To determine the role of recombinant human ciliary neurotrophic factor (rhCNTF) in myogenesis, we observed the effects of rhCNTF (0 10 ng/ml) on myoblast differentiation of adult human in vitro. The results showed that compared with the control group, the groups of 2.5 10 ng/ml rhCNTF treatment significantly inhibited myoblast differentiation (P<0.01), and the inhibition was dose-dependent and reversible. Western blots also indicated that compared with the control group, the expression of myogenin and p21, markers of myoblast differentiation phase, was significantly reduced (P<0.01), while Myf5 and desmin, markers of myoblast proliferative phase, significantly increased (P<0.01) in the groups of 2.5 10 ng/ml rhCNTF treatment. These findings demonstrate that exogenous rhCNTF can reversibly inhibit differentiation but permits proliferation of adult human myoblasts in vitro.
Adult ; Cell Differentiation ; drug effects ; Cell Proliferation ; Cells, Cultured ; Ciliary Neurotrophic Factor ; pharmacology ; Humans ; Middle Aged ; Muscle, Skeletal ; cytology ; Myoblasts ; cytology ; Recombinant Proteins ; pharmacology ; Young Adult

OBJECTIVETo study the regulatory effect of glucagon-like peptide-1 (GLP-1) on cell proliferation of skeletal myoblast strain L6 and its possible signal mechanism.

METHODSL6 cells cultured in DMEM high glucose culture medium containing 10% FBS were divided into control group (C, without addition), GLP-1 group (G, added with 10 nmol/L GLP-1), PI3K inhibitor group (W, added with 50 nmol/L PI3K specific inhibitor wortmannin), and GLP-1 + PI3K inhibitor group (GW, added with 10 nmol/L GLP-1 and 50 nmol/L wortmannin) according to the random number table. Cell proliferation activity was detected with MTT assay at post culture hour (PCH) 24, 48, 72 (denoted as absorbance value). At PCH 24, the change in cell cycle was evaluated with flow cytometer, the expression level of proliferating cell nuclear antigen (PCNA) was determined with immunohistochemical staining, the protein levels of phosphorylated PI3K (p-PI3K) and p-Akt were determined with Western blotting. Data were processed with multi-group analysis of variance.

RESULTS(1) The cell proliferation activity at PCH 48, 72 in G group was respectively 0.660 +/- 0.120, 0.870 +/- 0.240, all significantly higher than those in C group (0.530 +/- 0.060, 0.700 +/- 0.100, with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). The cell proliferation activity in W group at each time point was lower than that in C group. The cell proliferation activity in GW group at PCH 48, 72 was respectively 0.510 +/- 0.080, 0.740 +/- 0.160, all lower than those in G group (with F value respectively 5.46, 5.90, P < 0.05 or P < 0.01). (2) The percentage of S phase cell in G group at PCH 24 [(15.7 +/- 0.4)%] was significantly higher than that in C group [(13.6 +/- 0.6)%] and GW group [(10.1 +/- 0.6)%], while that in W group [(6.8 +/- 1.2)%] was lower than that in C group (with F values all equal to 15.39, P values all below 0.01). (3) PCNA level in G group at PCH 24 [(51.24 +/- 1.18)%] was markedly higher than that in C group [(36.72 +/- 1.56)%] and GW group [(25.90 +/- 1.22)%], and while in W group [(21.70 +/- 0.09)%] was lower than that in C group (with F values equal to 783.80, P values all below 0.05). (4) The protein level of p-Akt in G group at PCH 24 was significantly higher than that in the other 3 groups, while that in W group was lower than that in C group (with F values equal to 94.43, P values all below 0.01). There was no obvious difference in protein level of p-PI3K at PCH 24 among G, GW, and C groups ( F = 20.94, P > 0.05). The protein level of p-PI3K at PCH 24 in W group was lower than that in C group (F = 20.94, P < 0.05).

CONCLUSIONSGLP-1 can promote cell proliferation of skeletal myoblast by accelerating the progression of cell cycle and increasing the synthesis of DNA, which can be attributed to PI3K/Akt signal pathway.

Androstadienes ; pharmacology ; Animals ; Cell Cycle ; Cell Line ; Cell Proliferation ; drug effects ; DNA ; biosynthesis ; Glucagon-Like Peptide 1 ; pharmacology ; Myoblasts, Skeletal ; drug effects ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Rats ; Signal Transduction ; drug effects

Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. This study investigated the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 microgram/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group. A histological examination at the postoperative 7th day revealed that the rhEGF treatment increased the number of proliferating nuclear antigen immunoreactive cells in the epidermis layer. In addition, the immunoreactive area of alpha-smooth muscle actin and the expression of prolyl 4-hydroxylase were significantly higher than those of the control group. Overall, a topical treatment of rhEGF ointment promotes wound healing by increasing the rate of epidermal proliferation and accelerating the level of wound contraction related to myofibroblast proliferation and collagen deposition.
Actins/genetics/metabolism ; Administration, Topical ; Animals ; Cell Proliferation/drug effects ; Collagen/*biosynthesis ; Epidermal Growth Factor/*administration&dosage/*pharmacology ; Gene Expression Regulation ; Male ; Myoblasts, Skeletal/*drug effects ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Wound Healing/*drug effects

The aim of this study was to investigate the mechanism of deposition of extracellular matrix induced by TGF-β1 in skeletal muscle-derived stem cells (MDSCs). Rat skeletal MDSCs were obtained by using preplate technique, and divided into four groups: group A (control group), group B (treated with TGF-β1, 10 ng/mL), group C (treated with TGF-β1 and anti-connective tissue growth factor (CTGF), both in 10 ng/mL), and group D (treated with anti-CTGF, 10 ng/mL). The expression of CTGF, collagen type-I (COL-I) and collagen type-III (COL-III) in MDSCs was examined by using RT-PCR, Western blot and immunofluorescent stain. It was found that one day after TGF-β1 treatment, the expression of CTGF, COL-I and COL-III was increased dramatically. CTGF expression reached the peak on the day 2, and then decreased rapidly to a level of control group on the day 5. COL-I and COL-III mRNA levels were overexpresed on the day 2 and 3 respectively, while their protein expression levels were up-regulated on the day 2 and reached the peak on the day 7. In group C, anti-CTGF could partly suppress the overexpression of COL-I and COL-II induced by TGF-β1 one day after adding CTGF antibody. It was concluded that TGF-β1 could induce MDSCs to express CTGF, and promote the production of COL-I and COL-III. In contrast, CTGF antibody could partially inhibit the effect of TGF-β1 on the MDSCs by reducing the expression of COL-I and COL-III. Taken together, we demonstrated that TGF-β1-CTGF signaling played a crucial role in MDSCs synthesizing collagen proteins in vitro, which provided theoretical basis for exploring the methods postponing skeletal muscle fibrosis after nerve injury.
Animals ; Cell Differentiation ; drug effects ; physiology ; Cells, Cultured ; Fibrillar Collagens ; biosynthesis ; Male ; Myoblasts, Skeletal ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; drug effects ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo study the modulatory effect of insulin on apoptosis of skeletal myoblast (L6 cells) by serum of scalded rat and its mechanism.

METHODSL6 cells cultured with DMEM medium containing 10% FBS were divided into control (C, added with 20% normal rat serum), serum from rat with scald injury (S, added with 20% serum from scalded rat), insulin (I, added with 20% normal rat serum and 100 nmol/L insulin), and serum of scalded rat + insulin (SI, added with 20% serum of scalded rat + 100 nmol/L insulin) groups according to the random number table. After being cultured for 48 hours, apoptosis was observed with Hoechst 33258 staining and its number counted, annexin V -FITC/PI double-labeling method was used to assess apoptosis rate, the protein levels of phosphorylated (p-) Akt, p-PI3K, Bax, Bcl-2, and active caspase-3 were determined by Western blotting. Data were processed with grouped or paired t test.

RESULTS(1) The amount of apoptosis with typical morphological change in S group [(59.6 +/- 3.9) per visual field] was more than that in C, I, and SI groups [(4.9 +/- 2.6), (5.5 +/- 2.1), (19.7 +/- 2.3) per visual field, with t value respectively 28.53, 29.86, 21.53, P values all below 0.01]. (2) Apoptotic rate in S group was (18.5 +/- 1.8)%, which was markedly higher than that in C, I, and SI groups [(1.1 +/- 0.6)%, (1.5 +/- 0.3)%, (7.8 +/- 0.6)%, with t value respectively 22.41, 22.83, 13.92, P values all below 0.01]. (3) Compared with those in C group, the protein levels of Bax and active caspase-3 in S group were up-regulated (1.12 +/- 0.63 vs. 0.16 +/- 0.03, 2.15 +/- 0.51 vs. 0.21 +/- 0.03, with t value respectively 3.80, 10.69, P values all below 0.01), the protein level of p-Akt was lowered (0.20 +/- 0.03 vs. 0.42 +/- 0.07, t = -8.46, P < 0.01), and the protein levels of p-PI3K and Bcl-2 showed no statistical difference (0.19 +/- 0.03 vs. 0.26 +/- 0.09, 0.17 +/- 0.03 vs. 0.28 +/- 0.07, with t value respectively -2.73, - 1.14, P values all above 0.05). The protein levels of Bax (0.40 +/- 0.14) and active caspase-3 (0.83 +/- 0.18) in SI group were lowered (t = -3.23, P < 0.05; t = 6.66, P < 0.01) and the protein levels of p-Akt, Bcl-2, and p-PI3K in SI group were elevated (0.39 +/- 0.10, 0.78 +/- 0.03, 0.47 +/- 0.12, with t value respectively 4.07, 18.71, 5.05, P < 0.05 or P < 0.01) as compared with those in S group.

CONCLUSIONSSerum from scalded rat can induce apoptosis in skeletal myoblast, and the effect can be inhibited by insulin through PI3K/Akt signal pathway.

Animals ; Apoptosis ; drug effects ; Burns ; blood ; metabolism ; pathology ; Caspase 3 ; metabolism ; Cell Line ; Insulin ; pharmacology ; Male ; Myoblasts, Skeletal ; cytology ; drug effects ; pathology ; Rats ; Rats, Wistar ; Serum ; immunology ; Signal Transduction ; bcl-2-Associated X Protein ; metabolism

The present study was to explore the effects of insulin on proliferation of skeletal myoblast cells in rats. Separated and cultured primary skeletal myoblast cells from rats were treated by insulin. By means of the incorporation of (3)H-TdR, BrdU assay and MTT assay, the proliferation of skeletal myoblast cells was detected. Western blot was used to check the phosphorylation of Akt and ERK of myoblast cells. The results showed that insulin significantly promoted the incorporation of (3)H-TdR into cultured skeletal myoblast cells in a dose-dependent manner. MTT assay and BrdU assay also showed insulin promoted the proliferation of skeletal myoblast cells. The promotion of skeletal myoblast cells proliferation by insulin was inhibited by PI3K inhibitor wortmannin or MEK inhibitor U0126, and the same phenomenon was shown in L6 and C2C12 cells. Also, insulin increased the phosphorylation of Akt and ERK in myoblast cells. These results suggest that insulin may promote proliferation of skeletal myoblast cells through PI3K/Akt and MEK/ERK pathways.
Androstadienes ; pharmacology ; Animals ; Butadienes ; pharmacology ; Cell Proliferation ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Insulin ; pharmacology ; MAP Kinase Signaling System ; Myoblasts, Skeletal ; cytology ; drug effects ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats

OBJECTIVESTo evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.

METHODSThe expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.

RESULTSThe expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.

CONCLUSIONMyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Gene Expression Regulation ; drug effects ; physiology ; Glucose ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Insulin ; pharmacology ; Mice ; MicroRNAs ; biosynthesis ; genetics ; Multigene Family ; physiology ; Muscle Fibers, Skeletal ; cytology ; metabolism ; MyoD Protein ; genetics ; metabolism ; Myoblasts ; cytology ; metabolism ; Sweetening Agents ; pharmacology