Effects of recombinant retroviral vector mediated human insulin like growth factor-1 gene transfection on skeletal muscle growth in rat

Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hlGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochernistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghlGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. Results Recombinant retroviral plasmid vector pLghlGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening, hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast （P〈 0.05）. The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast （P〈 0.05）. Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myobiast group （P〈 0.05）. Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.

Flaxseed Lignans Promoted the Growth of Skeletal Muscle in Male Rats and Its Possible Mechanism

This study was aimed to determine whether flaxseed lignans could affect the growth of skeletal muscle in male animals and its possible mechanisms. The impact of flaxseed lignans on the skeletal muscle in male rats was determined in vivo. Flaxseed lignans （50 ppm） and daidzein （5 ppm） were added into the basal diets, respectively. The concentrations of serum lignans and daidzein were measured by high performance liquid chromatography （HPLC）, and the serum growth hormone and testosterone （T） levels were analyzed by radioimmunoassay （RIA）, and the expression of estrogen receptor β （ER β） in the soleus muscle and hypothalamus were determined by reverse-transcription polymerase chain reaction （RT-PCR）. Flaxseed lignans and daidzein could significantly improve the feed efficiency and facilitate the weight gain of the femoral muscle in male rats. The ratio of RNA to DNA in the muscles and serum T levels was remarkably increased, whereas, the urea nitrogen concentrations were significantly decreased by flaxseed lignan and/or its metabolites and daidzein. Meanwhile, the expression of ER β in soleus muscle and hypothalamus were both upgraded by the two phytoestrogens. Flaxseed lignan promoted the growth of male rats, and it might be by regulating serum T levels by binding to ER β in the hypothalamus. In turn, it depressed the catabolism of protein and promoted the hypertrophy of skeletal muscle ceils.

Different distributions of interstitial cells of Cajal and platelet-derived growth factor receptor-α positive cells in colonic smooth muscle cell/interstitial cell of Cajal/plateletderived growth factor receptor-α positive cell syncytium in mice

AIM To investigate the distribution and function of interstitialcells of Cajal(ICCs) and platelet-derived growth factor receptor-α positive(PDGFRα+) cells in the proximal and distal colon.METHODS The comparison of colonic transit in the proximal and distal ends was performed by colonic migrating motor complexes(CMMCs). The tension of the colonic smooth muscle was examined by smooth muscle spontaneous contractile experiments with both ends of the smooth muscle strip tied with a silk thread. Intracellular recordings were used to assess electrical field stimulation(EFS)-induced inhibitory junction potentials(IJP) on the colonic smooth muscle. Western blot analysis was used to examine the expression levels of ICCs and PDGFRα in the colonic smooth muscle.RESULTS Treatment with NG-nitro-L-arginine methyl ester hydrochloride(L-NAME) significantly increased the CMMC frequency and spontaneous contractions, especially in the proximal colon, while treatment with MRS2500 increased only distal CMMC activity and smooth muscle contractions. Both CMMCs and spontaneous contractions were markedly inhibited by NPPB, especially in the proximal colon. Accordingly, CyPPA sharply inhibited the distal contraction of both CMMCs and spontaneous contractions. Additionally, the amplitude of stimulationinduced nitric oxide(NO)/ICC-dependent slow IJPs(sIJPs) by intracellular recordings from the smooth muscles in the proximal colon was larger than that in the distal colon, while the amplitude of electric field stimulationinduced purinergic/PDGFRα-dependent fast IJPs(fIJPs) in the distal colon was larger than that in the proximal colon. Consistently, protein expression levels of c-Kit and anoctamin-1(ANO1) in the proximal colon were much higher, while protein expression levels of PDGFRα and small conductance calcium-activated potassium channel 3(SK3) in the distal colon were much higher.CONCLUSION The ICCs are mainly distributed in the proximal colon and there are more PDGFRα+ cells are in the distal colon, which generates a pressure gradient between the two ends of the colon to propel the feces to the anus.

Comparison of Muscle Fiber Growth from F1Generations of Large Yorkshire Pig andErhualian Pig by Reciprocal Cross

[ Objective] To compare the muscle fiber growth between F1 generations of large Yorkshire pig and Erhualian pig by reciprocal cross, and provide a theoretical basis for the integrated breeding of meat quality traits and growth performances by rational utilization of hybrid combination. [ Method] The hybrid pigs of large Yorkshire pig and Erhualian pig were fed and managed in the same conditions. The reciprocal combinations were Erhualian pig ♀ × Yorkshire pigd, and Yorkshire pig ♀ × Erhualian pig♂ , respectively. At the age of 20, 70, 120 and 180 d, the Iongissimus dorsi muscle and thigh muscle were collected and made into frozen sections for hematoxylin and eosin （HE） staining, and then the muscle fiber area was determined. [ Result] The muscle fiber was thickened gradually with increasing age; and the absolute growth curve of muscle fiber area was presented as ＂S＂ shape. The diameter, perimeter and area of Iongissimus dorsi muscle and thigh muscle fibers in the reciprocal combination of Yorkshire pig ♀ × Erhualian pig♂ were higher than those in the reciprocal combination of Erhualian pig ♀ × Yorkshire pig♂ （P 〈 0.01 ）. The thigh muscle grew faster than Iongissimus dorsi muscle at the age of 20 -120 d; but at the age of 120 -180 d, the growth rate of Iongissimus dorsi muscle was higher than that of thigh muscle; and at the age of 180 d, the muscle fiber area had no significant difference between the Iongissimus dorsi muscle and the thigh muscle. The muscle fiber area had extremely significant difference between the reciprocal combinations at the age of 20 d; but no significant difference was found at the age of 180 d. [ Conclusion] The reciprocal combinations can affect the muscle fiber growth of hybrid pigs of Yorkshire pig and Erhualian pig, but the affect degree reduces with the increasing age.

Integration of genome-wide association study and selection signatures reveals genetic determinants for skeletal muscle production traits in an F2 chicken population

Improving the production of broiler chicken meat has been a goal of broiler breeding programs worldwide for many years. However, the genetic architectures of skeletal muscle production traits in chickens have not yet been fully elucidated. In the present study, a total of 519 F_(2) birds, derived from a cross of Arbor Acres broiler and Baier layer, were re-sequenced(26 F_(0) individuals were re-sequenced at a 10-fold depth;519 F_(2) individuals were re-sequenced at a 3-fold depth) and the coupling of genome-wide association study(GWAS) and selection signatures(FST(fixation index) and θπ(nucleotide diversity)) was carried out to pinpoint the associated loci and genes that contribute to pectoral muscle weight(PMW) and thigh muscle weight(TMW). A total of 7 890 258 single nucleotide polymorphisms(SNPs) remained to be analyzed after quality control and imputation. The integration of GWAS and selection signature analyses revealed that genetic determinants responsible for skeletal muscle production traits were mainly localized on chromosomes 1(168.95–172.43 Mb) and 4(74.37–75.23 Mb). A total of 17 positional candidate genes(PCGs)(LRCH1, CDADC1, CAB39 L, LOC112531568, LOC112531569, FAM124 A, FOXO1, NBEA, GPALPP1, RUBCNL, ARL11, KPNA3, LHFP, GBA3, LOC112532426, KCNIP4, and SLIT2) were identified in these regions. In particular, KPNA3 and FOXO1 were the most promising candidates for meat production in chickens. These findings will help enhance our understanding of the genetic architecture of chicken muscle production traits, and the significant SNPs identified could be promising candidates for integration into practical breeding programs such as genome-wide selection(GS) to improve the meat yield of chickens.

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM:To investigate the role of insulin-like growth factor binding protein-7 (IGFBP-7) in the activation and transdifferentiation of hepatic stellate cells (HSC) in vitro.METHODS:Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor (TGF)-β1,IGFBP-7 or antiIGFBP-7 antibody for 24 h.The supernatant or a cytoplasm suspension was obtained from cultured HSC,followed by transfer of cells to form cell-coated dishes.Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin,collagen and α-smooth muscle actin (SMA).The pro-apoptotic effect of antiIGFBP-7 antibody was determined by flow cytometry.RESULTS:Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group.In addition,fibronectin,collagen and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent.Moreover,flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC,which is responsible for the development of liver fibrosis,and may represent a novel pathway and target for therapeutic intervention.CONCLUSION:IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC.AntiIGFBP-7 antibody may ameliorate liver fibrogenesis.

Association of growth rate with hormone levels and myogenic gene expression profile in broilers

Background： The growth rate often varies among individual broilers of the same breed under a common management condition. To investigate whether a variation in the growth rate is associated with a difference in hormone levels and myogenic gene expression profile in broilers, a feeding trial was conducted with 10,000 newly hatched Ross 308 chicks in a commercial production facility under standard management. At 38 d of age,30 fast-, 30 medium-, and 30 slow-growing broilers were selected among 600 healthy male individuals. The levels of insulin-like growth factor-1（IGF-1）, triiodothyronine（T3）, thyroxine（T4）, and growth hormone in the serum or breast muscle were assayed by ELISA or RIA kits, and the expression levels of several representative pro-and anti-myogenic genes in the breast muscle were also measured by real-time PCR.Results： Results showed that both absolute and relative weights of the breast muscle were in linear positive correlations with the body weight of broilers（P 〈 0.001）. Fast-growing broilers had higher concentrations of IGF-1 than slow-growing broilers（P 〈 0.05） in both the serum and breast muscle. The serum concentration of T3 was significantly higher in fast-growing birds than in slow-growing birds（P 〈 0.05）. However, no difference was observed in growth hormone or T4 concentration among three groups of birds. Additionally, a decreased expression of an anti-myogenic gene（myostatin） and increased expressions of pro-myogenic genes such as myogenic differentiation factor 1, myogenin, muscle regulatory factor 4, myogenic factor 5, IGF-1, and myocyte enhancer factor 2B, C, and D were observed in fast-growing broilers（P 〈 0.05）, relative to slow-growing broilers.Conclusions： Collectively, these findings suggested that the growth rate is linked to the hormone and myogenic gene expression levels in broiler chickens. Some of these parameters such as serum concentrations of IGF-1 and T3 could be employed to breed for enhanced growth.

IGF-1, bFGF EXPRESSION AND VASCULAR REGENERATION IN ACUTE INFARCTED CANINE MYOCARDIUM AFTER AUTOLOGUS SKELETAL MUSCLE SATELLITE CELL IMPLANTATION

Objective To study the cell growth factor secretion and vascular regeneration in acute in-farcted myocardium after autologous skeletal muscle satellite cell implantation. Methods Autologous skeletal muscle satellite cells from adult mongrel canine were implanted into the acute myocardial infarct site via the ligated left anterior descending (LAD) artery. Specimens were harvested at 2, 4 , 8 weeks after implantation for the expression of insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor ( bFGF) and the vascular density. Results The expression of IGF-1, bFGF and the vascular density in skeletal muscle satellite cell implant group were higher than that in the control group. Conclusion The skeletal muscle satellite cells, after being implanted into the acute myocardial infarction, not only showed myocardial regeneration, but also showed the ability to secrete the cell factors, hence representing a positive effect on the regeneration of the infarcted myocardium.

Artificial meat? Feasible approach based on the experience from cell culture studies

This short review is to list pros and cons which are based on the literature and personal experience in cell culture studies related to possible commercial production of artificial meat as functional food. The general view of muscle composition and determinants of meat quality are shortly described. Principles of muscle cell propagation in culture and mutual relationships between different cell types present in this organ are briefly discussed. Additionally, the effects of some cytokines and growth factors for muscle cell growth and muscle tissue development are indicated. Finally, conclusion remarks related to detrimental consequences of meat production to natural environment as well as personal opinion of author on the prospects of artificial meat production are declared.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder(Paralichthys olivaceus)

Follistatin （Fst） is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns ofFst gene at five different development stages of Japanese flounder （stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months）. The muscle tissue of Japanese flounder was obtained at different development stages in dais experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Dietary supplementation with succinic acid improves growth performance and flesh quality of adult Nile tilapia(Oreochromis niloticus)fed a high-carbohydrate diet

To evaluate the effects of dietary supplementation with succinic acid on growth performance,flesh quality,glucose,and lipid metabolism of Nile tilapia(Oreochromis niloticus)fed a high-carbohydrate diet(HCD),five iso-nitrogenous and iso-lipidic diets were prepared as follows:HCD(control group)consisting of 55%corn starch and HCD supplemented with 0.5%,1.0%,2.0%,and 4.0%succinic acid,respectively.Tilapia with an initial body weight of 204.90±1.23 g randomly assigned to 15 tanks with 3 replicates per group and 10 fish per tank fed for 8 weeks.Increasing dietary succinic acid supplementation resulted in significant second-order polynomial relationship in the weight gain rate(WGR),specific growth rate(SGR),feed conversion ratio(FCR),protein efficiency rate(PER),viscerosomatic index,condition factor,and contents of muscular crude lipid and glycogen(P<0.05).The hepatosomatic index,mesenteric fat index,liver glycogen content and crude lipid contents of the whole-body and liver demonstrated significantly linear and second-order polynomial relationship(P<0.05).Quadratic curve model analysis based on WGR,SGR,PER,and FCR demonstrated that optimal supplementation with succinic acid in the HCD of Nile tilapia ranged from 1.83%to 2.43%.Fish fed with 1.0%succinic acid had higher muscular hardness,increased the contents of alkali-soluble hydroxyproline in collagen,docosahexaenoic acid(DHA)and n-3 polyunsaturated fatty acid(n-3PUFA)in muscle,and lower total fatty acid content in muscle(P<0.05)compared with the control group.Compared to the control group,dietary supplementation with 1.0%succinic acid significantly increased the contents of total bounding amino acid(arginine,histidine,isoleucine,lysine,methionine,alanine,proline),total flavor amino acid(free aspartic acid),the catalase(CAT)activity and total antioxidant capacity,and the mRNA relative expression levels of CAT,superoxide dismutase(SOD),and nuclearfactor erythroidderived 2-like 2(Nrf2)in muscle(P<0.05).Furthermore,succinic acid supplementation significantly up-regulated mRNA relative expression levels of glycolysis genes(hexokinase 2[HK2],phosphofructokinase,muscle-A[PFKMA],and phosphofructokinase,muscle-B[PFKMB]),a key glycogen synthesis gene(glycogen synthase[GYS]),and lipid catabolism genes(carnitine palmitoyltransferase-1B[CPT1B],hormone sensitive lipase[HSL],and lipoprotein lipase[LPL]),while down-regulating the mRNA relative expression level of fatty acid synthase(FASN)in muscle(P<0.05).In conclusion,dietary supplementation with 1.83%to 2.43%succinic acid improved muscle quality by increasing muscle antioxidant capacity and hardness,changing muscle amino acid and fatty acid composition,and regulating muscle glucose and lipid metabolism.

Antifibrotic effect of aloe vera in viral infection-induced hepatic periportal fibrosis

AIM:To investigate the anti-oxidative and anti-fibrotic effects of aloe vera in patients with liver fibrosis.METHODS:Aloe vera high molecular weight fractions(AHM) were processed by patented hyper-dry system in combination of freeze-dry technique with microwave and far infrared-ray radiation.Fifteen healthy volunteers as the control group and 40 patients were included.The patients were randomly subdivided into two equal groups:the conventional group was treated with placebo(starch),and AHM group was treated with 0.15 gm/d AHM,both for 12 consecutive weeks.The patients were investigated before and after treatment.Serum activity of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),hyaluronic acid(HA),transforming growth factor-β(TGF-β) and matrixmetalloproteinase-2(MMP-2) were determined.The reduced glutathione(GSH) and malondialdehyde(MDA) levels in liver were assayed and the expression of hepatic α-smooth muscle actin(α-SMA) was identified by immunohistochemistry.RESULTS:At the start of the study,the hematoxylin and eosin staining revealed fibro-proliferated bile ductules,thick fibrous septa and dense inflammatory cellular infiltration in the patients before treatment.The use of AHM for 12 wk significantly ameliorated the fibrosis,inhibited the inflammation,and resulted in minimal infiltration and minimal fibrosis compared to the conventional group.The enzyme activities of the liver(ALT,AST and ALP) were attenuated after treatment in both groups,and the decrease in the AHM group was more significant as compared with the conventional group.Similar to the AST,the MDA levels were significantly higher before treatment,and were attenuated after treatment in both groups.In contrast,the hepatic glutathione content in the patients were decreased significantly in the AHM group compared to the controls.The serum levels of the fibrosis markers(HA,TGF-β and MMP-2) were also reduced significantly after treatment.The expression of α-SMA was modified in patients before and after treatment as compared with the normal controls.In the conventional group,there was only thin and incomplete parenchymal α-SMA positive septum joining the thickened centrilobular veins,while in the AHM group,few α-SMA positive cells were present in sinusoid and lobule after treatment.CONCLUSION:Oral supplementation with AHM could be helpful in alleviating the fibrosis and inflammation of hepatic fibrosis patients.

ACIDIC FIBROBLAST GROWTH FACTOR REDUCES RAT SKELETAL MUSCLE DAMAGE CAUSED BY ISCHEMIA AND REPERFUSION

Acute interruption of arterial blood flow to the extremities is often associated with significant morbidity and mortality. Broad spectrum mitogenic and non mitogenic activities of FGFs inspired us to study its protecting effects on tissue injuries in ischemia reperfusion condition. We found that systemic administration of aFGF after reperfusion onset prevented severe skeletal muscle injuries. In rats treated with aKGF, the tissue edema was reduced significantly, the tissue viability was increased, and the muscle fibers contained more succinate dehydrogenase (SDH) and adenosine triphosphatasc (ATPase). The pathological results supported the concept of improved prevention with aFGF treatment. The possible tissue protection by aFGF may come from its ability to regulate the concentration of evtra- and intracellular calcium ion. Besides, it may moderate other Ca2+ dependent enzyme conversion processes. Also, it may take part in the vascular tone regulation under ischemia and reperfusion conditions. These results suggest further study of tissue ischemia prevention with FGF and its possible mechanisms in the future.

Shexiang Baoxin Pill Regulates Intimal Hyperplasia,Migration,and Apoptosis after Platelet-Derived Growth Factor-BB-Stimulation of Vascular Smooth Muscle Cells via miR-451

Objective:To investigate the regulatory roles of Shexiang Baoxin Pill(SXBXW)in neointimal formation and vascular smooth muscle cells(VSMCs)invasion and apoptosis as well as the potential molecular mechanisms using cultured VSMCs model of vascular injury(platelet-derived growth factor(PDGF)-BBstimulated)in vitro.Methods:VSMCs were randomly assigned to 5 groups:blank,PDGF-BB(20 ng/mL+0.1%DMSO),SXBXW-L(PDGF-BB 20 ng/mL+SXBXW low dose 0.625 g/L),SXBXW-M(PDGF-BB 20 ng/mL+SXBXW medium dose 1.25 g/L)and SXBXW-H(PDGF-BB 20 ng/mL+SXBXW high dose 2.5 g/L)group.Cell proliferation was assessed using cell counting kit-8(CCK-8)assay and bromodeoxyuridine(BrdU)incorporation assay,the migration effects were detected by Transwell assay,cell apoptosis rate was measured by the Annexin V/propidium iodide(PI)apoptosis kit.The markers of contractile phenotype of VSMCs were detected with immunofluorescent staining.To validate the effects of miR-451 in regulating proliferation,migration and apoptosis treated with SXBXW,miR-451 overexpression experiments were performed,the VSMCs were exposed to PDGF-BB 20 ng/mL+0.1%DMSO and later divided into 4 groups:mimic-NC(multiplicity of infection,MOI=50),SXBXW(1.25 g/L)+mimic-NC,mimic-miR451(MOI=50),and SXBXW(1.25 g/L)+mimic-miR451,and alterations of proteins related to the miR-451 pathway were analyzed using Western blot.Results:PDGF-BB induced VSMCs injury causes acceleration of proliferation and migration.SXBXW inhibited phenotypic switching,proliferation and migration and promoted cell apoptosis in PDGF-BB-induced VSMCs.In addition,miR-451 was shown to be down-regulated in the VSMCs following PDGF-BB stimulation.SXBXW treatment enhanced the expression of miR-451 in PDGF-BB-induced VSMCs(P<0.05).Compared with SXBXW+mimic-NC and mimicmiR451 groups,the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta(Ywhaz)and p53 was further reduced in SXBXW+mimic-miR451 group,while activating transcription factor 2(ATF2)was increased in VSMCs(P<0.05).Conclusion:SXBXW regulated proliferation,migration and apoptosis via activation of miR-451 through ATF2,p53 and Ywhaz in PDGF-BB-stimulated VSMCs.

Enhanced hyperplasia in muscles of transgenic zebrafish expressing Follistatin1

Myostatin is a member of the transforming growth factor-β(TGF-β) super-family and functions as a negative regulator of muscle growth.Binding of the specific receptor,Activin receptor IIB(Act RIIB),with myostatin or other related TGF-β members,could be inhibited by the activin-binding protein follistatin(Fst) in mammals.Overexpressing Fst in mouse skeletal muscle leads to muscle hypertrophy and hyperplasia.To determine if Fst has similar roles in fish,we generated transgenic zebrafish expressing high levels of zebrafish Fst1 using the promoter of the zebrafish skeletal muscle-specific gene,myosin,light polypeptide 2,skeletal muscle(Mylz2).Independent transgenic zebrafish lines exhibited elevated expression levels of myogenic regulatory genes MyoD and Pax7 in muscle cells.Adult Fst1 overexpressing transgenic zebrafish exhibited a slight body weight increase.The high level of Fst1 expression dramatically increased myofiber numbers in skeletal muscle,without significantly changing the fiber size.Our findings suggest that Fst1 overexpression can promote zebrafish muscle growth by enhancing myofiber hyperplasia.

The double mutations of acvr2aa and acvr2ba leads to muscle hypertrophy in zebrafish

Activin A receptor,type II(Acvr2)is a member of the transforming growth factor beta receptor family and can function as a negative regulator of skeletal muscle mass.Acvr2 plays an important role in regulating muscle development that can inhibit skeletal muscle growth in mice.However,there is very little research reported on the function of acvr2 in muscle development of teleost.In this study,we analyzed the effect of acvr2aa and acvr2ba on muscle development in zebrafish.Growth rates of WT and acvr2a^(-/-b-/-)􀀀were measured from juvenile stage to adult stage.In addition,effects of acvr2 on skeletal muscle were tested in histological,protein and molecular levels.As a result,acvr2a^(-/-b-/-)􀀀exhibited a wider body trunk than WT and showed a significant increase in body weight and width from two months old.Histological analysis of skeletal muscle indicated that the size of muscle fiber in acvr2a^(-/-b-/-)􀀀(female:1809±123μm^(2);male:2261±130μm^(2))was larger than that in WT(709.8±49μm^(2);815±53μm^(2)).In addition,western blot of fast MyLc protein showed the protein synthesis of acvr2a^(-/-b-/-)􀀀are increased.Besides,Histological analysis of heart showed the ventricle area is aslo increased in acvr2a^(-/-b-/-)􀀀.Our results demonstrated acvr2 attends the development of muscle fiber and will cause muscle hypertrophy when they were knocked out in zebrafish.In conclusion,acvr2 in zebrafish can control the development of muscle fibers during posthatch growth.

Dietary fishmeal replacement by Clostridium autoethanogenum protein meal influences the nutritional and sensory quality of turbot(Scophthalmus maximus)via the TOR/AAR/AMPK pathways

Clostridium autoethanogenum protein(CAP)is a promising protein source for aquaculture;however,how CAP influences fish quality is worth extensive research.We randomly allocated 630 turbot with initial body weights of about 180 g into 6 groups,with fishmeal-based control diet or diet with CAP replacing 15%(CAP15),30%(CAP30),45%(CAP45),60%(CAP60),or 75%(CAP75)of fishmeal protein.After a 70-d feeding trial,the fillet yield(P=0.015)and content of protein(P=0.017),collagen(P 0.05).By contrast,the muscle hardness increased linearly with increasing CAP(P=0.004),accompanied by linear reduction of muscle fiber area(P=0.003)and expression of myogenesis-related genes,including cathepsin D(ctsdP<0.001)and muscle ring finger protein 1(murf 1,P<0.001).Phosphorylation of protein kinase B(Akt,P<0.001),target of rapamycin(TOR,P=0.001),eukaryotic initiation factor 4E-binding protein 1(4E-BP1,P<0.001),and ribosomal protein S6(S6,P<0.001)decreased linearly;however,phosphorylation of AMP-activated protein kinase(AMPK,P<0.001),eukaryotic initiation factor 2α(eIF2α,P<0.001),and the abundance of activating transcription factor 4(ATF4,P<0.001)increased with increasing CAP,suggesting that the TOR signaling pathway was inhibited,and the amino acid response(AAR)and AMPK pathways were activated.Additionally,expression of genes related to protein degradation,including myogenic factor 5(myf 5,P<0.001),myogenic differentiation(myod,P<0.001),paired box 7(pax 7,P<0.001),and ctsd(P<0.001),decreased linearly with increasing CAP.In conclusion,CAP could be used to replace up to 15%of fishmeal without negatively impacting turbot quality.However,higher levels of CAP decreased fillet yield,muscle protein content,and muscle fiber diameter while increasing muscle hardness,which could be attributed to the inhibition of the TOR pathway and activation of the AAR and AMPK pathways.

Effect of glial gro wth factor on functional recovery ofperipheral nerve after crush injury

Objective: To evaluate in vivo the effect of the recombinant human glial growth factor 2 (rhGGF 2) on the recovery of motor function of rat sciatic nerve following crush injury. Methods: Seventy three normal SD rats were randomly divided into three groups. A sham operation was performed in Group Ⅰ (n=5), and a 5 mm segment of the sciatic nerve was subjected to a 100 g crush load for 2 hours of duration in Groups Ⅱ (n=34) and Ⅲ (n=34). Group Ⅲ was treated with rhGGF 2 (1 mg/kg by subcutaneous injection after nerve crush, and then injected daily for the subsequent 4 days), and Group Ⅱ was given vehicle of the same volume. The motor functional recovery of never was assessed by calculating the sciatic functional index (SFI) and measuring the tetanic contractile strength of the extensor digitorum longus (EDL) of the hind limb. Results: Nerve function in Group Ⅲ began to recover at day 11 whereas the nerves in Group Ⅱ were still paralyzed after crush injury. The tetanic contractile strength of EDL was generally stronger in Group Ⅲ than that in Group Ⅱ, with a significant difference at 70 and 100 Hz stimulus frequencies from day 4 to day 14. Histological sections revealed less axonal degeneration and earlier regeneration of nerve fibers in Group Ⅲ. Conclusions: It suggests that rhGGF 2 is effective in promoting nerve regeneration and can significantly improve the functional recovery of rat sciatic nerve following crush injury.