| Chronic renal failure (Chronic renal failure, CRF) patients is with progressive malnutrition, manifested as serum albumin, anemia, skeletal muscle atrophy, body protein reserves decline. China has about 20% to 50% of CRF patients with malnutrition. Malnutrition is closely associated with a poor prognosis, is one of the main causes of mortality in patients with CRF increased. Malnutrition is not only one of the most important complications of CRF, is closely related to other complications, such as harm to cardiovascular health, increasing the risk of infection, etc., seriously affecting the quality of life of patients. The etiology and pathogenesis of CRF malnutrition is complex. Reduced food intake, gastric mucosal damage caused by toxins, acidosis, endocrine disorders, dialysis missing protein, amino acids, inflammation and many other reasons can lead to malnutrition. At present, the major clinical use to treat CRF malnutrition is compound α- keto acid, but its price is more expensive, there are limitations in the clinical application. So, how cost-effective prevention and treatment of CRF malnutrition, clinical and medical research needed to solve the problem.As one of the key evaluation indicators, the loss of skeletal muscle mass and weight caused by skeletal muscle atrophy in CRF malnutrition, is the key point of this study. Root cause of skeletal muscle atrophy is metabolic imbalance of protein. Protein synthesis reduction and catabolism increased of various causes play a leading role in the CRF malnutrition. In terms of molecular biology, many signaling pathways are involved on the regulation of skeletal muscle atrophy and hypertrophy. Within metabolic acidosis, uremic toxins, micro-inflammatory status of skeletal muscle can be activated ubiquitin-proteasome (ubiquitin-proteasome pathway, UPP) pathway for protein degradation and muscle atrophy, and severe inhibition of protein synthesis pathway protein kinase B/rapamycin to protease (Akt/mTOR) signaling pathway significantly reduced. In addition to the reasons for the imbalance of protein metabolism, skeletal muscle atrophy in skeletal muscle regeneration process of the existence of damage repair mechanisms. Skeletal muscle satellite cells (skeleton satellite cell, MSC) activation, proliferation and differentiation of muscle regeneration played a decisive role. Under the CRF nutrition environment uremic toxins, metabolic and endocrine disorders, micro-inflammation, nutrient deficiency, such as MSC can inhibit the activation, growth and differentiation processes. And after the MSC death of long-term malnutrition there is non-renewable alternative. According to this change, many scholars believe that the number of MSC reduced or even completely exhausted is an important cause of chronic renal failure caused by malnutrition, muscle shrinking. Wnt7a can increase the amount of muscle tissue in the MSC, in turn, can accelerate the growth and repair of skeletal muscle, and through its receptor Fzd7, activation of protein kinase B/mammalian target of rapamycin (AKT/mTOR) signaling pathway, promoting myotube hypertrophy and increased muscle fibers. Moreover, skeleton muscle atrophy also reduces the number of muscle related nucleus, while the number of muscle cell nuclei is mainly lost by apoptosis pathway. Under the skeletal muscle contraction, protein expression of anti-apoptotic Bcl-2 down-regulated pro-apoptotic protein Bax, caspases increased expression, indicating that the catabolic state, via the mitochondrial apoptosis plays an important role. Autophagy is the cell’s own use of lysosomal degradation damaged cells and macromolecules process. Now it’s believed that cellular autophagy involved in the majority of the degradation of the long half-life proteins. Autophagy activation can lead to protein breakdown and muscle atrophy. Although a large number of studies have shown that intracellular protein synthesis pathway Wnt7a-Akt/mTOR signaling pathway impaired muscle cell apoptosis and autophagy is an important cause of increased skeletal muscle atrophy, but their role and mechanism of muscle atrophy in the CRF is still lacking study.CRF malnutrition leads to skeletal muscle atrophy, belonging to Chinese medicine, "Consumption", "Weizheng" and other areas. CRF pathogenesis based virtual reality, false is mixed. This virtual function that spleen and stomach, kidney deficiency, phlegm real standard for wet toxic cloud. Spleen is acquired, the source of qi and blood, "focus bullied juice changes in red, is that blood," Valley of the stomach by a water diet, transport and absorption through the spleen, will spend the transportation of refined material to generate the blood, so that sufficient to support the organs and meridians, limb bones, skin veins camp guard. In CRF, because of the spleen and stomach weakness, Mizutani subtle blocked qi and blood, phlegm from wet toxic cloud. Thus, CRF malnutrition disease located in the kidney, the key lies in the spleen. In the whole process of CRF malnutrition stomach disorders consistent. If the spleen and stomach function improved, the recovery operation of the absorption, after days of returning talent, so that blood biochemical active, kidney yuan resume, it will help to improve the nutritional status. Treatment on it, when supplementation and attack, both good and evil, treatment with spleen and kidney, nourishing blood, wet exhaust vent muddy, righting without leaving the evil, not to hurt the positive Quxie. Shenshuai Yingyang Capsule (SSYYJN) is Professor Wei according to many years of clinical experience in "Ginseng Yang Rong Tang" based on the screening of drug prescriptions, and by the scientific production technology development. It has determining efficacy in clinical applications for many years, for the prevention and treatment of malnutrition CRF. Clinical observations show that the prescription can enhance the patient’s appetite, increase plasma albumin levels, anemia, weight gain, improve the quality of life of patients, but its role in the treatment affect protein metabolism on the molecular mechanism of action is unclear, pending further the study.This issue observes the affects of SSYYJN to tumor necrosis factor-α (TNF-α) induced the formation of C2C12 mouse skeletal muscle cells atrophy model, detects Wnt7a-Akt/mTOR signaling pathway, apoptosis signaling pathway and autophagy signaling pathway in skeletal muscle C2C12 cells, and explore SSYYJN promote protein synthesis in skeletal muscle, antagonistic MSC damage, thereby improving the prevention of skeletal muscle contraction and related molecular mechanisms, and for SSYYJN prevention and treatment of CRF malnutrition and skeletal muscle atrophy of clinical applications, provide an objective basis for the theory test. As follows:Chapter 1 Effect of SSYYJN on C2C12 myotubes atrophy through Wnt7a-Akt/mTOR signalingObjective:To observe the protective effect of SSYYJN on TNF-a-induced C2C12 muscle myotubes atrophy and explore its mechanisms.Methods:First, with 2% horse serum high-density cultures of C2C12 skeletal muscle cells to differentiate into C2C12 myotubes. The cultured C2C12 myotubes were divided into 4 groups, one group for the normal serum group, the other three groups with medium containing 20 ng/mL TNF-a cultured skeletal muscle C2C12 myotubes established model of skeletal muscle atrophy, and are divided into:TNF-a model group, TNF-α+10% SSYYJN containing serum group, TNF-α+10% KA with serum containing group. Each group after 24h incubation, cells were fixed with eosin-hematoxylin (HE) staining method for each group, with 200 times the light microscope to take pictures of each group, to calculate the average diameter of each group myotubes with Image Pro-Plus software; and to detect the expression of p-Akt, p-mTOR, Wnt7a, frizzled-7 content by Western blot. Internal reference uses GAPDH.Results:Compared with the control group, myotubes average diameter significantly reduced in TNF-a model group, SSYYJN and KA interventions can improve the degree of reducement in diameter, were statistically significant between the groups (P<0.05), but there was no difference of muscle tube diameter between SSYYJN group and KA group; p-Akt, p-mTOR, were significantly or scattered expression in normal group, expression of p-Akt, p-mTOR, Wnt7a, frizzled-7 significantly reduced in TNF-a model group, while the SSYYJN and KA can restore the abnormal signal factor expression of the model group and were statistically significant between the groups (P<0.05), but p-Akt, p-mTOR, Wnt7a, frizzled-7 expression levels did not differ between the SSYYJN and KA groups.Conclusion:TNF-a decreases skeletal muscle contraction and muscle tube diameter, Wnt7a-Akt/mTOR signaling pathway was significantly inhibited. SSYYJN can effectively recover Wnt7a-Akt/mTOR signal, increasing muscular tube diameter, thereby improving skeletal muscle contraction.Chapter 2 Effect of SSYYJN on C2C12 myoblasts through Bcl-2/Bax apoptotic pathwayObjective:To observe the effect of SSYYJN to TNF-α-induced apoptosis in C2C12 myoblasts, and to explore its mechanisms.Methods:The cultured C2C12 myoblasts divided into four groups, one group was normal serum group, the other three groups containing 20 ng/mL TNF-a in the culture medium, and distributed:TNF-α model group, TNF-α+10% SSYYJN containing serum group, TNF-α+10% KA serum containing group. After 24h incubation with Hoechst 33342 staining C2C12 myoblast apoptosis of each group; for each group Cleaved caspase 3, B-cell lymphoma 2 gene related proteins (Bcl-2), Bcl-2 associated X protein (Bax) expression levels were detected by Western blot.Results:Compared with the control group, TNF-a model group the apoptosis rate of C2C12 muscle cells increased significantly, compared with the TNF-a model group, after SSYYJN and KA intervention can significantly reduce the apoptosis, the above comparisons were statistically significant (P<0.05); Compared with normal control group, the expression of Cleaved caspase 3, Bax significant increase in TNF-a model group, while Bcl-2 expression reduction. Relative to the TNF-a model group, expression of Cleaved caspase 3, Bax significantly reduced, Bcl-2 expression levels recover in SSYYJN and KA group, the above comparisons were statistically significant (P<0.05).Conclusion:C2C12 cell apoptosis induced by TNF-α can be reduced by SSYYJN, its mechanism may be through regulating Bcl-2/Bax apoptotic pathway.Chapter 3 Effect of SSYYJN on C2C12 myoblasts autophagyObjective:To observe the inhibitory effect of SSYYJN on TNF-α-induced autophagy in C2C12 myoblasts and explore its mechanisms.Methods:The cultured C2C12 myoblasts divided into four groups, one group was normal serum group, the other three groups containing 20 ng/mL TNF-a in the culture medium, and distributed:TNF-α model group, TNF-α+10% SSYYJN containing serum group, TNF-α+10% KA with serum containing group. After 24h incubation to detect the C2C12 myoblasts autophagy situation with transmission electron microscopy; for each group LC3B-Ⅰ, LC3B-Ⅱ, beclin 1-expression levels were detected by Western blot.Results:Compared with the control group, the rate of autophagy in TNF-α model group of C2C12 myoblasts significantly increased; compared with the TNF-α model group, the SSYYJN and KA intervention can significantly reduce autophagy, the above comparisons were statistically significant (P<0.05); Compared with normal control group, LC3B-Ⅱ/Ⅰ, beclin-1 expression was significantly increased in the TNF-α model group; relative to the TNF-α mode group, the SSYYJN and KA group significantly reduced LC3B-II/I, beclin-1 expression, the above comparisons were statistically significant (P<0.05).Conclusion:SSYYJN inhibited TNF-a-induced C2C12 myoblasts autophagy, the mechanism may be regulate autophagy related protein levels. |
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