Effects of nitric oxide on C2C12 cell inflammatory responses and notexin-induced muscle injury / Efectos del óxido nítrico en las respuestas inflamatorias de las células C2C12 y la lesión muscular inducida por notexina

SUMMARY: Skeletal muscle injury is an acute inflammatory condition caused by an inflammatory response. To reduce inflammatory cell infiltration and relieve skeletal muscle injury, efficient treatment is urgently needed. Nitric oxide is a free radical molecule reported to have anti-inflammatory effects. In this study, we showed that NO could inhibit the inflammatory response of C2C12 cells in vitro and protect rat skeletal muscle injury from notexin in vivo. NO synthase inhibitor (L-NG-Nitroarginine Methyl Este?L-NAME) and NO donor (sodium nitroprusside dehydrate ?SNP) were used to explore the vital role of lipopolysaccharides (LPSs) in LPS-stimulated C2C12 myoblasts.The expression of IL-18 and IL-1b was upregulated by L-NAME and downregulated by SNP, as indicated by the ELISA results. NO can reduce ASC, Caspase-1, and NLRP3 mRNA and protein levels. Furthermore, NO was detected in the rat model. The results of immunohistochemical staining showed that the production of DMD decreased. We conducted qRT-PCR and western blotting to detect the expression of Jo-1, Mi-2, TLR2, and TLR4 on day 6 post injury following treatment with L-NAME and SNP. The expression of Jo-1, Mi-2, TLR2, and TLR4 was upregulated by L-NAME and significantly reversed by SNP. NO can alleviate C2C12 cell inflammatory responses and protect rat skeletal muscle injury from notexin.

RESUMEN: La lesión del músculo esquelético es una afección inflamatoria aguda causada por una respuesta inflamatoria. Para reducir la infiltración de células inflamatorias y aliviar la lesión del músculo esquelético es necesario un tratamiento eficaz. El óxido nítrico es una molécula de radicales libres que tiene efectos antiinflamatorios. En este estudio, demostramos que el ON podría inhibir la respuesta inflamatoria de las células C2C12 in vitro y proteger la lesión del músculo esquelético de rata de la notexina in vivo. El inhibidor de ON sintasa (L-NG-nitroarginina metil este, L-NAME) y el donante de ON (nitroprusiato de sodio deshidratado, SNP) se utilizaron para explorar el papel vital de los lipopolisacáridos (LPS) en los mioblastos C2C12 estimulados por LPS. La expresión de IL- 18 e IL-1b fue regulada positivamente por L-NAME y regulada negativamente por SNP, como indican los resultados de ELISA. El ON puede reducir los niveles de proteína y ARNm de ASC, Caspasa-1 y NLRP3. Además, se detectó ON en el modelo de rata. Los resultados de la tinción inmunohistoquímica mostraron que disminuyó la producción de DMD. Realizamos qRT-PCR y transferencia Western para detectar la expresión de Jo-1, Mi-2, TLR2 y TLR4 el día 6 después de la lesión después del tratamiento con L-NAME y SNP. La expresión de Jo-1, Mi-2, TLR2 y TLR4 fue regulada positivamente por L- NAME y significativamente revertida por SNP. El ON puede aliviar las respuestas inflamatorias de las células C2C12 en ratas, y proteger la lesión del músculo esquelético de la notexina.

Silencing of Myh3 Gene by siRNA Inhibits Glycolysis in C2C12 Cells / 中国生物化学与分子生物学报

The Myh3 (myosin heavy chain 3) gene is a marker gene of muscle cell differentiation and regulates the utilization of energy in muscle cells, but whether it affects the glycolysis process of muscle cells in different states is rarely reported. In this paper, the expression patterns of Myh3 and glycolysis-related genes Pkm (M-type pyruvate kinse), Prkag3 (protein kinase adenosine monophosphate-activated γ3-subunit), and Gsk3β (glycogen synthase kinase-3β) were studied by the qRT-PCR (quantitative-Real-Time-PCR) method using C2C12 cells at different stages of myoblast and adipogenic differentiation as models. It was found that in the process of myoblast differentiation of C2C12 cells, the relative expression trends of Myh3 and glycolysis genes Prkag3 and Pkm were basically the same, and the relative expression levels first increased, reached the peak on the second day of differentiation, and then decreased; glycogen synthase the expression trend of the inhibitory gene Gsk3β was relatively stable. In the process of adipogenic differentiation of C2C12 cells, the relative expression trend of Myh3 and glycolysis genes Prkag3 and Pkm remained basically the same, and the relative expression gradually increased, reaching the highest value on the 8th day of differentiation; glycogen synthase inhibitory gene Gsk3β expression remained stable. In the myogenic differentiation state of C2C12 cells, qRT-PCR and Western blotting were used to detect the effects of interfering Myh3 on the mRNA and protein expressions of glycolysis-related genes Pkm, Prkag3, and Gsk3β. The results showed that after interfering with Myh3, the mRNA expressions of glycolysis genes Pkm and Prkag3 were significantly decreased (P 0.05). The protein levels of Myh3 and Pkm were significantly lower than those in the blank group and NC group. Under the adipogenic differentiation state of C2C12 cells, after interfering with Myh3, the mRNA levels of glycogen synthase inhibitor Gsk3β and glycolysis gene Prkag3 were significantly increased (P<0.01), and the mRNA level of glycolysis gene Pkm was decreased; the protein levels of Myh3 and Pkm in the Myh3 interference group were also lower than those in the blank group and NC group. Based on the above studies, there are significant differences in the levels of glycolysis in C2C12 cells in the myogenic and adipogenic states, and the expression patterns of Myh3 and glycolysis genes are similar. Further results showed that Myh3 suppression could inhibit the glycolysis of C2C12 cells in the myogenic state without affecting the glycogen synthesis. Unlike in the myogenic state, interfering expression of Myh3 in the adipogenic state of C2C12 cells inhibited both glycogen synthesis and glycolysis.

Efecto de extractos de frutas y hortalizas en la incorporación de glucosa en células musculares in vitro / Effect of fruit and vegetable extracts on the incorporation of glucose in muscle cells "in vitro"

Skeletal muscle appears to play a central role in the development of insulin resistance (IR) and consequently the metabolic syndrome due to high-fat diets, obesity, and aging. Recent evidence suggests that some bioactive compounds present in natural products can affect blood glucose, possibly due to interactions between the compounds and glucose transporters. As an objective, to evaluate the effect of the extract of the green bean (PV, Phaseolus vulgaris) and apple of small fruit of thinning (Malus domestica, MAF and MIT extracts) on the incorporation of glucose in C2C12 muscle cells. For this, the cytotoxic effect of the extracts on the cells was determined by detecting formazan. Subsequently, glucose incorporation was determined using a fluorescent glucose analog in cells treated with the extracts. Finally, the effect of the extracts on IL-6 and TNFα production was evaluated by ELISA. Results: PV and MAF decreased 50% of viability at 1000µg / mL while MIT only decreased 10% at that concentration. PV had no significant effect on glucose incorporation and the MAF and MIT extract extracts significantly increased glucose incorporation at 100 µg / mL (13500 and 18000 URF respectively). PV increases the secretion of IL-6 and TNF-α, MAF and MIT only increase the expression of IL-6. Conclusion: These results make it possible to establish natural extracts derived from thinning small fruit apple can be used as a possible treatment for pathologies with high blood glucose levels.

Effects of N-Oleoylglycine and Oleate on Mitochondrial UCP1-independent Thermogenesis / 中国生物化学与分子生物学报

Besides UCP1-dependent thermogenesis pathways, UCP1-independent thermogenesis pathways also could increase heat production in adipose tissue to combat obesity. N-Acyl amino acids (NAAs) have been suggested as novel endogenous uncouplers to induce mitochondria UCP1-independent thermogenesis in adipose tissue. Here, we use mouse skeletal muscle C2C12 cells which lack of UCP1 as UCP1 negative cell models. Comparing with its corresponding common fatty acid—oleate, one of the NAAs—N-Oleoylglycine (NOGly), which is highly expressed in the plasma of HFD mice, is selected to study their effects and mechanisms on mitochondrial thermogenesis. We found that 60 μmol / L oleate could induce mitochondrial oxidative phosphorylation protein levels, as well as increase mitochondria thermogenesis-related genes (COX8b, DIO2, UCP3) expression (P < 0. 05) . However, 60 μmol / L NOGly damaged the production and oxidative phosphorylation of mitochondria, significantly down-regulated expression of thermogenic genes (PGC1a, COX8b, COX2, DIO2, UQCRFS1and UCP3) (P< 0. 01), induced the production of reactive oxygen species (ROS) in the mitochondria, and enhanced the oxidative stress in cells. Our study found that oleate can induce UCP1-independent thermogenesis under 60 μmol / L addition dose, whereas NOGly does not due to the induction of oxidative stress in cells.

Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes

Objective: To investigate the in vivo and in vitro antidiabetic potential of Chrysophyllum albidum. Methods: The effects of oral treatment with hydro-ethanolic extract (125, 250 and 500 mg/kg) of the stem bark of Chrysophyllum albidum and glibenclamide for 21 d on glucose level, serum enzyme markers for liver function, lipid profile, total protein, serum urea, serum creatinine, and body weight were evaluated in experimental diabetic rats administered with 45 mg/kg of streptozotocin. In vitro assays including glucose uptake in C2C12 cells and 3T3-L1 adipose tissues, α-glucosidase and α-amylase inhibition were employed to evaluate the possible mechanism of hypoglycemic action of the extract. DPPH and nitric oxide radical antioxidant activity of the extract was also measured. Results: The increased levels of blood glucose, triglycerides, low-density lipoprotein, total cholesterol, serum aspartate, and alanine transaminases, creatinine, and urea in the diabetic animals were reduced significantly (P<0.01) after treatment with Chrysophyllum albidum extract. The decreased total protein and high-density lipoprotein concentrations were normalized after treatment. In addition, the extract significantly (P<0.01) increased the transport of glucose in 3T3-L1 cells and C2C12 myotubes and exhibited considerable potential to inhibit α-amylase and α-glucosidase. It also demonstrated potent antioxidant action by scavenging considerably DPPH and nitric oxide radicals. Conclusions: Chrysophyllum albidum stem bark extract exhibits considerable antidiabetic effect by stimulating glucose uptake and utilization in C2C12 myotubes and 3T3-L1 adipocytes as well as inhibiting the activities of α-amylase and α-glucosidase.

Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes

Objective: To investigate the in vivo and in vitro antidiabetic potential of Chrysophyllum albidum. Methods: The effects of oral treatment with hydro-ethanolic extract (125, 250 and 500 mg/kg) of the stem bark of Chrysophyllum albidum and glibenclamide for 21 d on glucose level, serum enzyme markers for liver function, lipid profile, total protein, serum urea, serum creatinine, and body weight were evaluated in experimental diabetic rats administered with 45 mg/kg of streptozotocin. In vitro assays including glucose uptake in C2C12 cells and 3T3-L1 adipose tissues, α-glucosidase and α-amylase inhibition were employed to evaluate the possible mechanism of hypoglycemic action of the extract. DPPH and nitric oxide radical antioxidant activity of the extract was also measured. Results: The increased levels of blood glucose, triglycerides, low-density lipoprotein, total cholesterol, serum aspartate, and alanine transaminases, creatinine, and urea in the diabetic animals were reduced significantly (P<0.01) after treatment with Chrysophyllum albidum extract. The decreased total protein and high-density lipoprotein concentrations were normalized after treatment. In addition, the extract significantly (P<0.01) increased the transport of glucose in 3T3-L1 cells and C2C12 myotubes and exhibited considerable potential to inhibit α-amylase and α-glucosidase. It also demonstrated potent antioxidant action by scavenging considerably DPPH and nitric oxide radicals. Conclusions: Chrysophyllum albidum stem bark extract exhibits considerable antidiabetic effect by stimulating glucose uptake and utilization in C2C12 myotubes and 3T3-L1 adipocytes as well as inhibiting the activities of α-amylase and α-glucosidase.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca²⁺ signaling of differentiated C2C12 myotubes

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca²⁺]i transient and reduced sarcoplasmic reticulum (SR) Ca²⁺ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca²⁺-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca²⁺ signaling by downregulating the expression of DHPR and SERCA proteins.

The Mechanisms of Cobaltous Chloride-Induced Hypoxia on Muscle Atrophy / 中国运动医学杂志

Objective To study the regulating mechanisms of cobaltous chloride (CoCl2)induced hypoxia on muscle atrophy.Methods The mouse skeletal C2C12 myotube line was used as the cell model and divided into a control group,a CoCl2 group,a 3-Methyladenine(3MA)group and a CoCl2+3MA group.The control group was not given any treatment,the CoCl2 group was treated with 200 μM CoCl2 to induce hypoxia,and the 3MA group or CoCl2+3MA group was treated with 5 mM 3MA in the absence or presence of 200 μM CoCl2.The Giemsa stain was conducted to observe the morphology of myotubes.The multifunctional microplate reader was utilized to quantify the reactive oxygen species (ROS)expression.The autophagosome was observed by using the transmission electron microscopy.The mRNA and protein expression of hypoxia-inducible factor-1α(HIF-1α),Bcl2/adenovirus E1B19kDa interacting protein 3(BNIP3),microtubule associated protein1 light chain 3(LC3)were detected using the quantitative real time polymerase chain reaction (QRT-PCR)and Western blotting (WB).Moreover,3-Methyladenine(3MA)was used to suppress autophagy and the expression of muscle atrophy F-box(MAF-bx)was detected.Results More spindly ring-shaped myotubes were formed in the control group,while CoCl2 induced myotubes atrophy and rupture.The expression of ROS increased significantly in the CoCl2 group compared with the control group(t=-4.965,P=0.008).Transmission electron microscopy results showed autophagosome formation induced by CoCl2,and significant improvement in the HIF-1α,BNIP3 and LC3 in the CoCl2 group compared with the control group(P＜0.05).The protein expression of MAFbx decreased when co-cultured with CoCl2 and 3MA(F=18.246,P=0.001).Conclusions The skeletal muscle atrophy caused by CoCl2 induced hypoxia may be associated with autophagy promoted by the HIF-1α/BNIP3 signal pathway,and inhibition of hypoxia induced autophagy can partly reduce the atrophy in skeletal C2C12 myotubes.

Effects of laser stimulation with different power on oxygen consumption rate of CC myoblasts in mice / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of 650 nm laser irradiation on cell oxygen consumption rate in CC myoblasts following different doses.@*METHODS@#CC cells were irradiated with 650 nm laser(λ=650 nm, p=5 mW) with energy densities of 0, 0.4, and 0.8 J/cm. Cell oxidative function was measured by oxygen consumption rate kit. Protein expression of myogenic determination factor (MyoD), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), mammalian target of rapamycin (mTOR) and its phosphorylation were detected by Western blot.@*RESULTS@#Compared to the control group, the expression levels of MyoD, PGC-1α protein were increased and cell oxygen consumption rate was promoted in the low dose group(<0.05). MyoD and PGC-1α protein expressions were also increased(<0.05), the ratio of mTOR and its phosphorylationwere decreased significantly in the high dose group(<0.05).@*CONCLUSIONS@#650 nm laser irradiation that dose is 0.4 J/cm enhances cell oxidative function, it related to that proper dose laser irradiation promoted the expression of PGC-1a protein.

Syringaresinol-4---d-glucoside alters lipid and glucose metabolism in HepG2 cells and C2C12 myotubes

Syringaresinol-4---d-glucoside (SSG), a furofuran-type lignan, was found to modulate lipid and glucose metabolism through an activity screen of lipid accumulation and glucose consumption, and was therefore considered as a promising candidate for the prevention and treatment of metabolic disorder, especially in lipid and glucose metabolic homeostasis. In this study, the effects of SSG on lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes were further investigated. Treatment with SSG significantly inhibited lipid accumulation by oil red O staining and reduced the intracellular contents of total lipid, cholesterol and triglyceride in HepG2 cells. No effect was observed on cell viability in the MTT assay at concentrations of 0.1-10 μmol/L. SSG also increased glucose consumption by HepG2 cells and glucose uptake by C2C12 myotubes. Furthermore, real-time quantitative PCR revealed that the beneficial effects were associated with the down-regulation of sterol regulatory element-binding proteins-1c, -2 (), fatty acid synthase (), acetyl CoA carboxylase () and hydroxyl methylglutaryl CoA reductase (), and up-regulation of peroxisome proliferator-activated receptors alpha and gamma (and). SSG also significantly elevated transcription activity oftested by luciferase assay. These results suggest that SSG is an effective regulator of lipogenesis and glucose consumption and might be a candidate for further research in the prevention and treatment of lipid and glucose metabolic diseases.

Liraglutide increases FNDC5 expression in C2C12 myotubes via activa-tion of CAMKK2/AMPK signaling pathways / 中国病理生理杂志

AIM:To investigate the effect of liraglutide ( LG) on the expression of fibronectin type Ⅲdomain-containing protein 5 (FNDC5) in the C2C12 myotubes.METHODS:The C2C12 mouse myoblast cell line was induced to differentiation.Differentiated cells were stimulated with gradient concentrations (1 ~1000 nmol/L) of LG for different time (0 ~24 h).The effects of LG on the expression of FNDC5 and the activation of adenosine 5'-monophosphate ( AMP)-activated protein kinase ( AMPK) signaling pathway were determined .After pretreated with glucagon-like peptide-1 ( GLP-1 ) receptor antagonist exendin 9-39 , the inhibitor of Ca 2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2), STO609, or the inhibitor of AMPK, Compound C, the LG-induced FNDC5 expression in C2C12 myotubes was examined.The expression of FNDC5 and the activation of AMPK were determined by Western blot .RESULTS: In C2C12 myotubes, LG promoted the expression of FNDC5 in a dose-and time-dependent manner .LG also activated AMPK signaling pathway .These effects of LG were partly abolished by exendin 9-39 , STO609 and Compound C .CONCLUSION:LG promotes the expression of FNDC5 via GLP-1 receptor in the C2C12 myotubes possibly through activation of the CAMKK2/AMPK signaling pathways .

Effects of the Electrical Stimulation on the Free Radicals and Nrf2/ARE Signaling Pathway in C2C12 Cells of Skeletal Muscles / 中国运动医学杂志

Objective To discuss the effect of the electric stimulation on the free radicals and Nrf2 signaling pathway in skeletal muscle myoblasts(C2C12) of mice.Methods After 6 days of differentiation,skeletal muscle myoblasts(C2C12) cells of mice were randomly divided into a control group and an electrical stimulation group.The control group was not stimulated,while the electrical group was stimulated 30,45,60,75,90,120 and 150 minutes respectively with the electrical parameter as 20 ms,45 V and 5 Hz.The reactive oxygen species(ROS),glutathione peroxidase(GSH-Px) and nuclear factor E2 related factor 2(Nrf2) protein expression in C2C12 muscle tubes were determined.Results Compared with the control group,the amount of ROS produced in the C2C12 muscle tube increased significantly in the stimulation group(P＜0.05),except those stimulated for 30 and 150 minutes,with greatest increase for those stimulated for 60 and 120 minutes(P＜0.01).Compared with the control group,the activity of GSH-Px and the expression of Nrf2 nuclear protein increased significantly in all the stimula tion group(P＜0.05),except those stimulated for 30 minutes and with the greatest increase for those stimulated for 75 and 150 minutes(P＜0.01).Conclusion The electrical stimulation of 45 V,20 ms and 5 Hz can cause oxidative stress in the C2C12 muscle tube.Based on the variation of ROS production with the time of electric stimulation,it is found that only when ROS increases to a certain amount,can it activate the Nrf2 signaling system and related genes to make the GSH-Px play an antioxidant role.However,with the increase of stimulation time,the anti-oxidative ability decreases slightly,and then it continues to maintain its high level.

Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition

The present study investigated the effect of silibinin, the principal potential anti-inflammatory flavonoid contained in silymarin, a mixture of flavonolignans extracted from Silybum marianum seeds, on palmitate-induced insulin resistance in C2C12 myotubes and its potential molecular mechanisms. Silibinin prevented the decrease of insulin-stimulated 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) uptake and the downregulation of glutamate transporter type 4 (GLUT4) translocation in C2C12 myotubes induced by palmitate. Meanwhile, silibinin suppressed the palmitate-induced decrease of insulin-stimulated Akt Ser473 phosphorylation, which was reversed by wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K). We also found that palmitate downregulated insulin-stimulated Tyr632 phosphorylation of insulin receptor substrate 1 (IRS-1) and up-regulated IRS-1 Ser307 phosphorylation. These effects were rebalanced by silibinin. Considering several serine/threonine kinases reported to phosphorylate IRS-1 at Ser307, treatment with silibinin downregulated the phosphorylation of both c-Jun N-terminal kinase (JNK) and nuclear factor-κB kinase β (IKKβ), which was increased by palmitate in C2C12 myotubes mediating inflammatory status, whereas the phosphorylation of PKC-θ was not significantly modulated by silibinin. Collectively, the results indicated that silibinin prevented inhibition of the IRS-1/PI3K/Akt pathway, thus ameliorating palmitate-induced insulin resistance in C2C12 myotubes.

Effect of different concentrations of calpain inhibitor ALLN on proliferation and apoptosis of C2C12 myoblasts / 中华临床营养杂志

Objective To explore the effect of different concentrations of ALLN on proliferation and apoptosis of C2C12 myoblasts.Methods After intervention with Ca2+ and ALLN,methyl thiazolyl tetrazolium and flow cytometry were used to determine the effect of Ca2+ and ALLN on the proliferation and apoptosis of C2C12 cells,respectively.The morphological changes of C2C12 myoblasts were observed using Giemsa staining.Results The absorbance of Ca2 + group was significantly lower than that of the control group (P ＜0.05).After 6,12,24,36 hours of intervention,the absorbance in ALLN groups 1 to 7 (cultured in serum-free media containing 16 mmol/L Ca2+ and ALLN at final concentrations of 3.125,6.25,12.5,25,50,100,200 μmol/L) were all significantly higher than that in the 16 mmol/L Ca2+ group (after 6 hours:0.449±0.024,0.472±0.022,0.513 ±0.008,0.540±0.014,0.588±0.016,0.607±0.030,0.700±0.020 vs.0.355 ±0.012,all P =0.000; after 12 hours:0.407 ±0.007,0.414 ±0.006,0.434 ±0.004,0.441 ±0.003,0.460 ±0.010,0.484 ± 0.006,0.525 ± 0.006 vs.0.368 ± 0.027,all P =0.000; after 24 hours:0.436±0.005,0.431 ±0.015,0.441 ±0.006,0.459 ±0.013,0.527 ±0.009,0.581 ±0.005,0.599 ±0.011 vs.0.386 ± 0.007,all P =0.000 ; after 36 hours:0.464 ± 0.022,0.460 ± 0.018,0.461 ± 0.007,0.434 ± 0.020,0.454 ± 0.028,0.479 ± 0.006,0.524 ± 0.011 vs.0.379 ± 0.011,all P =0.000),while no significant differences were observed after 48-72 hours of intervention.After treatment for 36 hours,the apoptosis rate in ALLN 10,50,100,and 200 μmol/L groups were (6.00 ± 1.20) ％,(5.02 ± 1.13) ％,(4.89±1.11)％,and (2.71 ± 1.15)％,all significantly lower than that in the Ca2+ group [(13.70 ±2.30)％] (all P =0.000).Giemsa staining showed apoptotic morphological changes in the Ca2+ group,which were obviously alleviated in the ALLN group.Conclusions Ca2+ at a concentration of 16mmol/L can induce apoptosis of C2C12 cells.In contrast,ALLN can inhibit cell apoptosis and promote proliferation in a time-and dose-dependent manner.

Cigarette smoke extract promotes senescence by reducing expression of HDAC2 in C2C12 myoblasts / 中国病理生理杂志

AIM:To investigate whether the cigarette smoke extract (CSE) causes senescence of C2C12 myo-blasts and the relationship between senescence and histone deacetylase 2(HDAC2).METHODS: Murine C2C12 cells were induced to differentiate into myoblasts.The HDAC2 activator and inhibitor were used to investigate the effects of CSE in the myoblasts on cell senescence and the expression of HDAC2.The expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blot, respectively, and the positive cell rate of β-galactosidase staining for cell senescence was also detected.RESULTS:The optimal concentration of CSE was 60 mL/L and the intervention time was 24 h.After the intervention of CSE, the positive cell rate ofβ-galactosidase staining was increased, accompanied with the reduction of HDAC2 expression at mRNA and protein levels.The expression of HDAC2 at mRNA and protein levels was increased by 4, 5, 6, 7-tetrabromobenzotriazole (TBB), accompanied with the reduction of positive cell rate ofβ-galacto-sidase staining.Furthermore, when HDAC2 expression at mRNA and protein levels was reduced by HDAC2 inhibitor valp-roic acid, the positive cell rate of β-galactosidase staining was increased.CONCLUSION: CSE promotes the senescence by reducing the expression of HDAC2 in C2C12 myoblasts.

Chemical Composition, Antioxidant Activities and Protective Effects of Sideritis italica Extract on C2C12 Oxidative Stress.

Aims: Sideritis italica is a medicinal plant used for medical purposes mainly based on experiences rather than scientific evidence. Biological properties, composition of primary and secondary metabolites as well as the antioxidant capacity were investigated on samples from wild plant. Methodology: The ultrastructure of aerial parts and quantitative distribution of pigments, including chlorophylls and amino acids, as well as the main class of secondary metabolites (phenols, flavonoids, flavonols and proanthocyanidins) were investigated. The extracts were tested by radical scavenging assays (DPPH, ABTS) and pharmacological assays (antiproliferative activity, effects on ROS production and protective effects against DNA damage induced by hydrogen peroxide) for their effects on C2C12 cell line. Results: Scanning electron microphotography confirms the presence of pharmacognostic characteristics, such as glandular and non-glandular trichomes on aerial parts. The chemical analysis indicates that the leaves are the most important part of the plant, and ethanol/water 70/30 is the preferable extraction solvent. The highest concentration of all metabolites was found in 70% ethanol extract of leaves. The antiradical assays and the in vitro tests on mouse myoblast cells C2C12 confirm the biological activities of the extract. C2C12 culture medium supplemented with extract, at doses (5-200μg/ml) not interfering with cell viability, was seen to modulate the ROS production and balance the increased oxidative stress induced by hydrogen peroxide. The treatment of C2C12 cells with 200 μg/ml of extract results in a percentage reduction of ROS of -60% and -71%, compared to untreated and H2O2 treated groups, respectively, P<.05. The quantitative reduction of 8- hydroxy-2’-deoxyguanosine (8-OH-dG), which is a biomarker of free radical DNA damage, confirms the protective effect of S. italica extract on oxidative stress at basal condition as well as in presence of exogenous stimuli (-11 and -7%, at 20μg/ml, respectively versus untreated and H2O2 groups, P<.05). Conclusion: The results obtained in the present study support the rational base for the medicinal use of plant and extracts in modulating the free radical metabolism and balancing the oxidative stress.

Effects of myostatin propeptide gene tranfection on glucose metabolism in cultured C2C12 cells / 中华内分泌代谢杂志

Objective To investigate the effects of recombinant adeno-associated virus-mediated myostatin propeptide (MPRO) on uptake and oxidation of glucose,and glycogen synthesis in C2C12 myotubes,as well as the associated molecular mechanism.Methods Mature C2C12 myotubes were assigned to the following 6 groups:control,insulin,green fluorescent protein (GFP),insulin + GFP,MPRO,and insulin + MPRO groups.Glucose uptake,glucose oxidation,and glycogen synthesis were detected by counting radioactivity of 14CO2 or 14C labeled glycogen derived from 2-deoxy-[1-14 C] glucose.The activity of insulin signal pathway was evaluated by Western blot.Results Compared with control group,glucose uptake and glycogen synthesis were significantly increased in insulin and insulin+GFP groups,and further increased in insulin+MPRO group as compared with insulin alone(all P＜ O.05).However,MPRO and insulin had no effect on glucose oxidation.The phosphorylations of insulin receptor (IR) β,insulin receptor substrate 1 (IRS-1),protein kinase B (Akt),glycogen synthase kinase-3 β (GSK-3β),and the expressions of phosphatidylinositol 3-kinase (PI3K) and glucose transporter 4 (Glut4) in membrane were significantly increased in insulin and insulin+GFP groups compared with control group(all P＜0.05),and were further increased after MPRO transfection (all P ＜ 0.05).Conclusion MPRO may increase insulin-stimulated glucose uptake and glycogen synthesis in C2C12 cells by activating the IRS/PI3K/Akt signal pathway.

The Stimulatory Effect of Essential Fatty Acids on Glucose Uptake Involves Both Akt and AMPK Activation in C2C12 Skeletal Muscle Cells

Essential fatty acid (EFA) is known to be required for the body to function normally and healthily. However, the effect of EFA on glucose uptake in skeletal muscle has not yet been fully investigated. In this study, we examined the effect of two EFAs, linoleic acid (LA) and alpha-linolenic acid (ALA), on glucose uptake of C2C12 skeletal muscle cells and investigated the mechanism underlying the stimulatory effect of polyunsaturated EFAs in comparison with monounsaturated oleic acid (OA). In palmitic acid (PA)-induced insulin resistant cells, the co-treatment of EFAs and OA with PA almost restored the PA-induced decrease in the basal and insulin-stimulated 2-NBDG (fluorescent D-glucose analogue) uptake, respectively. Two EFAs and OA significantly protected PA-induced suppression of insulin signaling, respectively, which was confirmed by the increased levels of Akt phosphorylation and serine/threonine kinases (PKCtheta and JNK) dephosphorylation in the western blot analysis. In PA-untreated, control cells, the treatment of 500 microM EFA significantly stimulated 2-NBDG uptake, whereas OA did not. Phosphorylation of AMP-activated protein kinase (AMPK) and one of its downstream molecules, acetyl-CoA carboxylase (ACC) was markedly induced by EFA, but not OA. In addition, EFA-stimulated 2-NBDG uptake was significantly inhibited by the pre-treatment of a specific AMPK inhibitor, adenine 9-beta-D-arabinofuranoside (araA). These data suggest that the restoration of suppressed insulin signaling at PA-induced insulin resistant condition and AMPK activation are involved at least in the stimulatory effect of EFA on glucose uptake in C2C12 skeletal muscle cells.

miR-376b-3p promotes Runx2-induced early osteogenic differentiation of C2C12 cells / 中国组织工程研究

BACKGROUND: The transcription factor Runx2 is the key factor that regulates osteogenic differention and bone development. It has been reported that the C2C12 mesenchymal cells can be induced to differentiate into osteoblasts by Runx2 overexpression, but the molecular mechanism of induction is stil largely unclear. OBJECTIVE: To investigate the role of the members of the miR-376 family during Runx2-induced osteogenic differentiation in C2C12 cells. METHODS: The expression of the members of the miR-376 family was detected by real-time quantitative PCR at different time points using C2C12/Runx2Dox sub-line with conditional Runx2 expression. In miR-376b-3p-transfected C2C12/Runx2Dox cells, the expression of osteoblast markers, such as alkaline phosphatase and osteocalcin, was detected by real-time quantitative PCR, and the alkaline phosphatase activity was also examined by alkaline phosphatase staining. The putative miR-376b-3p targets were commonly predicted by online tools (miRanda, miRWalk and TargetScan). The functional classification of these putative targets was performed by DAVID Bioinformatics Resources database. RESULTS AND CONCLUSION: The expression of miR-376b-3p was significantly increased during Runx2-induced osteogenic differentiation of C2C12 cells, but the expression of other members was not changed. Transfection of miR-376b-3p mimic upregulated alkaline phosphatase expression, but had no effect on osteocalcin expression. The alkaline phosphatase activity was also increased by transfection of miR-376b-3p. The functional classification of miR-376b-3p putative targets showed that miR-376b-3p is involved in the skeleton development, indicating the role of miR-376b-3p in osteoblast differentiation. Taken together, these results suggest that Runx2 promotes early osteogenic differentiation in C2C12 cells by regulating the expression of genes related to osteogenic differentiation through upregulation of miR-376b-3p.