Screening and characterization of ovalbumin active peptides for muscle repair: Computer simulation and in vitro activity validation.

BackgroundSkeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel.MethodsMyogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10 weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20 μL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80 mg/L) and fed to the mice (50 mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT‐qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining.ResultsDuring myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P < 0.001) and the Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.05), and conversely, knockdown of Wnt5a inhibited their expression (P < 0.001). The Wnt5a pathway mediated the opening of Ca2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P < 0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P < 0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated.ConclusionsCurcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.

Introduction: Paralytic strabismus involves a functional loss of extraocular muscles resulting from muscular or neuronal disorders. Currently, only a limited number of drugs are available for functional repair of extraocular muscles. Here, we investigated the effects of a novel drug, flavonoids sophoranone, on the differentiation of extraocular muscles as assessed in bothin vivo and in vitro models. Materials and Methods: The effect of flavonoids sophoranone on C2C12 cells was examinedin vitro as evaluated with use of apoptosis, reactive oxygen species (ROS), and cell viability assays. Then, both in vivo and in vitro effects of this drug were examined on the differentiation of C2C12 and satellite cells within extraocular muscles in rabbits. For these latter experiments, RT-PCR and Western blot assays were used to determine expression levels of markers for myogenic differentiation. Results: With use of flavonoids sophoranone concentrations ranging from 0 to 10 μM, no effects were observed upon cell apoptosis, ROS, and cell cycle in C2C12 cells. Based on MTT assay results, flavonoids sophoranone was shown to increase C2C12 cell proliferation. Moreover, flavonoids sophoranone promoted the differentiation of C2C12 and satellite cells within extraocular muscles in rabbits, which were verified as based on cell morphology and expression levels of mRNA and protein markers of myogenic differentiation. Finally, flavonoids sophoranone treatment also increased gene expressions of Myh3, Myog, and MCK. Conclusion: The capacity for flavonoids sophoranone to upgrade the differentiation of both C2C12 and satellite cells within extraocular muscles in rabbits at concentrations producing no adverse effects suggest that this drug may provide a safe and effective means to promote repair of damaged extraocular muscles.

Osteoporosis (OP) is a systemic bone metabolic disorder, which negatively affects the quality of life in the elders and postmenopausal females. Healthy volunteers and postmenopausal females with OP were enrolled in the present study. Bone densitometry (BMD) was detected by dual-energy X-ray absorptiometry (DXA). CD14+PBMCs and C2C12 cells were cultured to induce osteoclast differentiation and osteoblast differentiation, respectively. The interaction between miR‑140-3p and PTEN was predicted and verified by TargetScan 7.2 and dual luciferase reporter assay, respectively. miRNA/RNA level and protein level were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Cell proliferation and apoptosis were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining and flow cytometry, respectively. Cell differentiation of CD14+PBMCs and C2C12 cells were detected by tartrate-resistant acid phosphatase (TRAP) staining and alizarin red staining, respectively. The activity of alkaline phosphatase (ALP) was detected by ALP assay. Differences were observed in age, body mass index (BMI), and BMD between the OP group and the control group. Higher miR‑140-3p level and lower PTEN level were found in PBMCs of OP group compared to control group; there was a negative correlation between them in the serum of OP group. miR-140-3p targeted and downregulated the expression of PTEN. miR-140-3p inhibitor inhibited cell proliferation, differentiation, and promoted cell apoptosis of CD14+PBMCs; while promoted cell proliferation, differentiation and inhibited cell apoptosis of C2C12 cells, by targeting PTEN and inactivating PTEN/PI3K/AKT signaling pathway. These findings suggested a potential therapeutic role of miR-140-3p in the treatment of patients with OP.

Objective To investigate the alteration of the cytoskeleton of airway smooth muscle cells in young asthmatic rats with airway remodeling and the effect of RhoA/ROCK signal pathway. Methods Airway smooth muscle cells (ASMCs) were primary cultured and purified from Sprague-Dawley(SD) rats that were induced by ovalbumin (OVA) inhalation for 8w, then incubated by Pho kinase inhibitor Y27632. Real time quantitative polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry were used to measure the alteration of F-actin, and α-tubulin in the cytoskeleton of airway smooth muscle. Results ⑴ The asthma group showed a high average gray value of F-actin in ASMC than control groups, especially 8 weeks; and were significantly down in the group after adding Y27632(P<0.01). ⑵ The intension and intensity of fluorescence signal of α-tubulin in asthma groups in 8 weeks were higher than control group(P<0.01), and were significantly decreased in Y27632 group. ⑶ A higher expression of α-tubulin protein was shown in the asthma group in 8 weeks relative to control group(P<0.01), and was significantly down-regulated in Y27632 group(P<0.05). Conclusions Alteration of the cytoskeleton of airway smooth muscle exists in young asthmatic rats and the RhoA/ROCK signal pathway possibly plays a significant role. Key words: rho-associated kinases/AI/ME; Asthma/PP/DT; Trachea/PP/DE/ME; Myocytes, smooth muscle/PA/DE/ME

Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (-791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.

BackgroundGlucocorticoid-induced osteoporosis (GIOP) is the most common secondary osteoporosis. Patients with GIOP are susceptible to fractures and the subsequent delayed bone union or nonunion. Thus, effective drugs and targets need to be explored. In this regard, the present study aims to reveal the possible mechanism of the anti-GIOP effect of all-trans retinoic acid (ATRA).MethodsBone morphogenetic protein 9 (BMP9)-transfected mesenchymal stem cells (MSCs) were used as an in vitro osteogenic model to deduce the relationship between ATRA and dexamethasone (DEX). The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin were detected using real-time quantitative polymerase chain reaction, Western blot, and immunofluorescent staining assay. ALP activities and matrix mineralization were evaluated using ALP staining and Alizarin Red S staining assay, respectively. The novel genes associated with ATRA and DEX were detected using RNA sequencing (RNA-seq). The binding of the protein–DNA complex was validated using chromatin immunoprecipitation (ChIP) assay. Rat GIOP models were constructed using intraperitoneal injection of dexamethasone at a dose of 1 mg/kg, while ATRA intragastric administration was applied to prevent and treat GIOP. These effects were evaluated based on the serum detection of the osteogenic markers osteocalcin and tartrate-resistant acid phosphatase 5b, histological staining, and micro-computed tomography analysis.ResultsATRA enhanced BMP9-induced ALP, RUNX2 expressions, ALP activities, and matrix mineralization in mouse embryonic fibroblasts as well as C3H10T1/2 and C2C12 cells, while a high concentration of DEX attenuated these markers. When DEX was combined with ATRA, the latter reversed DEX-inhibited ALP activities and osteogenic markers. In vivo analysis showed that ATRA reversed DEX-inhibited bone volume, bone trabecular number, and thickness. During the reversal process of ATRA, the expression of retinoic acid receptor beta (RARβ) was elevated. RARβ inhibitor Le135 partly blocked the reversal effect of ATRA. Meanwhile, RNA-seq demonstrated that serine protease inhibitor, clade A, member 3N (Serpina3n) was remarkably upregulated by DEX but downregulated when combined with ATRA. Overexpression of Serpina3n attenuated ATRA-promoted osteogenic differentiation, whereas knockdown of Serpina3n blocked DEX-inhibited osteogenic differentiation. Furthermore, ChIP assay revealed that RARβ can regulate the expression of Serpina3n.ConclusionATRA can reverse DEX-inhibited osteogenic differentiation both in vitro and in vivo, which may be closely related to the downregulation of DEX-promoted Serpina3n. Hence, ATRA may be viewed as a novel therapeutic agent, and Serpina3n may act as a new target for GIOP.

BackgroundCardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy.MethodsHuman and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR.ResultsThe mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy.ConclusionsOur findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

Fibrodysplasia ossificans progressiva (FOP), a rare genetic and catastrophic disorder characterized by progressive heterotopic ossification, is caused by a point mutation, c.617G>A; p.R206H, in the activin A receptor type 1 (ACVR1) gene, one of the bone morphogenetic protein type I receptors (BMPR-Is). Although altered BMP signaling has been suggested to explain the pathogenesis, the molecular consequences of this mutation are still elusive. Here we studied the impact of ACVR1 R206H mutation on BMP signaling and its downstream signaling cascades in murine myogenic C2C12 cells and HEK 293 cells. We found that ACVR1 was the most abundant of the BMPR-Is expressed in mesenchymal cells but its contribution to osteogenic BMP signal transduction was minor. The R206H mutant caused weak activation of the BMP signaling pathway, unlike the Q207D mutant, a strong and constitutively active form. The R206H mutant showed a decreased binding affinity for FKBP1A/FKBP12, a known safeguard molecule against the leakage of transforming growth factor (TGF)-beta or BMP signaling. The decreased binding affinity of FKBP1A to the mutant R206H ACVR1 resulted in leaky activation of the BMP signal, and moreover, it decreased steady-state R206H ACVR1 protein levels. Interestingly, while WT ACVR1 and FKBP1A were broadly distributed in plasma membrane and cytoplasm without BMP-2 stimulation and then localized in plasma membrane on BMP-2 stimulation, R206H ACVR1 and FKBP1A were mainly distributed in plasma membrane regardless of BMP-2 stimulation. The impaired binding to FKBP1A and an altered subcellular distribution by R206H ACVR1 mutation may result in mild activation of osteogenic BMP-signaling in extraskeletal sites such as muscle, which eventually lead to delayed and progressive ectopic bone formation in FOP patients.

Matrix Metalloproteinase-1 Promotes Muscle Cell Migration and Differentiation

HIPK2 is an evolutionarily conserved serine/threonine kinase and is considered a co-regulator of an increasing number of transcription factors modulating a variety of cellular processes, including inflammation, proliferation and fibrosis. Skeletal muscle injuries repair is an overlapping event between inflammation and tissue repair. There are no reports about HIPK2 expression in skeletal muscles after trauma. A foundational study on distribution and time-dependent expression of HIPK2 was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR, which is expected to obtain a preliminary insight into the functions of HIPK2 during the repair of contused skeletal muscle in mice. An animal model of skeletal muscle contusion was established in 50 C57B6/L male mice. Samples were taken at 1, 3, 5, 7, 9, 14, 17, 21 and 28 days after contusion, respectively (5 mice at each posttraumatic interval). 5 mice were employed as control. No HIPK2-positive staining was detected in uninjured skeletal muscle. Intensive immunoreactivties of HIPK2 were observed in polymorphonuclear cells, round-shaped mononuclear cells, regenerated multinucleated myotubes and spindle-shaped fibroblastic cells in the contused tissue. The HIPK2-positive cells were identified as neutrophils, macrophages and myofibroblasts by double immunofluorescent procedure. HIPK2 protein and mRNA expression were remarkably up-regulated after contusion by Western blotting and qPCR analysis. The results demonstrated that the expression of HIPK2 is distributed in certain cell types and is time-dependently expressed in skeletal muscle after contusion, which suggested that HIPK2 may participate in the whole process of skeletal muscle wound healing, including inflammatory response, muscle regeneration and fibrogenesis.

Background2,6-Dimethoxy-1,4-benzoquinone (DMBQ), a natural phytochemical present in fermented wheat germ, has been reported to exert anti-cancer, anti-inflammatory, and anti-adipogenic effects. However, the effect of DMBQ on muscle hypertrophy and myoblast differentiation has not been elucidated. PurposeWe investigated the effect of DMBQ on skeletal muscle mass and muscle function and then determined the possible mechanism of DMBQ. MethodsTo examine myogenic differentiation and hypertrophy, confluent C2C12 cells were incubated in differentiation medium with or without various concentrations of DMBQ for 4 days. In animal experiments, C57BL/6 mice were fed DMBQ-containing AIN-93 diet for 7 weeks. Grip strength, treadmill, microscopic evaluation of muscle tissue, western blotting, and quantitative real-time PCR were performed. ResultsDMBQ significantly increased fusion index, myotube size, and the protein expression of myosin heavy chain (MHC). DMBQ increased the phosphorylation of protein kinase B (AKT) and p70 ribosomal protein S6 kinase (S6K), whereas the phosphorylation of these proteins was abolished by the phosphoinositide 3-kinase inhibitor LY294002 in C2C12 cells. In addition, DMBQ treatment increased peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), which programs mitochondrial biogenesis, protein levels compared with control C2C12 cells. DMBQ significantly increased maximal respiration and spare respiratory capacity in C2C12 cells. In animal experiments, DMBQ increased skeletal muscle weights and skeletal muscle fiber size compared with the control group values. In addition, the DMBQ group showed increased grip strength and running distance on an accelerating treadmill. The protein expression of total MHC, MHC1, MHC2A, and MHC2B in skeletal muscle was upregulated by DMBQ supplementation. We found that DMBQ increased the phosphorylation of AKT and mammalian target of rapamycin (mTOR), as well as downstream S6K and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in skeletal muscle. DMBQ also stimulated mRNA expression of PGC1α, accompanied by an increase in mitochondrial DNA content, oxidative phosphorylation (OXPHOS) proteins, and oxidative enzyme activity. ConclusionCollectively, DMBQ was shown to increase skeletal muscle mass and performance by regulating the AKT/mTOR signaling pathway and enhancing mitochondrial function, which might be useful for the treatment and prevention of skeletal muscle atrophy.

Bone Morphogenetic Protein/SMAD Signaling Orients Cell Fate Decision by Impairing KSRP-Dependent MicroRNA Maturation

Background and objectiveSympathetic activity plays an important role in the proliferation and differentiation of stem cells, and it changes over time, thereby exerting differential effects on various stem cell types. Aging causes sympathetic hyperactivity in aged tissues and blunts sympathetic nerves regulation, and sympathetic abnormalities play a role in aging-related diseases. Currently, the effect of sympathetic activity on skeletal muscle stem cells, namely satellite cells (SCs), is unclear. This study aimed to investigate the effects of skeletal muscle sympathetic activity on SC aging and skeletal muscle repair.Materials and methodsTo evaluate skeletal muscle and fibrotic areas, numbers of SCs and myonuclei per muscle fiber, β2-adrenoceptor (β2-ADR) expression, muscle repair, and sympathetic innervation in skeletal muscle, aged mice, young mice that underwent chemical sympathectomy (CS) were utilized. Mice with a tibialis anterior muscle injury were treated by barium chloride (BaCl2) and clenbuterol (CLB) in vivo. SCs or C2C12 cells were differentiated into myotubes and treated with or without CLB. Immunofluorescence, western blot, sirius red, and hematoxylin–eosin were used to evaluate SCs, myogenic repair and differentiation.ResultsThe number of SCs, sympathetic activity, and reparability of muscle injury in aged mice were significantly decreased, compared with those in young mice. The above characteristics of young mice that underwent CS were similar to those of aged mice. While CLB promoted the repair of muscle injury in aged and CS mice and ameliorated the reduction in the SC number and sympathetic activity, the effects of CLB on the SCs and sympathetic nerves in young mice were not significant. CLB inhibited the myogenic differentiation of C2C12 cells in vitro. We further found that NF-κB and ERK1/2 signaling pathways were activated during myogenic differentiation, and this process could be inhibited by CLB.ConclusionNormal sympathetic activity promoted the stemness of SCs to thereby maintain a steady state. It also could maintain total and self-renewing number of SCs and maintain a quiescent state, which was correlated with skeletal SCs via β2-ADR. Normal sympathetic activity was also beneficial for the myogenic repair of injured skeletal muscle.

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.