| Malnutrition is chronic renal failure (Chronic renal failure, CRF) common major complications, is one of the leading cause of end-stage renal disease (ESRD) and increased mortality in patients with CRF. In China, about20%to50%of patients with CRF malnutrition can occur. CRF complex pathogenesis of malnutrition and various complications and immune dysfunction, infections, anemia, cardiovascular events are closely related, and even lead to multiple organ failure, seriously affecting the quality of life of patients. Insulin currently used in the clinical treatment of major growth factor-1(Insulin-like growth factor1, IGF-1) injection and compound a-keto acid (kai tong), its application has been limited so expensive. Therefore, cost-effective prevention and treatment of malnutrition CRF in the medical field has become an extremely important issue.CRF malnutrition mediated by multiple factors, reduced intake of nutrients, metabolic and endocrine disorders, protein lost during dialysis, anemia, inflammation can lead to various reasons such as malnutrition. Skeletal muscle mass and weight loss caused by skeletal muscle atrophy is an important indicator. Protein metabolic imbalance is the root cause skeletal muscle atrophy, including anabolic various causes reduction and increased catabolism, its molecular mechanism is very complex. Protein catabolism pathway mediated primarily by four:lysosomal pathway (lysosomal pathway), calcium-dependent pathway (Ca2+-dependent pathway), cysteinyl aspartate pathway proteolytic enzyme (caspase-dependent pathway) and Pan hormone-dependent proteasome pathway (ubiquitin-proteasome pathway, UPP). And IGF-1as opposed to be kinase B/Rapamycin the protease (Akt/mTOR) signaling pathway, phosphorylation of downstream protein kinases into the nucleus to activate various nuclear transcription factors, to increase related protein mRNA translation by thereby increasing protein synthesis. Recent studies have confirmed that malnutrition in CRF process, not only the existence of protein catabolism, which also severely inhibited anabolic, muscle Akt, mTOR expression levels were significantly reduced. Therefore, to explore new ways of malnutrition through activation of CRF Akt/mTOR signaling pathway therapy, find its role is more specific and effective regulatory factors or aspects, has important clinical significance.Skeletal muscle satellite cells (Muscle Satellite Cell, MSC) is located in a single core muscle fiber membrane and basilar membrane between pluripotent stem cells, because of their proliferation after activation can differentiate into new muscle fibers, broad participation and repair of skeletal muscle tissue regeneration in recent years become a hot research. MSC cells resting state under less chromatin condensation, cells and lack of realization of the stationary transcriptional activity. When the body is subjected to trauma, such as skeletal muscle stimulation, resting muscle cytoplasm occurs MSC expansion, activation, myoblast formation and further differentiation, fuse to form myotubes, and thus participate in the repair of skeletal muscle. However, in CRF nutrition environment, this process is subject to many influences, including uremic toxins, metabolic acidosis, micro-inflammatory state, lack of nutrients, such as MSC can inhibit the activation, growth and differentiation processes. Which oxidative stress (Oxidative Stress, OS) reaction prevalent in these factors, the MSC is also causing damage to an important part. CRF nutritional environment arising under excess ROS (Reactive Oxygen Species, ROS), which react with phospholipids, enzymes and membrane receptors on muscle cell membrane and into the MSC, the oxidation of polyunsaturated fatty acids, the body side chain and nucleic acids and other macromolecules, cell membrane damage, organelles, nucleus and various metabolic enzyme function, eventually leading to injury and apoptosis MSC and myoblasts, loss of repair capacity. Can be inferred, clear machine excess body ROS, reduced OS response levels, and promote MSC activation points, process, and repair of skeletal muscle atrophy delay is significant.CRF-induced skeletal muscle atrophy malnutrition, are medicine’s "Consumption","Weizheng" category. Spleen Qi deficiency is its incidence foundation. Spleen is acquired, the source of qi and blood. Therefore,"Spleen Qi" law has an important role in the traditional medicine of skeletal muscle atrophy caused by malnutrition CRF understanding and treatment. Medicinal Astragalus Astragalus is a legume film (Astragalus membranaceus (Fisch) Bge) and Mongolia Astragalus (Astragalus membranaceus Bge.var.mongholicus (Bge) Hsiao) root, sweet, slightly warm, is one of the common medicines. China Pharmacopoeia records it has Spleen, Yi Wei solid form, diuretic swelling, care drugs myogenic effect, is also the ancient physicians in the treatment of "Consumption", while "Weizheng" common, reusable goods. Astragalus polysaccharides (Astragalus polysaccharides, APS) as Astragalus polysaccharide water extraction is the main bioactive ingredients Astragalus. Experimental and clinical studies indicate that in vivo outside the modern, APS on immune dysfunction, inflammation, oxidative stress has a role in improving the regulation, which states and skeletal muscle atrophy in the etiology and mechanism of extensive contact, some studies have further confirmed the APS can Akt expression levels increase insulin resistance in skeletal muscle cells in state, to maintain the level of skeletal muscle anabolism. Based on this we can guess by APS through the promotion of skeletal muscle protein synthesis, antagonistic MSC oxidative damage, thereby preventing and improving skeletal muscle contraction. Therefore, this issue through in vitro experiments, observed improvement APS on skeletal muscle atrophy, while exploring the molecular mechanism to provide experimental evidence for its clinical application. Follows: Chapter1APS improve atrophy on C2C12myotubes atrophyObjective:To observe the APS dexamethasone (Dexamethasone, DEX) C2C12skeletal muscle tubular atrophy induced protective effect, and explore its mechanism.Methods:First, culture containing low concentrations of serum high-density C2C12skeletal myoblasts to differentiate into C2C12myotubes. After use with culture medium containing1μM Dex C2C12skeletal myotubes established models of skeletal muscle atrophy, and divided into three groups:Dex model group, Dex+APS Group, Dex+APS+LY group and normal control group and APS controls. Dex+APS group and the control group APS APS experiments were set amount of0.2mg/mL.Dex+APS+LY group and add another phosphatidylinositol3-kinase (PI3K) inhibitor LY294002(LY), the concentration of10μM. Each group after24h incubation cells were fixed with eosin-hematoxylin (HE) staining method for each group, with200times the light microscope to take pictures of each group, calculate the average diameter of each group myotubes with Image Pro-Plus software; by Western blot to detect the phosphorylation of Akt, mTOR, ribosomal protein S6kinase (P70s6k), ribosomal protein S6(rpS6), fork head transcription factor1(FoxO1), fork head transcription factor3a (FoxO3a) expression levels. The reference to the use of GAPDH.Results:Compared with normal control group, Dex model group myotubes average diameter significantly reduced, APS intervention can improve the degree of reduced diameter, LY294002could significantly antagonize the effect of this improvement, reducing the diameter of C2C12myotubes above group were statistically significant between (P<0.05), but there was no difference between the muscular tube diameter APS control group and normal control group; phosphorylated Akt, mTOR, P70s6k, rpS6, FoxO1, FoxO3a, rpS6normal group were significantly or fragmented expression, Dex model group phosphorylated Akt, mTOR, P70s6k, significantly reduced the expression of rpS6significantly increased expression levels of FoxO1and FoxO3a, and APS can restore the abnormal expression of the signal factor than the untreated group, LY294002antagonized except FoxO3a compared to all APS signal recovery factor for the role, were statistically significant between these groups (P<0.05), while the single plus between APS group and normal control group, Akt, mTOR, P70s6k, rpS6, FoxO1, Fox03a, no difference in expression levels of rpS6.Conclusion:The skeletal muscle contraction pipe diameter decreases, Akt/mTOR signaling pathway was inhibited. APS can effectively regulate restore Akt/mTOR signal, increasing muscular tube diameter, thereby improving skeletal muscle contraction.Chapter2APS for proliferation of C2C12myoblastsObjective:To observe the effects of APS and Dex on C2C12myoblast proliferation rate.Methods:To observe the effect on the concentration gradient APS C2C12myoblast proliferation rate:The cultured C2C12myoblasts were divided into six groups, each with a culture medium containing different concentrations of APS (0.05mg/mL,0.1mg/mL,0.2mg/mL,0.5mg/mL, lmg/mL,2mg/mL), a separate control group were incubated for24h; influence on the observed temporal gradient APS C2C12myoblast proliferation rate:The cultured C2C12myoblasts into four group, containing0.2mg/mL APS media were incubated8h,12h,24h,48h, separate each group a set of blank control; influence on the observed Dex C2C12myoblast proliferation rate:The cultured C2C12myoblasts Cells were divided into five groups, each with a culture medium containing different concentrations of Dex (O.1μM,1μM,10μM,100μM,1000μM), a separate control group were incubated for24h; observe the APS on hydrogen peroxide-induced activity of C2C12myoblasts improve Injury: The cultured C2C12myoblasts into blank control group, hydrogen peroxide group, APS Group, the amount of hydrogen peroxide experiments set to200μM, APS group on this basis with a0.2mg/mL drug intervention, according to8h,12h,24h incubation time was repeated three times. Detection above groups C2C12proliferation rate and activity of muscle cells by MTT assay. Results:The effect of concentration gradient APS on C2C12myoblast proliferation rate:Compared with normal control group,0.05mg/mL～0.5mg/mL of APS on C2C12myoblasts have a significant multiplier effect, which0.05mg/mL～0.2mg/mL segment in a concentration-effect relationship,2mg/mL the APS on C2C12myoblast proliferation inhibition, the above comparisons were statistically significant (P<0.05); time gradient APS on C2C12myoblast proliferation rate Impact:Compared with normal control group,12h～48h APSC2C12myoblast proliferation has significant effects, which12h～24h segment in time-effect relationship, the above comparisons were statistically significant (P<0.05); Dex on C2C12impact of myoblast proliferation rate:Compared with the normal group,100μM and1000μM Dex could significantly inhibit the proliferation of C2C12myoblasts, was statistically significant (P<0.05), and0.1μM,1μM,10μM Dex on C2C12myoblasts Cell proliferation had no effect; APS to improve the role of hydrogen peroxide-induced activity of C2C12myoblasts injury:Compared with the normal group, hydrogen peroxide,12h and24h after the intervention can reduce the activity of C2C12myoblasts, APS hydrogen peroxide intervention effect was inhibited, the above comparisons were statistically significant (P<0.05)Conclusion:APS at a certain concentration and time on the proliferation of C2C12myoblasts can promote and improve the hydrogen peroxide-induced damage C2C12myoblasts activity, Dex concentration on C2C12myoblast proliferation without affecting less than lOμM.Chapter3Anti-apoptotic and anti-oxidation effect of APS on C2C12myoblastsObjective:To observe the improvement APS hydrogen peroxide-induced apoptosis in C2C12muscle and into the OS, and explore its mechanism.Methods:The improvement was observed on the APS into C2C12cell apoptosis induced by hydrogen peroxide muscle:C2C12myoblasts cultured cells were divided into four groups, with culture medium containing200μM and500μM hydrogen peroxide, respectively, and0.2mg/mL APS intervention as a drug control group, with another two as the control group, after24h incubation C2C12myoblasts apoptosis detection status of each group with PI/Annexin V staining; explore the molecular mechanisms of the anti-apoptotic APS:The culture The C2C12myoblasts into hydrogen peroxide model group and APS intervention group, the amount of hydrogen peroxide in the model group is set to200μM, APS intervention group amount set0.2mg/mL, and normal control group and the control group APS incubated24h later by Western blot for each group Cleaved caspase8, Cleaved caspase3, B-cell lymphoma2gene related protein (Bcl-2), Bcl-2associated X protein (Bax) expression levels were detected; observed on ROS APS Effect:The cultured C2C12myoblasts into four groups containing200μM and500μM hydrogen peroxide culture medium, and were used to intervene as a pharmaceutical0.2mg/mL and0.5mg/mL APS control, a separate blank the control group, after30min incubation with DCFH-DA probe to detect ROS concentrations.Results:APS improvement of C2C12into hydrogen peroxide-induced apoptosis of muscle:Compared with normal control group, hydrogen peroxide200μM and500μM group of C2C12myoblasts apoptosis was significantly increased compared with the model group than, after the APS intervention can significantly reduce apoptosis, the above comparisons were statistically significant (P<0.05); explore the molecular mechanism of the anti-apoptotic APS:Compared with normal control group, model group, hydrogen peroxide, Cleaved caspase8, Cleaved caspase3, Bax expression was significantly increased, decreased Bcl-2expression. Relative to the untreated group, APS significantly reduced Cleaved caspase8, Cleaved caspase3, Bax expression, restored expression of Bcl-2, the above comparisons were statistically significant (P<0.05); impact on ROS APS:the normal control group Compared,200μM and500μM hydrogen peroxide group increased significantly ROS concentrations,0.2mg/mL and0.5mg/mL APS can effectively reduce ROS concentrations above were statistically significant (P<0.05)Conclusion:APS hydrogen peroxide-induced apoptosis in C2C12myoblasts and OS both have a role in improving the mechanism may be by regulating caspase8/ caspase3and two Bcl-2/Bax apoptosis pathway. |
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