Myoprotective effects of bFGF on skeletal muscle injury in pressure-related deep tissue injury in rats

Abstract

Background: Pressure ulcers (PUs) are a major clinical problem that constitutes a tremendous economic burden on healthcare systems. Deep tissue injury (DTI) is a unique serious type of pressure ulcer that arises in skeletal muscle tissue. DTI arises in part because skeletal muscle tissues are more susceptible than skin to external compression. Unfortunately, few effective therapies are currently available for muscle injury. Basic fibroblast growth factor (bFGF), a potent mitogen and survival factor for various cells, plays a crucial role in the regulation of muscle development and homeostasis. The main purpose of this study was to test whether local administration of bFGF could accelerate muscle regeneration in a rat DTI model.

Methods: Male Sprague Dawley (SD) rats (age 12 weeks) were individually housed in plastic cages and a DTI PU model was induced according to methods described before. Animals were randomly divided into three groups: a normal group, a PU group treated with saline, and a PU group treated with bFGF (10 μg/0.1 ml) subcutaneously near the wound.

Results: We found that application of bFGF accelerated the rate of wound closure and promoted cell proliferation and tissue angiogenesis. In addition, compared to saline administration, bFGF treatment prevented collagen deposition, a measure of fibrosis, and up-regulated the myogenic marker proteins MyHC and myogenin, suggesting bFGF promoted injured muscle regeneration. Moreover, bFGF treatment increased levels of myogenesis-related proteins p-Akt and p-mTOR.

Conclusions: Our findings show that bFGF accelerated injured skeletal muscle regeneration through activation of the PI3K/Akt/mTOR signaling pathway and suggest that administration of bFGF is a potential therapeutic strategy for the treatment of skeletal muscle injury in PUs.

Keywords: PI3K/Akt/mTOR; Pressure ulcer; Regeneration; Skeletal muscle injury; bFGF.

Fig. 1

bFGF improved muscle regeneration and inhibited fibrosis. a HE staining. The elevation of area of interstitial space (star), structural fractured (arrow), and mild edema were found in injured muscle tissues on the 0th day. Numerous of inflammatory cells infiltrating (triangle) were found in saline group, while bFGF inhibited inflammatory cells infiltrating (triangle) on the 4th day. bFGF increased centro-nucleated myofibers (arrow) on the 7th and 14th days. b Observation of fibrosis distributed in injured muscle tissue by Masson’s trichrome staining. Injured muscle tissues containing collagen deposition (blue) and myofibers (red) were analyzed. Injured muscle tissues treated with bFGF showed a significant reduction in collagen deposition and increased myofibers within the areas of injury compared with saline group on the 7th, 14th, and 21st days

Fig. 2

Enhancement of proliferation and angiogenesis by bFGF administration. a Photographs showing PCNA immunohistochemistry in saline group and bFGF group (×200). b Photographs showing CD31 immunohistochemistry (arrow) in two groups (×200). c, d Graph of PCNA and CD31 immunohistochemistry in two groups. e Photographs showing CD31 immunofluorescence (arrow) on the 7th and 14th days in two groups (×200). f Graph of CD31 immunofluorescence in two groups. Data are expressed as mean ± SEM (n = 6). *P < 0.05 compared with saline group

Fig. 3

bFGF increased expression of myogenic markers myogenin and MyHC. a Western blot analysis of myogenin and MyHC protein expression in skeletal muscle tissues of saline group and bFGF group at indicated time. Actin was used as the loading control and for band density normalization. All of the experiments were repeated three times. b, c The optical density analysis of myogenin and MyHC protein. Data are expressed as Mean ± SEM (n = 3). d Photographs showing myogenin immunofluorescence (green, red arrow in merge pictures) on the 7th and 14th days in two groups (×200). e Graph of myogenin immunofluorescence in two groups. Data are expressed as mean ± SEM (n = 6). *P < 0.05 compared with saline group

Fig. 4

bFGF up-regulated the phosphorylation of Akt and mTOR. a Western blot analysis of phosphorylation of Akt and mTOR levels in skeletal muscle tissues of saline group and bFGF group at indicated time. Actin was used as the loading control, and total Akt and mTOR were used for band density normalization. All of the experiments were repeated three times. b, c The optical density analysis of phosphorylation of Akt and mTOR levels. Data are expressed as mean ± SEM (n = 3). *P < 0.05 compared with saline group