FIGURE 5

Expression levels of IL-1β and IL-6 in gastrocnemius were detected using ELISA kits. Both IL-1β and IL-6 expressions were increased in the denervated mice muscle, and the elevated level of both cytokines was lower in the Den-FCL. mice (n = 4 mice in each group). (A) IL-1β expression was determined. (B) IL-6 expression was determined. **p < 0.01 and *p < 0.05 versus Control. ##p < 0.01 versus Den-Saline.

FIGURE 6

FCL. attenuates skeletal muscle atrophy via PPARα/NF-κB signaling. (A,B) Elevated expression of PPARα in both denervated group mice were determined by western blotting. PPARα elevation level of Den-FCL group mice was much higher than the Den-saline group. (C) PPARA mRNA levels determined by qPCR showed a trend similar to that of the protein. (D–F) p65-NF-κB activity was increased after muscle denervation, but was reduced by FCL. extract as compared to saline treatment. Meanwhile, the Den-FCL. group mice showed higher IκBα expression level than other groups (n = 4 mice in each group). **p < 0.01 and *p < 0.05 versus Control. ##p < 0.01 and #p < 0.05 versus Den-Saline.

FIGURE 7

A scheme for FCL. extract attenuating denervated muscle atrophy by inhibiting the inflammation response through PPARα/NF-κB signaling pathways.