Objective To observe the expressions of tumor necrosis factor alpha (TNF-α),matrix metalloproteinase 2 (MMP-2) and collagen in local skin tissue of pressure ulcer of rats,and to explore the possible mechanism of the pathogenesis of pressure ulcer.Methods Forty male SD rats were divided into normal control group,3 d compression group,5 d compression group,7 d compression group,and 9 d compression group according to the random number table,with 8 rats in each group.The rats in normal control group did not receive any treatment,whereas the rats in the latter 4 groups were established the deep tissue injury model (3 d compression group) and pressure ulcer model (the other 3 groups) on the gracilis muscle on both hind limbs using a way of cycle compression of ischemia-reperfusion magnet.The rats in 3 d compression group received only three cycles of compression,while the compressed skin of the rats in 5 d compression group,7 d compression group,and 9 d compression group were cut through and received pressure to 5,7 and 9 cycles after three cycles of compression,respectively.The rats in 3 d compression group were sacrificed immediately after receiving compression for 3 d (the rats in normal control group were sacrificed at the same time),and the rats in the other 3 groups were respectively sacrificed after receiving compression for 5,7,and 9 d,and the skin tissue on the central part of gracilis muscle on both hind limbs were harvested.The morphology of the skin tissue was observed with HE staining.The expression of collagen fiber was observed with Masson staining.The expressions of collagen type Ⅳ and MMP-2 were detected by immunohistochemical method.The expressions of TNF-α and phosphorylated NF kappa B (NF-κB) were determined by Western blotting.Data were processed with one-way analysis of variance and LSD test.Results (1) In normal control group,the skin tissue of rats was stratified squamous epithelium,with the clear skin structure,and there was no obvious infiltration of inflammatory cells.In 3 d compression group,the skin layers of rats were clear,with quite a few fibroblasts,and the inflammatory cells began to infiltrate.In 5 d compression group,7 d compression group,and 9 d compression group,the epidermis of rats thickened,with the number of fibroblasts reduced,and the infiltration of inflammatory cells enhanced with the compressed time prolonging.(2) In normal control group,the collagen fibers in skin tissue of rats were arranged in order,with rich content.In 3 d compression group,the collagen fibers in skin tissue of rats were arranged orderly,with high expression level,which was similar to that in normal control group (P ＞ 0.05).In 5 d compression group and 7 d compression group,the collagen fibers in skin tissue of rats were arranged in disorder,with the expression level gradually reduced,which were significantly lower than that in normal control group (with P values below 0.01).In 9 d compression group,the expression of collagen fiber in skin tissue of rats was a little higher than that in 7 d compression group,but it was still significantly lower than that in normal control group (P ＜ 0.01).(3) The expressions of collagen type Ⅳ in skin tissue of rats in normal control group,3 d compression group,5 d compression group,7 d compression group,and 9 d compression group were respectively 11.0 ± 2.8,9.0 ± 1.7,8.3 ± 2.8,5.1 ± 1.8,and 5.4 ± 1.2.The expression of collagen type Ⅳ in skin tissue of rats in 3 d compression group was similar to that in normal control group (P ＞ 0.05).The expressions of collagen type Ⅳ in skin tissue of rats in 5 d compression group,7 d compression group,and 9 d compression group were significantly lower than that in normal control group (P ＜ 0.05 or P ＜0.01).The expression of MMP-2 in skin tissue of rats in 3 d compression group was similar to that in normal control group (P ＞ 0.05).The expressions of MMP-2 in skin tissue of rats in 5 d compression group,7 d compression group,and 9 d compression group were significantly higher than that in normal control group (P ＜0.05 orP ＜0.01).(4) The expression of TNF-α in skin tissue of rats in normal controlgroup was 0.48 ± 0.11,and the expressions of TNF-α in skin tissue of rats in 3 d compression group,5 d compression group,7 d compression group,and 9 d compression group were respectively 0.84 ± 0.08,1.13 ± 0.19,1.34 ± 0.16,and 1.52 ± 0.23,which were all significantly higher than that in normal control group (with P values below 0.01).The expressions of phosphorylated NF-κB in skin tissue of rats in 3 d compression group and 9 d compression group were similar to that in normal control group (with P values above 0.05),and the expressions of phosphorylated NF-κB in skin tissue of rats in 5 d compression group and 7 d compression group were significantly higher than that in normal control group (P ＜ 0.05 or P ＜ 0.01).Conclusions The high expression of MMP-2 and reduction of collagen induced by inflammatory reaction mediated by the high expression of TNF-α in local skin tissue of pressure ulcer of rats may be one of the important reasons for the formation of pressure ulcer.

OBJECTIVETo investigate the effect of exogenous recombinant human basic fibroblast growth factor (rhbFGF) on the healing of muscles in rats after deep tissue injury of pressure ulcers.

METHODSForty-eight SD rats were randomly divided into normal control group, injury control group, post injury day (PID) 4 group, PID 7 group, PID 14 group, PID 21 group according to the random number table, with 8 rats in each group. The rats in normal control group did not receive any treatment, whereas the rats in the latter 5 groups were established the deep tissue injury of pressure ulcer model on both sides of the gracilis muscle on the hind limb. The rats in injury control group did not receive any treatment after injury, while the rats in the latter 4 groups were given subcutaneous injection of 0.1 mL rhbFGF to the left gracilis in a dosage of 100 µg/mL immediately after injury, and an equal volume of normal saline (NS) was injected to right gracilis, once every other day. The rats in injury control group were sacrificed immediately after injury, and the rats in normal control group were sacrificed at the same time point. The rats in the other 4 groups were sacrificed on PID 4, 7, 14, 21, and the gracilis muscles on both sides were harvested respectively. The morphology of the gracilis muscle was examined after HE staining. The expression of myogenin in the tissues was detected by immunofluorescence method. The levels of muscle structural proteins myosin heavy chain (MyHC), phosphorylated protein kinase B (Akt), and phosphorylated mammalian target of rapamycin (mTOR) were determined by Western blotting. Data were processed with one-way analysis of variance and LSD test.

RESULTS(1) In normal control group, the nuclei of graciles cells were in uniform size, and they were closely arranged with clear structure, and there were no significant infiltration of inflammatory cells. In injury control group, the nuclei of graciles cells showed signs of pyknosis, dissolution, fracture and structural disorder. Swelling of muscle cells, inflammation infiltration, structural disorder and other pathological signs of injury phenomena in graciles of PID 4 group, PID 7 group, PID 14 group, PID 21 group after rhbFGF treatment were milder compared with those after NS treatment. In addition, the numbers of regenerated myocytes in graciles of PID 4 group, PID 7 group, PID 14 group, PID 21 group after rhbFGF treatment were higher than those after NS treatment. (2) The numbers of graciles myogenin positive cells in normal control group and injury control group were respectively 28 ± 17 and 42 ± 28. The numbers of graciles myogenin positive cells in PID 4 group, PID 7 group, PID 14 group after NS treatment were 100 ± 50, 196 ± 87, 460 ± 110 respectively, while the numbers of graciles myogenin positive cells in PID 4 group, PID 7 group, PID 14 group after rhbFGF treatment were 174 ± 34, 717 ± 182, 613 ± 122 respectively, and the numbers of graciles myogenin positive cells after rhbFGF treatment were significantly higher than those after NS treatment in each group(P < 0.05 or P < 0.01). The number of graciles myogenin positive cells in PID 21 group after rhbFGF treatment was 109 ± 34, which was significantly lower than that after NS treatment (218 ± 71, P < 0.05). (3) The expression of MyHC in graciles in normal control group was high, which was decreased in injury control group. Both the expressions of MyHC in graciles in PID 4 group, PID 7 group, PID 14 group, PID 21 group after treatment of NS and rhbFGF showed a trend of gradual elevation, while the expressions of MyHC in graciles after rhbFGF treatment were significantly higher than those after NS treatment (P < 0.05 or P < 0.01). The expression of MyHC in graciles in PID 21 group showed a high level, and it was similar to that of the normal control group (P > 0.05). The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of normal control group were low, and the expression of phosphorylated Akt in graciles increased in injury control group, while the expression of phosphorylated mTOR in graciles decreased in injury control group. The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of PID 4 group, PID 7 group, PID 14 group, PID 21 group after treatment with rhbFGF showed a trend of elevation in the beginning but declined afterwards. The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of PID 4 group after rhbFGF treatment were significantly lower than those after NS treatment (P <0 .05 or P < 0.01). The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of PID 7 group, PID 14 group, PID 21 group after rhbFGF treatment were significantly higher than those after NS treatment (P < 0.05 or P < 0.01).

CONCLUSIONSExogenous rhbFGF may effectively facilitate the healing of muscle structure and accelerate the regeneration of muscles in rats after deep tissue injury of pressure ulcers, and its mechanism may be related to the improvement of the expression of myogenin and enhancement of the expression of protein of muscle growth-related signaling pathways.

Animals ; Disease Models, Animal ; Fibroblast Growth Factor 2 ; metabolism ; Humans ; Myogenin ; metabolism ; Phosphorylation ; Pressure Ulcer ; therapy ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Wound Healing