【Abstract】 Objective To construct tissue engineered skeletal muscle in vivo using glial cell derived neurotrophic factor (GDNF) genetically modified myoblast (Mb) on acellular collagen sponge with hypoglossal nerve implantation, and to observe whether structural or functional connection could be established between engineered tissue and motor nerve or not. Methods Mbs were isolated from 7 male Lewis rats at age of 2 days, cultured and genetically modified by recombinant adenovirus carrying GDNF cDNA (MbGDNF). Calf skin-derived acellular collagen sponge was used as scaffold; cell adhesion was detected by scanning electron microscope after 24 hours. Hypoglossal nerve was implanted into Mb-scaffold complex (Mb group, n=27) or MbGDNF-scaffold complex (MbGDNF group, n=27) in 54 female Lewis rats at age of 8 weeks. HE staining was performed at 1, 6, and 12 weeks postoperatively, and immunohistochemistry staining and fluorescence in situ hybridization were used. Results MbGDNF could highly expressed GDNF gene. Mb and MbGDNF could adhere to the scaffold and grew well. HE staining showed tight junctions between implant and peripheral tissue with new muscle fiber and no distinguished line at 12 weeks in 2 groups. Immunohistochemistry staining showed that positive cells of myogenin and slow skeletal myosin were detected, as well as positive cells of actylcholine receptor α1 at 1, 6, and 12 weeks. The positive cells of Y chromosome decreased with time. At 1, 6, and 12 weeks, the positive neurons were 261.0 ± 6.6, 227.3 ± 8.5, and 173.3 ± 9.1, respectively in MbGDNF group, and were 234.7 ± 5.5, 196.0 ± 13.5, and 166.7 ± 11.7, respectively in Mb group; significant differences were found between 2 groups at 1 and 6 weeks (P lt; 0.05), no significant difference at 12 weeks (P gt; 0.05). Conclusion Connection can be established between engineered tissue and implanted hypoglossal nerve. Recombinant GDNF produced by MbGDNF might play a critical role in protecting central motor neurons from apoptosis by means of retrograde transportation.
To explore the role of cell apoptosis in denervated skeletal muscle atrophy in rats and the effect of losartan on it. Methods Forty-two Sprague Dawley rats were randomly divided into 3 groups: group I (n =14, normal control group), group II (n =14, denervated group) and group III (n =14, losartan group). The rats were not treated in group I, and were made denervated gastrocnemius models in groups II and III. In group III, the rats were treated with losartan 10 mg /kg• d by gavage and with normal sal ine in groups I and II. After 4 weeks, gastrocnemius mass to body mass ratio (GAS/BM) served as the degree of muscle atrophy. Apoptotic cells in gastrocnemius were stained in situ by using TUNEL. Gastrocnemius Bcl-2 and Bax protein were quantified by immunohistochemistry and Western blot. Bax /Bcl-2 served as the degree of apoptosis. Results The ratio of apoptosis was higher in group II than that in group I (11.32% ± 4.51% vs 0.56% ± 0.21%, P lt; 0.05). The ratio of apoptosis was lower in group III than that in group II (7.21% ± 2.05% vs 11.32% ± 4.51%, P lt; 0.05). The atrophy of skeletal muscle（GAS/BM） in group II was more serious than that in group I (11.68 ± 1.98 vs 12.86 ±0.74, P lt; 0.05), there was no significant difference between group III and group II (12.11 ± 0.65 vs 11.68 ± 1.98, P gt; 0.05). The expression of Bcl-2 in group II (18.3% ± 4.9%) was significantly lower than that in group I (27.5% ± 2.8%) and group III (25.5% ± 3.5%); there was no significant difference between group III and group I (P gt; 0.05). The expression of Bax in group II (24.1% ± 3.1%) was significantly higher than that in group I (22.1% ± 3.6%) and group III (21.7% ± 2.3%); there was no significant difference between group III and group I (P gt; 0.05). Western blot results showed that: the expressions of Bcl-2 were 122.5 ± 14.6 in group II, 135.3 ± 6.2 in group I and 139.2 ± 16.2 in group III; showing significant diffeerences between group II and group I, between group III and group II (P lt;0.05). The expressions of Bax were 107.1 ± 15.8 in group II, 89.3 ± 8.4 in group I, and 94.2 ± 9.5 in group III; showing significant diffeerences between group II and group I, between group III and group II (P lt; 0.05). There was no significant difference in the expression of Bcl-2 and Bax between group Ⅲ and group I (P gt; 0.05). Conclusion Cell apoptosis plays an important role in denervated skeletal muscle atrophy in rats and may be one of the factors causing skeletal muscle atrophy. Losarton can decrease skeletal muscle cell apoptosis through regulating the ratio of Bax / Bcl-2.
Objective To investigate the effect of bFGF on denervated skeletal muscle in accelerating muscle satell ite cell prol iferation, supplying neurotrophic factors and reducing muscle atrophy. Methods Twenty-eight Wistar male rats weredivided into the experimental group and the control group randomly, whose left lower l imb sciatic nerve was excised to make animal models of denervated skeletal muscle. The sil ia gel tubes containing 0.1 g bFGF and normal sal ine were implanted into gastrocnemius in the experimental and control groups, respectively. After 14 and 30 days of operation, gross appearance was observed; muscle wet weight and potential ampl itude of gastrocnemius fibrillation were measured; histological observation and electron microscope observation were made. Results At 14 and 30 days after operation, gastrocnemius atrophy and adhesion were more obvious in the control group than those in the experimental group. At 30 days after operation, the potential amplitude of gastrocnemius fibrillation and muscle wet weight were experimental group (0.220 6 ± 0.301 0) μm and (2.475 7 ± 0.254 6) g in the experimental group, and (0.155 2 ± 0.050 3) μm and (1.459 1 ± 0.642 5) g in the control group. There was a significant difference between two groups (P lt; 0.05). At 14 and 30 days after operation, HE staining showed more muscle satell ite cell nucleiin gastrocnemius of the experimental group than that of the control group; Mallory staining showed more blue connective tissues in the control group than in the experimental group; PCNA staining showed more PCNA positive cell nuclei in the experimental group than in the control group; and the AgNO3 staining testified more grains of vitamin C and less connective tissue proliferation in the experimental group than in the control group. At 30 days after operation, the fiber diameter and the fiber area were (66.368 6 ± 12.672 7) μm and (2 096.112 9 ± 311.563 9) μm2 in the experimental group, (55.504 0 ± 4.945 0) μm and (1 418.068 0 ± 264.953 7) μm2 in the control group. The PCNA positive cell nuclei number was 116.200 ± 5.357 in the experimental group and 53.000 ± 3.937 in the control group, showing statistically significant difference between the two groups (P lt; 0.05). At 14 and 30 day after operation, ompared with control group, the muscle fiber in the experimental group arrangedly more regularly and had lessatrophy fiber and the connective tissue proliferation. Conclusion bFGF can stimulate the proliferation of muscle satell ite cells in denervated gastrocnemius, delay the muscle fiber atrophy and inhibit connective tissues proliferation in muscle fibers.
Objective To investigate the delay of the denervated skeletal muscle atrophy with the method of restraining the increment of the connective tissues by tetrandrine and hormone. Methods The left hind limbs of 42 male adult SD rats were made into models of the denervated gastrocnemius, and then the rats were randomly divided into 3 groups, with 14 rats in each. In Group A, tetrandrine (8 mg/L）was injected into the denervated gastrocnemius; in Group B, triamcinolone acetonide（1.6 g/L） was injected; in Group C (the control group),normal saline was injected. Enough samples were obtained according to the different observation indexes at 30 days after operation. Electromyography, muscle wet weight measurement, light microscopy,electron microscopy,and microimage analysis were performed. ResultsThe fibrillation potential amplitude was 0.195 8±0.041 9 μV in Group A and 0.185 2±0.050 3 μV in Group B, and there was no significant difference betweenthe two groups (Pgt;0.05). However,in Group C the fibrillation potential amplitude was 0.137 7±0.058 9μV. The fibrillation potential amplitude was significantly greater in Group A than in Group C（Plt;0.05）. The muscle wet weight was 1.740 0±0.415 9 g in Group A and 1.940 1±0.389 4 gin Group B, and there was no significant difference between the two groups(Pgt;0.05).However, in Group C the muscle wet weight was 0.800 0±0.100 0 g. The muscle wet weight was significantly greater in Group A than in Group C（Plt;0.05）.The microscopy showed that more remarkable atrophy occurred in the control group. The muscle fibers were more complete, thicker and larger, with more nuclei and clearer cross-lines. More connective tissue and flat cells could be observed in Groups A and B. The myogenic protein amount was 440.124 2±46.135 6 in Group A and 476.211 4±41.668 8in Group B, and there was no significant difference between the two groups(Pgt;0.05).However, in Group C the amount was 380.040 0±86.315 9.The myogenic protein amount was significantly greater in Group A thanin Group C（Plt;0.05）. The muscle fiber number, diameter, cross section, and connective tissue increment were all significantly greater in Group A than in Group C（Plt;0.05）; however, there wasno significant difference between Groups A and B (Pgt;0.05). The electron microscopy showed that there were more degeneration changes, such as muscle silk disorder, chondriosome disappearance, and hepatin reduction, could be observed inGroup C than in Groups A and B. Conclusion Tetrandrine and hormone can delay the denervated skeletal muscle atrophy by restraining the increment of the connective tissues.
Skeletal muscle and metabolic function are important factors affecting the health status of the elderly. Branched-chain amino acids (BCAA) can improve muscle recovery, reduce muscle soreness after exercise, and BCAA can also enhance metabolic health, helping to regulate blood sugar levels and improve insulin sensitivity in the elderly. In addition, BCAA can improve cognitive function, reducing the risk of age-related cognitive decline. This article reviews the relationship between BCAA and aging, skeletal muscle, and metabolic diseases, explaining how BCAA can support and promote muscle mass and function in the elderly, as well as have a positive impact on metabolic health and cognitive function.
Objective To investigate the role of cysteinyl aspartate specific proteinase-3 (Caspase-3)/ gasdermin-E (GSDME)-mediated pyroptosis in skeletal muscle atrophy induced by cigarette smoke in mice.Methods To construct a mouse model of COPD, C57BL/6 mice were exposed to cigarette smoke (CS) for 24 weeks. HE staining was used to observe the changes in the morphology of the gastrocnemius muscle in mice. Immunohistochemistry was used to detect the expression of pyroptosis-related proteins in gastrocnemius muscle. To construct a model of skeletal muscle cell atrophy in vitro, C2C12 myoblasts were induced to differentiate into skeletal muscle cells with 2% horse serum, and then skeletal muscle cells were treated with cigarette smoke extract (CSE). Skeletal muscle cells were further treated with the caspase-3 inhibitor Z-DEVD-FMK and the GSDME inhibitor Dimethyl fumarate (DMF) to explore the effects of inhibition of caspase-3/GSDME on CSE-induced skeletal muscle cell atrophy. To observe the effects of TNF-α on the expression of caspase-3 and GSDME proteins as well as the impact on myotubes, skeletal muscle cells were stimulated with tumor necrosis factor-alpha (TNF-α). Western blotting was applied to detect protein expression levels of caspase-3 and GSDME in skeletal muscle cells. Hoechst 33342/ Hoechst33342/ Propidium Iodide (PI) staining was applied to detect the PI-positive rate of skeletal muscle cells. The lactate dehydrogenase (LDH) release of C2C12 myotubes was measured by LDH release test. Immunofluorescence was used to detect changes in myotube diameter. Results CS-induced skeletal muscle atrophy was observed in mice, accompanied by increased pyroptosis- associated proteins (c-caspase-3 and GSDME-N) (P<0.05). CSE also induced elevated c-caspase-3 and GSDME-N expression in C2C12 cells , resulting in increased LDH release, positive ratio of PI, along with reduced myotube diameter (P<0.05). In addition, TNF-α promotes myotube atrophy and the expression of cleaved-caspase-3 and GSDME-N proteins in skeletal muscle cells. ConclusionCS can induce skeletal muscle atrophy through activated TNF-α/Caspase-3/GSDME-mediated pyroptosis.