Skeletal Muscle Contusion Research Articles

SAT1/ALOX15 Signaling Pathway Is Involved in Ferroptosis After Skeletal Muscle Contusion

Skeletal muscle contusion (SMC) is common in daily life and clinical practice, but the molecular mechanisms underlying SMC healing are unclear. Ferroptosis, a regulated cell death type, has gained attention recently. We observed iron overload in skeletal muscle following contusion through HE and Perls staining. Abnormal iron levels are highly likely to induce ferroptosis. Therefore, we aimed to explore whether iron overload after contusion leads to ferroptosis in skeletal muscle and the underlying mechanisms, which will help us understand the effects of iron abnormalities on skeletal muscle repair. Initially, we searched SMC gene expression profiles from the GEO database and used bioinformatics analysis to reveal ferroptosis occurrence. Then, we identified the gene sat1 plays an important role in this process. We further established a rat SMC model and treated rats with ferroptosis inhibitors (Ferrostatin-1, Deferoxamine). Our findings confirmed iron overload from SMC can lead to ferroptosis in rats. We also demonstrated that SAT1 can regulate ferroptosis by affecting ALOX15. Moreover, we constructed a ferroptosis L6 cell model and found that SAT1 knockdown significantly inhibited ALOX15 expression and reduced cellular lipid peroxidation. In conclusion, these results indicated ferroptosis can occur following SMC, and SAT1, as a key regulator, affects skeletal muscle injury healing by mediating high ALOX15 expression, which in turn regulates lipid peroxidation.

Development of a screening system of gene sets for estimating the time of early skeletal muscle injury based on second-generation sequencing technology.

The present study is aimed to address the challenge of wound age estimation in forensic science by identifying reliable genetic markers using low-cost and high-precision second-generation sequencing technology. A total of 54 Sprague-Dawley rats were randomly assigned to a control group or injury groups, with injury groups being further divided into time points (4h, 8h, 12h, 16h, 20h, 24h, 28h, and 32h after injury, n = 6) to establish rat skeletal muscle contusion models. Gene expression data were obtained using second-generation sequencing technology, and differential gene expression analysis, weighted gene co-expression network analysis (WGCNA) and time-dependent expression trend analysis were performed. A total of six sets of biomarkers were obtained: differentially expressed genes at adjacent time points (127 genes), co-expressed genes most associated with wound age (213 genes), hub genes exhibiting time-dependent expression (264 genes), and sets of transcription factors (TF) corresponding to the above sets of genes (74, 87, and 99 genes, respectively). Then, random forest (RF), support vector machine (SVM) and multilayer perceptron (MLP), were constructed for wound age estimation from the above gene sets. The results estimated by transcription factors were all superior to the corresponding hub genes, with the transcription factor group of WGCNA performed the best, with average accuracy rates of 96% for three models' internal testing, and 91.7% for the highest external validation. This study demonstrates the advantages of the indicator screening system based on second-generation sequencing technology and transcription factor level for wound age estimation.

Detection of multiple biomarkers associated with satellite cell fate in the contused skeletal muscle of rats for wound age estimation.

From the perspective of forensic wound age estimation, experiments related to skeletal muscle regeneration after injury have rarely been reported. Here, we examined the time-dependent expression patterns of multiple biomarkers associated with satellite cell fate, including the transcription factor paired box 7 (Pax7), myoblast determination protein (MyoD), myogenin, and insulin-like growth factor (IGF-1), using immunohistochemistry, western blotting, and quantitative real-time PCR in contused skeletal muscle. An animal model of skeletal muscle contusion was established in 30 Sprague-Dawley male rats, and another five rats were employed as non-contused controls. Morphometrically, the data obtained from the numbers of Pax7 + , MyoD + , and myogenin + cells were highly correlated with the wound age. Pax7, MyoD, myogenin, and IGF-1 expression patterns were upregulated after injury at both the mRNA and protein levels. Pax7, MyoD, and myogenin protein expression levels confirmed the results of the morphometrical analysis. Additionally, the relative quantity of IGF-1 protein > 0.92 suggested a wound age of 3 to 7days. The relative quantity of Pax7 mRNA > 2.44 also suggested a wound age of 3 to 7days. Relative quantities of Myod1, Myog, and Igf1 mRNA expression > 2.78, > 7.80, or > 3.13, respectively, indicated a wound age of approximately 3days. In conclusion, the expression levels of Pax7, MyoD, myogenin, and IGF-1 were upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting the potential for using them as candidate biomarkers for wound age estimation in skeletal muscle.

Contusion concomitant with ischemia injury aggravates skeletal muscle necrosis and hinders muscle functional recovery.

Contusion concomitant with ischemia injury to skeletal muscles is common in civilian and battlefield trauma. Despite their clinical importance, few experimental studies on these injuries are reported. The present study established a rat skeletal muscle contusion concomitant with ischemia injury model to identify skeletal muscle alterations compared with contusion injury or ischemia injury. Macroscopic and microscopic morphological evaluation showed that contusion concomitant with ischemia injury aggravated muscle edema and hematoxylin-eosin (HE) injury score at 24 h postinjury. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels, together with gastrocnemius muscle (GM) tumor necrosis factor-alpha (TNF-α) content elevated at 24 h postinjury too. During the 28-day follow-up, electrophysiological and contractile impairment was more severe in the contusion concomitant with ischemia injury group. In addition, contusion concomitant with ischemia injury decreased the percentage of larger (600-3000 μm2) fibers and increased the fibrotic area and collagen I proportion in the GM. Smaller proportions of Pax7+ and MyoD+ satellite cells (SCs) were observed in the contusion concomitant with ischemia injury group at 7 days postinjury. In conclusion, contusion concomitant with ischemia injury to skeletal muscle not only aggravates early muscle fiber necrosis but also hinders muscle functional recovery by impairing SC differentiation and exacerbating fibrosis during skeletal muscle repair.

Quantitative assessment of changes in skeletal muscle injury by computer-aided analysis based ontwo-dimensional ultrasonography combined with contrast-enhanced ultrasonography and estimated by a modified semi-quantitative scoring system: Anexperimental study in a contusion model.

The aim of this study was to investigate the potential application of computer-aided analysis in the quantitative assessment of changes in skeletal muscle injury in the rabbit contusion model. Forty healthy rabbits were randomly divided into control (n=5) and contusion (n=35) groups. Rabbits in the contusion group were used to construct a muscle contusion model induced by a hammer hitting the right gastrocnemius, while the muscles of rabbits in the control group were non-injured. Two-dimensional ultrasound (2D US) and contrast-enhanced ultrasonography (CEUS) were performed on the rabbits that had received skeletal muscle contusion injury at 1h, and 1, 3, 7, 14, 21 and 28 days after injury. Afterwards, a multiscale blob feature (MBF) method was used to extract the textural features from the 2D US, and the muscle injuries were quantitatively evaluated. The eight textural parameters of skeletal muscle analysed by MBF at 1h, and 1, 3 and 7 days post-injury were found to be significantly higher in the contusion group than in the control group (p < .05). On Day 14, the textural parameters (e.g., greyscale mean [Mean], greyscale standard deviation [SDev], number of blobs, average size of blobs, homogeneity of distribution, periodicity of distribution [POD] and irregularity) were also evidently higher in the contusion group than in the control group (p < .05). On Day 28, Mean, SDev and POD in the contusion group were markedly higher (p < .05). After that, the microcirculation in the injured areas increased from Day 7 to Day 21 after injury, but decreased on Day 28 after injury. Thus the quantitative assessment of changes in skeletal muscle injury (SMI) using computer-aided analysis allowed us to describe the geometric features of injured muscle fibres and the microperfusion changes estimated by the modified semi-quantitative scoring system. This provides a scientific basis for the development of a novel approach for the evaluation of SMI and rehabilitation process.

Biomimetic Design and Biocompatibility of Biomimetic Calcium Carbonate Nanocomposites for Skeletal Muscle Injury Repair

As an important part of the human body, skeletal muscle is easily damaged in the process of exercise. The purpose of this study is to investigate the controllable construction and biocompatibility of biomimetic calcium carbonate nanocomposites in exercise skeletal muscle repair. In this study, adult healthy mice were selected as the research objects. First, the mice were anesthetized with 2.5% pentobarbital sodium. Then, the mice were fixed and hit with steel balls on the middle part of the gastrocnemius muscle abdomen to establish a skeletal muscle contusion model. The mice were killed by cervical dislocation under anesthesia. The injured gastrocnemius muscles were quickly removed to prepare the cell suspension. 0.8600 g chitosan was weighed and dissolved in 200 ml deionized water. Then, 0.0660 g of calcium chloride dihydrate was added and stirred to dissolve to prepare chitosan calcium carbonate composite. The calcium carbonate composite material with a length of 8.0 cm is cut into a square sheet of stainless steel. The stainless steel plate was sterilized and then placed in a 12 culture plate to dilute the full cells in the culture bottle. The cells were seeded in 12 well culture plates, and 100 μL cells and 600 μL medium were added to each well. The samples were cultured in the incubator (there were three control holes in each group for SEM test, three control holes for fluorescence measurement, and six control holes for MTT measurement). The plates were taken out and placed in a new 24 plate. 1000 μL medium was added. Then. 100 μL MTT solution was added into each well. The biocompatibility test was conducted for 4 h. The GSH PX level in the chitosan calcium carbonate composite group was lower than that in the natural recovery group (P < 0.01). The results showed that chitosan calcium carbonate composite could promote the growth of skeletal muscle cells.

Estimating the time of skeletal muscle contusion based on the spatial distribution of neutrophils: a practical approach to forensic problems.

The study aimed to explore the neutrophil's spatial distributions used to estimate the histological age of contused skeletal muscle, and assessed the accuracy of various indicators, such as the proportion of neutrophils, "neutrophil mean distance," and distribution of neutrophils in areas of "contiguous contour lines." Fifty-five Sprague-Dawley rats were divided randomly into a control group and contusion groups at 1, 1.5, 2, 3, 4, and 6h, as well as 1, 3, 5, and 15days, post-injury (n = 5 per group). Nuclei and neutrophils were detected by hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining. At 0-24h after injury, the distribution of neutrophils at distances of 100, 200, 300, 400, 500, and 600µm from adjacent blood vessels was determined, and the best samples were screened to estimate wound age. To estimate wound age as accurately as possible, Fisher discriminant analysis (FDA) of the proportion of neutrophils, neutrophil mean distance, and distribution of neutrophils was performed, and 100.0% and 95.0% of the original and cross-validated cases were correctly classified, respectively. The spatial distribution of neutrophils at different distances from adjacent blood vessels showed a strong correlation with the histological age of contusion skeletal muscle, and the combination of the proportion of neutrophils, neutrophil mean distance, and distribution of neutrophils could be used to accurately estimate wound age.

Bone Marrow Stromal Cell-Derived Exosomes Promote Muscle Healing Following Contusion Through Macrophage Polarization.

Skeletal muscle contusion is among the most common injuries in traumatology and clinics of sports medicine. The injured muscle is vulnerable to re-injury owing to fibrosis formation. Given that the bone marrow stromal cell-derived exosomes (BMSC-Exos) displayed promising therapeutic effect for various tissues, we used BMSC-Exos to treat skeletal muscle contusion and investigated its effects on muscle healing. In this study, the in vivo model of skeletal muscle contusion was established by subjecting the tibialis anterior of young male mice to hit injury, and the in vitro inflammation model was established by lipopolysaccharide treatment on macrophages. Macrophage depletion model was built by intraperitoneal injection with clodronate-containing liposomes. Exosomes were isolated and purified from the supernatant of BMSCs using gradient centrifugation. Nanoparticle tracking analysis, transmission electron microscope, and western blot were used to identify the exosomes. HE stain, Masson stain, immunofluorescence, and biomechanical testing were carried out on the muscle tissue. In addition, enzyme-linked immunosorbent assay (ELISA) assays, real-time qPCR, flow cytometry, and PKH67 fluorescence trace were conducted in vitro. Intramuscular injection of BMSC-Exos to mice after muscle contusion alleviated inflammation level, reduced fibrosis size, promoted muscle regeneration, and improved biomechanical property. After macrophages depletion, the effects of BMSC-Exos were inhibited. In vitro, PKH-67 fluorescence was internalized into macrophages. BMSC-Exos promoted M2 macrophages polarization both in vivo and in vitro. At the same time, BMSC-Exos reduced the production of inflammatory cytokines under the inflammatory microenvironment and upregulated anti-inflammatory factors expression. In conclusion, BMSC-Exos attenuated muscle contusion injury and promoted muscle healing in mice by modifying the polarization status of macrophages and suppressing the inflammatory reaction.

Sequential Changes of Serum Biomarkers after Skeletal Muscle Contusion in Rats

Objective To screen serum biomarkers after skeletal muscle contusion in rats based on gas chromatography-mass spectrometry （GC-MS） metabolomics technology, and support vector machine （SVM） regression model was established to estimate skeletal muscle contusion time. Methods The 60 healthy SD rats were randomly divided into experimental group （n=50）, control group （n=5） and validation group （n=5）. The rats in the experimental group and the validation group were used to establish the model of skeletal muscle contusion through free fall method, the rats in experimental group were executed at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 96 h, 144 h and 240 h, respectively, and the rats in validation group were executed at 192 h, while the rats in the control group were executed after three days' regular feeding. The skeletal muscles were stained with hematoxylin-eosin （HE）. The serum metabolite spectrum was detected by GC-MS, and orthogonal partial least square-discriminant analysis （OPLS-DA） pattern recognition method was used to discriminate the data and select biomarkers. The SVM regression model was established to estimate the contusion time. Results The 31 biomarkers were initially screened by metabolomics method and 6 biomarkers were further selected. There was no regularity in the changes of the relative content of the 6 biomarkers with the contusion time and the SVM regression model can be successfully established according to the data of 6 biomarkers and the 31 biomarkers. Compared with the injury time ［（55.344±7.485） h］ estimated from the SVM regression model based on the data of 6 biomarkers, the injury time ［（195.781±1.629） h］ estimated from the SVM regression model based on the data of 31 biomarkers was closer to the actual value. Conclusion The SVM regression model based on metabolites data can be used for the contusion time estimation of skeletal muscles.

Effects of Ultrasound and Stretching on Skeletal Muscle Contusion in Rats: Immunohistochemistry Analysis

Effects of Ultrasound and Stretching on Skeletal Muscle Contusion in Rats: Immunohistochemistry Analysis

Hydrogen Sulfide Alleviates Skeletal Muscle Fibrosis via Attenuating Inflammation and Oxidative Stress.

The purpose of this study was to investigate the effect of exogenous hydrogen sulfide (H2S) treatment on skeletal muscle contusion. We established a skeletal muscle contusion model (S group) and an H2S treated of skeletal muscle contusion model (H2S group). Gastrocnemius muscles (GMs) were collected at day 1, day 5, day 10, and day 15 after injury, and comprehensive morphological and genetic analyses was conducted. H2S treatment reduced M1 macrophage (CD68), profibrotic cytokines (TGF-β), pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6), chemokines (CCL2, CCR2, CCL3, CCL5, CXCL12, and CXCR4), matrix metalloproteinases (MMP-1, MMP-2, MMP-9, and MMP-14) and oxidative stress factor (gp91phox) expression levels, improved M2 macrophage (CD206) level. Thus, exogenous H2S treatment reduced inflammation and oxidative stress, attenuated skeletal muscle fibrosis, and partly improved skeletal muscle injury.

Role of miR-128, Sp1 and CDKN1A mRNA in Repairing Muscle Injury in a Rat Skeletal Muscle Injury Model

In order to detect the changes in expression of miR-128 during skeletal muscle repair in a rat model of muscle injury and to provide a basis for treating skeletal muscle injury, a rat skeletal muscle injury model was established. Rats were randomly divided into the quiet control group, 0 h after exercise group, 6 h after exercise group, 12 h after exercise group, 24 h after exercise group, 48 h after exercise group, 72 h after exercise group, 1 w after exercise group and 2 w after exercise groups to explore the expression pattern of miR-128, Sp1, CDKN1A during skeletal muscle contusion repair. Results showed that muscle fibers in the control group were polygonal, closely arranged and regular in shape. The myocyte membrane was intact and the nucleus was located under the myometrium. At 0 h after exercise, cells were swollen and round, cell volume increased and the cell gap increased. At 6, 12, 24 and 48 h after exercise, the cells continued to swell and the intercellular space became smaller than that in group C. In the 72 h after exercise group, cell swelling was very high and there were a large number of infiltrated inflammatory cells and exudation of blood cells. In 1 w after exercise group, inflammatory cell infiltration decreased since exercise. When it reaches to 2w, the morphology and structure of the cells return to normal, and the repair process was completed. Successful establishment of the rat skeletal muscle repair model, it is found that the expression of muscle-specific miR-128 did not change significantly during the process of muscle repair. miR-128 might be involved in the process of regeneration after skeletal muscle injury.

Expression and role of lncRNAs in the regeneration of skeletal muscle following contusion injury.

Studies performed previously have indicated that long non-coding RNAs (lncRNAs) may be involved in skeletal muscle regeneration; however, the roles of lncRNAs during the repair of skeletal muscle contusion remain unclear. The present study established a mouse skeletal muscle contusion injury model to identify the roles of lncRNAs that are specifically enriched in the skeletal muscle, namely metastasis-associated lung adenocarcinoma transcript 1 (Malat1), H19, myogenesis-associated lnc (lnc-mg), long intergenic non-protein coding RNAs (linc)-muscle differentiation 1 (linc-MD1), linc-yin yang 1 (linc-YY1) and sirtuin 1-antisense (Sirt1-AS). Morphological analyses revealed that fibrotic scars and regenerating myofibers were formed in the muscle following contusion injury. Gene expression was analyzed by reverse transcription-quantitative polymerase chain reaction. The data revealed that the expression of inflammatory cytokines, myogenic regulatory factors and angiogenic factors increased significantly following skeletal muscle contusion. Additionally, various lncRNAs, including Malat1, H19, lnc-mg, linc-MD1, linc-YY1 and Sirt1-AS were also upregulated. Correlation was also observed between lncRNAs and regulatory factors for skeletal muscle regeneration including transforming growth factor-β1, myogenic differentiation, myogenin, myogenic factor 5 (myf5), myf6, hypoxia-inducible factor-1α and angiopoietin 1. In conclusion, lncRNAs may serve important roles in the regeneration of skeletal muscle following contusion injury, which provides a promising therapy avenue for muscle injury.

Relationship between Changes of Pericyte Number and Wound Age during Repair of Skeletal Muscle Contusion in Mice.

Objective To observe the change pattern of pericyte number at different time periods after mice skeletal muscle contusion and discuss its role in wound age estimation. Methods A mice gastrocnemius muscle contusion model was established. The form and number changes of pericytes at 1, 3, 5, 7, 9, 14, and 28 d post-injury were detected by multiple immunofluorescence staining. Results Compared with the slender shape of pericytes in normal skeletal muscles, pericytes in the contusion area had increased volume, rounder form and a round nuclei. Part of pericytes were found to express satellite cell markers paired-box transcription factor （Pax7） or myoblast determination 1 （MyoD1）. The changes of pericyte number in skeletal muscles after contusion were time-dependant, and showed unimodal distribution with the extension of wound age. In the central contusion area, the number of pericytes peaked at 5 d post-injury while in the peripheral contusion area, the number of pericytes peaked at 5 d and 7 d post-injury. Conclusion The number of pericytes in contusion area varies time-dependently after skeletal muscle contusion in mice and might be a reference index for muscle wound age estimation, and is involved in the repair and regeneration of skeletal muscle injury.

Relationship between the Degeneration and Regeneration of Neuromuscular Junction and Wound Age during the Repair of Mouse Skeletal Muscle Contusion.

Objective To investigate the morphological changes in the degeneration and regeneration of neuromuscular junctions （NMJ） during the repair of mouse skeletal muscle contusion and discuss the correlation between the degeneration and regeneration of NMJ and wound age. Methods A total of 50 healthy adult male mice were randomly divided into 10 groups, including 9 experimental groups and 1 control group. Immunofluorescent staining was applied, and neurofilament was marked with neurofilament protein-H （NF-H）, presynaptic membrane was marked with synaptophysin （Syn）, presynaptic membrane was marked with acetylcholine receptor （AChR）. Morphological changes of NMJ regeneration at different time points after mouse skeletal muscle contusion were detected. Results The neurofilament and presynaptic membrane of NMJ at the junction of contusion zones began to degrade after contusion, and completed degradation at about 3 d post-injury. Then they gradually regenerated, roughly completing the regeneration at about 21 d and basically reaching the control group level. The ratio of presynaptic membrane quantity to presynaptic membrane quantity showed a trend of decreasing then rising and finally reaching the control level. Conclusion During the repair of mouse skeletal muscle contusion, the morphological changes and wound age of the NMJ at the junction of contusion zones have a close correlation, which is expected to be one of the biological indicators for forensic skeletal muscle wound age estimation.

Effect of electroacupuncture of acupoints on the healthy limb (opposing needling) on acute skeletal muscle contusion in rats

To observe the therapeutic effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Ashi"-point on the healthy side (opposing needling) on muscular injury and expression of myogenin (myoG) and fast myosin skeletal heavy chain (Fast MyHC) proteins in the gastrocnemius muscle (GM) tissues in skeletal muscle contusion rats，so as to explore its mechanism underlying improvement of skeletal muscle injury. A total of 54 male SD rats were divided into normal control (n ＝ 6)，model (n＝24) and opposing needling (EA, n＝24) groups. The latter two groups were further randomized into 3, 5, 7 and 14 d subgroups (n＝6 per subgroup). The skeletal muscle contusion model of the hind-limb was established by using a self-made striking device. EA (1 Hz/3 Hz，1－2 mA) was applied to ST36 and "Ashi"-point on the uninjured side of the hind-limb for 15 min every time, once a day for 3, 5, 7 and 14 days, respectively. The injured GM was harvested on the 3rd, 5th, 7th and 14th day after muscular contusion. The morphological changes of the injured GM and the mean cross-sectional areas (CSAs) of the neonatal muscle cells were observed by microscope after H．E. staining. The immunoactivity of desmin protein (myogenic marker protein of myoblast cell) of GM was detected by immunofluorescence stain on the 7th day after injury, and the expression levels of myoG (on the 3rd and 5th day after injury) and fast MyHC protein of GM tissues (on the 7thand 14th day after injury) were detected by Western blot. H．E. staining of GS tissue showed fewer neuronal myocytes with disordered arrangement at different sizes, and appearance of some collagenous fibers among the mesenchyme on day 7 and 14 after muscular contusion, which was relatively milder in the EA group. In the EA group, the CSA values of the neonatal muscle cells were significantly larger than those in the model group on the day 7th (P<0.05), 14th (P<0.001) after injury. On day 7 after muscular contusion, the desmin was found to express on the cellular membrane of GM in the normal control group, while in the model group, the desmin expressed mainly in the cellular plasma in the model group, and on the cellular membrane of neonatal myocytes in the EA group, respectively. The desmin positive myocytes showed disordered arrangement and different sizes after muscular contusion, whereas the situations of the EA group were close to those of the normal control group. Desmin expression was up-regulated in the EA group compared with the model group which was not significant difference (P>0.05). On the 3rd and 5th day after muscular contusion, the expression level of myoG protein was significantly up-regulated in the model group compared with the normal control group (P<0.001), and significantly up-regulated in the EA group than that in the model group (P<0.001). On the 7th and14th day after contusion, the expression level of fast MyHC protein was significantly down-regulated in the model group relevant to the normal control group (P<0.001), and markedly up-regulated in the EA group relevant to the model group (P<0.01)．. EA of ST36 and "Ashi"-point on the contralateral limb can up-regulate the expression of myoG and fast MyHC proteins of GM in acute skeletal muscle contusion rats, which may contribute to its effect in promoting the repair of skeletal muscle injury.

Melittin – A bee venom component – Enhances muscle regeneration factors expression in a mouse model of skeletal muscle contusion

Melittin is a major peptide component of sweet bee venom that possesses anti-allergic, anti-inflammatory, anti-arthritis, anti-cancer, and neuroprotective properties. However, the therapeutic effects of melittin on muscle injury have not been elucidated. We investigated the therapeutic effects of melittin on muscle injury in a mouse model of muscle contusion. The biceps femoris muscle of the mice was injured using drop mass method, and the animals were treated with melittin (4, 20, or 100 μg/kg) for 7 days. Melittin significantly increased: locomotor activity in open field test, and treadmill running activity in a dose-dependent manner to level comparable to the positive control, diclofenac (30 mg/kg). Melittin treatment attenuated the pro-inflammatory cytokine MCP-1, TNF-α and IL-6. The expression of muscle regeneration biomarkers, including MyoD (muscle differentiation marker), myogenin, smooth muscle actin, and myosin heavy chain was markedly increased in the injured muscle tissue of melittin-treated mice, as determined by western blotting and quantitative real-time polymerase chain reaction. These results demonstrate that melittin inhibits inflammatory response and improves muscle damage by regenerating muscles in a mouse model of muscle contusion. Taken together, the results of present study suggest that melittin is a promising candidate for the muscle injury treatment.

Combination of mRNA of Repair-related Genes in Rat Skeletal Muscles for Wound Age Estimation.

Objective To investigate the estimation of early and mid-term wound age by a combination of four mRNAs, the DNA polymerase delta-interacting protein 3 （POLDIP3） mRNA, regulator of chromosome condensation 1 like （RCC1L） mRNA, proline-rich 5 （PRR5） mRNA, and ribonucleic acid export 1 （RAE1） mRNA in rats skeletal muscles. Methods The model of rat skeletal muscle contusion was established, and then contusion area muscle tissue was extracted 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 h after injury. Histomorphological changes during the repair process after rat skeletal muscle contusion were observed. The relative expressions of Poldip3, Rcc1l, Prr5 and Rae1 mRNAs were detected by reverse transcription real-time quantitative polymerase chain reaction （RT-qPCR）. Different stages of wound age were classified by using the expression patterns of four genes at various time points after injury. The accuracy of the results was verified by Fisher discriminant analysis. Results Histomorphological results showed that the repair process after skeletal muscle contusion occurred with the prolonging of time. Through combination of the expression trends of the four kinds of mRNAs, the 48 h after injury could be divided into three periods, 4-12 h, 16-28 h and 32-48 h. The Fisher discrimination method showed that the classification accuracy rates of the three periods were 83.3%, 75.0% and 73.3%, respectively. Conclusion The classification discrimination based on the relative expression of every gene has a higher accuracy, and the accuracy of wound age estimation with combination of mRNA relative expressions is higher than that with a single indicator. By combining with Fisher discrimination method, this method can be used for early and mid-term wound age estimation.

Expressions of TGF-β1 and EⅢA-FN after Rat Skeletal Muscle Contusion and Wound Age Estimation.

Objective To study the expressions of transforming growth factor-β1 （TGF-β1） and EⅢA-fibronectin （EⅢA-FN） at different time points of antemortem injury, antemortem injury postmortem expression and postmortem injury and to explore their application value in wound age estimation. Methods A model of rat skeletal muscle contusion was established. The rats were randomly divided into normal control group （n=5）, antemortem contusion group （n=40）, antemortem contusion postmortem expression group （n=110） and postmortem injury group （n=25）. The expressions of TGF-β1 and EⅢA-FN after rat skeletal muscles antemortem contusion were detected with immunohistochemical staining. Expression changes of TGF-β1 and EⅢA-FN mRNA in each group were analyzed with real-time fluorescence quantitative PCR. Results Immunohistochemical staining results showed that a large number of polymorphonuclear leukocyte, mononuclear cells and fibroblastic cells showed a strong expression of TGF-β1 in wounded zones 12 h-14 d after antemortem contusion. EⅢA-FN was mainly distributed in the extracellular matrix, 3 to 7 d post-traumatic. Real-time fluorescence quantitative PCR results showed that TGF-β1 and EⅢA-FN mRNA in antemortem injury group reached the peak at 3 and 5 d post-traumatic respectively. The expressions of TGF-β1 and EⅢA-FN mRNA in antemortem contusion postmortem expression group peaked at 6 h and 12 h postmortem. The expression of TGF-β1 and EⅢA-FN mRNA in postmortem injury group 0.5-12 h postmortem was significantly lower than those of the normal control group and the antemortem contusion group. Conclusion TGF-β1 and EⅢA-FN might become a reference index for skeletal muscle wound age estimation.

Wound Age Estimation by Neutrophil Migration Distance.

Objective To explore the application of neutrophil migration distance for wound age estimation of skeletal muscles in rats, and to provide methodological basis for follow-up study in future. Methods The skeletal muscle contusion model was established in rats, and the control group and the 2, 4, 6 h post-traumatic groups were set. The law of response of neutrophils that participated in the inflammation after injury was detected by immunohistochemical staining, and the relationship between neutrophil migration distance and injury time was detected by TissueFAXS PLUS software. Results The skeletal muscle was obviously infiltrated with neutrophils 2-6 h after injury. The positive rate of neutrophil was （28.75±0.94）% at 2 h post-traumatic, and reached the peak （45.50±3.63）% at 4 h post-traumatic, then decreased to （31.92±1.56）% at 6 h post-traumatic. The neutrophil migration distances increased with the progress of inflammation, and reached （124.80±12.32） μm, （229.03±21.45） μm and （335.04±16.75） μm at 2 h, 4 h and 6 h, respectively. Conclusion There is a relationship of neutrophil infiltrated number and migration distance and wound age within the 2-6 h after skeletal muscle injury, which could be used for the inference of skeletal muscle wound age.