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

Pathological cardiac hypertrophy induced by angiotensin II (AngII) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngII-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosinone (25, 50, 100, and 200 μmol/L) or AngII (1 μmol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngII. The mRNA expression of ANP, BNP and β-MHC was obviously elevated in AngII-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang II-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.
Angiotensin II ; physiology ; Animals ; Cardiotonic Agents ; pharmacology ; Cell Line ; Cell Size ; drug effects ; Hypertrophy ; metabolism ; pathology ; MAP Kinase Signaling System ; Myoblasts, Cardiac ; cytology ; drug effects ; metabolism ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Sesquiterpenes ; pharmacology ; TOR Serine-Threonine Kinases ; genetics ; metabolism

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

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

OBJECTIVETo investigate whether Astragaloside IV(AST) protects H9c2 cells against H2O2-induced oxidative injury partly through ERK1/2 signaling pathway.

METHODSH9c2 cells oxidative injury was induced by 200 tmol/L H2O2 for 6 hours to establish the H2O2-induced injury model of H9c2 cells. The viability of H9c2 cells was detected using MTf method. Activity of lactate dehydrogenase(LDH), total-superoxide dismutase (T-SOD), manganese-superoxide dismutase (Mn-SOD) and content of MDA (malondialdehyde) in the culture medium were detected using colorimetric method. Western blot was performed to exam expression of p-ERK1/2 and ERK1/2 in H9c2 cells respectively.

RESULTSUnder 200 micromol/L H2O2 treatment for 6 hours, the vaibility of H9c2 cells was suitable for the following study. Compared with H2O2 group, the cell viability was increased significantly in AST10 + H2O2 and AST2O + H2O2 groups (P < 0.01). The activity of LDH in the culture medium was decreased significantly (P < 0.01). The activity of T-SOD and Mn-SOD was increased significantly (P < 0.01), the content of MDA was decreased significantly (P < 0.01). Treated with 10 mg/L or 20 mg/L of AST, expression of p-ERK1/2 in H9c2 cells injured from H2O2 was increased significantly (P < 0.01), when PD98059 (inhibitor of ERK1/2) was added, the effects of AST were cancelled.

CONCLUSIONAST protects H9c2 cells against H2O2-induced oxidative injury partly through ERK1/2 signaling pathway.

Animals ; Antioxidants ; pharmacology ; Cell Line ; Hydrogen Peroxide ; toxicity ; MAP Kinase Signaling System ; physiology ; Myoblasts, Cardiac ; drug effects ; metabolism ; pathology ; Oxidative Stress ; drug effects ; Protective Agents ; pharmacology ; Rats ; Saponins ; pharmacology ; Triterpenes ; pharmacology

Phospholipase D (PLD) hydrolyzes phosphocholine into choline and phosphatide acid, and these metabolites play an important role in regulating cell physiology and biochemistry. To study the biological function of phospholipase D3 (PLD3) during the insulin stimulation in C2C12 myoblasts, we constructed PLD3 over-expressed cell lines (C2C12/pPLD3) and investigated the phosphorylation of Akt. The results showed that the level of phosphorylated Akt (P-Akt) was significantly increased in control C2C12 cells when insulin concentration was elevated during cell treatment, whereas the level of P-Akt in C2C12/pPLD3 cells was not changed. When extending the time of insulin treatment, P-Akt level in C2C12/pPLD3 cells was increased around 2 folds, but the total level of P-Akt in C2C12/pPLD3 was still lower than that in control group. 1-Butanol, a PLD inhibitor, could completely block Akt phosphorylation in C2C12 cells that even stimulated by insulin. However, 1-Butanol did not inhibit the Akt phosphorylation in C2C12/pPLD3 cells, but increased the phosphorylation up to 6 folds higher than control cells. The level of Akt phosphorylation in control C2C12 cells was increased significantly when stimulated by phosphatidic acid (PA), while there was no change in C2C12/pPLD3 cells with the similar treatment. When cells simulated by both PA and insulin, P-Akt level in both C2C12/pPLD3 cells and C2C12 cells were down regulated. Our observations indicated that PLD3 over expression may inhibit Akt phosphorylation and further block the transduction of insulin signaling in C2C12 cells.
Cell Line ; Humans ; Insulin ; pharmacology ; Myoblasts ; cytology ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phospholipase D ; biosynthesis ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; chemistry ; drug effects ; Signal Transduction

OBJECTIVEThe present study is to observe in vitro the proliferation ability of the muscle cells from permanent myopathy (PM) patients of nomokalaemic periodic paralysis (normKPP), which is caused by mutations of Met1592Val in the skeletal muscle voltage gated sodium channel (SCN4A) gene on chromosome 17q23.1. We also evaluate the possible effect of the foreign basic fibroblast growth factor (bFGF) in preventing and curing PM.

METHODSThe gastrocnemius muscle cells were taken from two male patients with PM of the same Chinese family with Met1592Val mutation of SCN4A, determined by gene screening. Four male patients suffering from the skeletal injury without PM were taken as control. All preparations were protogenerationally cultured in vitro. Proliferation of the cultured preparations was measured by MTT. Activities of the lactic dehydrogenase (LDH), creatine kinase (CK), and protein content in these cells were also detected. The effects of bFGF with different doses (10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 120 ng/mL and 160 ng/mL) on the above mentioned parameters were also evaluated.

RESULTSCells from both PM and control subjects were successfully cultured in vitro. The cultivation of the muscle cells from PM patients in vitro was not yet seen. Results indicated the obvious stimulation of bFGF on cell proliferation, activities of LDH and CK, protein synthesis, in a dose dependent manner. The optimal dose of bFGF was 120 ng/mL (P<0.05), beyond which greater dose caused a less effect. The effect of bFGF on 160 ng /mL was stronger than that on 80 ng/mL, but there was no significant difference (P>0.05).

CONCLUSIONMyoblastic cells from patients with PM had a weaker ability of developing into the myotubules, thus they were unable to perform effective regeneration, which resulted in a progressive necrosis. The exogenous bFGF could promote the division and proliferation of the muscle cells in vitro. These results shield a light on bFGFos potential role in preventing and treating PM.

Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Creatine Kinase ; metabolism ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Male ; Methionine ; genetics ; Middle Aged ; Muscle Development ; genetics ; physiology ; Muscular Diseases ; genetics ; pathology ; Mutation ; genetics ; Myoblasts ; drug effects ; NAV1.4 Voltage-Gated Sodium Channel ; Sodium Channels ; genetics ; Valine ; genetics

OBJECTIVETo investigate the relation between transdifferentiation of the airway myofibroblasts and the expression level of (trypsinogen16, TG16) in vitro and explore the mechanism of airway basement membrane thickening.

METHODSThe total lung proteins were extracted from normal and OVA-induced asthmatic mice and the protein expression profiles were analyzed with SDS-PAGE. The differentially expressed proteins were isolated for analysis with liquid chromatography-mass spectrometry. TG16 was cloned from mouse lung tissue and subcloned into the expression vector pcDNA3.0 to generate a pcDNA3-TG16 plasmid. The vectors were transfected into mouse embryonic fibroblast 3T3 cells and cultured in MEM in the presence of transforming growth factor-beta1 (TGF-beta1). The mRNA levels of alpha-actin and the housekeeping GAPDH gene were analyzed with RT-PCR. Using RNA interference, TG16 expression was suppressed and the resultant alpha-actin or GAPDH protein levels were analyzed using Western blotting.

RESULTSIn the total lung proteins from OVA-induced mice, a 25 000 Da protein was significantly enhanced in comparison with the protein profiles of normal mice. The protein band was identified to represent the protein of TG16. With TGF-beta1 stimulation, transfection with the plasmid pcDNA3-TG16 significantly suppressed the mRNA expression of alpha-actin (alpha-actin/GAPDH=1.78-/+0.50) in 3T3 cells as compared with the expression in cells transfected with pcDNA3.0 (3.20-/+1.36); transfection of the cells with TG16 stealth RNAi oligonucleotide to decrease TG16 mRNA level upregulated the protein level of alpha-actin (3.60-/+0.44) as compared with the alpha-actin protein level in 3T3 cells transfected with control oligonucleotide (2.78-/+0.50).

CONCLUSIONTG16 can inhibit the expression of alpha-actin in fibroblasts, which might be a protective mechanism in the progression of airway remodeling in asthma.

3T3 Cells ; Actins ; biosynthesis ; genetics ; Animals ; Cell Transdifferentiation ; drug effects ; genetics ; physiology ; Chromatography, Liquid ; Fibroblasts ; cytology ; drug effects ; metabolism ; Lung ; cytology ; metabolism ; Male ; Mass Spectrometry ; Mice ; Mice, Inbred BALB C ; Myoblasts ; cytology ; drug effects ; metabolism ; Proteomics ; methods ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Transforming Growth Factor beta1 ; pharmacology ; Trypsinogen ; genetics ; metabolism