Rat Skeletal Muscle Research Articles

Metformin Protects Rat Skeletal Muscle from Physical Exercise-Induced Injury.

Metformin (Met) is a drug commonly prescribed in type 2 diabetes mellitus. Its efficacy is due to the suppression of hepatic gluconeogenesis, enhancement of peripheral glucose uptake and lower glucose absorption by the intestine. Recent studies have reported Met efficacy in other clinical applications, such as age-related diseases. Despite the wide clinical use of Met, its mechanism of action on muscle and its effect on muscle performance are unclear. We investigated the effects of Met combined with training on physical performance (PP) in healthy rats receiving Met for 8 weeks while undergoing daily moderate exercise. We evaluated the following: PP through graded endurance exercise test performed before the beginning of the training protocol and 48 h before the end of the training period; blood ALT, AST, LDH and CK-MB levels in order to address muscle damage; and several blood and muscle myokines and the expression of factors believed to be involved in muscle adaptation to exercise. Our data demonstrate that Met does not improve the positive effects of exercise on performance, although it protects myocytes from exercise-induced damage. Moreover, given that Met positively affects exercise-induced muscle adaptation, our data support the idea of the therapeutic application of Met when muscle function and structure are compromised.

Exploring the optimal impact force for chronic skeletal muscle injury induced by drop-mass technique in rats.

Introduction: Skeletal muscle injuries are widespread in sports, traffic accidents and natural disasters and some of them with poor prognoses can lead to chronic skeletal muscle damage in the clinic. We induced a chronic skeletal muscle injury by controlling time and contusion force using an acute blunt trauma model that will help us better comprehend the pathological features of chronic skeletal muscle injury. Methods: Several levels of injury were induced by repeatedly striking in 5, 10, and 15 times the gastrocnemius muscle from the same height with 200g weights. After injury, the markers of muscle injury were assessed at 2 and 4 weeks by serum elisa. Electron microscopy, histologic and immunohistochemical staining, and mRNA analysis were used to evaluate the ultrastructure, inflammation, extracellular matrix decomposition, and anabolism of injured muscle in 2 and 4 weeks. Results: All three different kinetic energies can result in skeletal muscle injuries. However, the injured skeletal muscles of rats in each group could not recover within 2 weeks. After 4 weeks, tissue self-repair and reconstruction caused the damage induced by 5J kinetic energy to almost return to normal. In contrast, damage induced by 10J kinetic energy displayed slight improvement compared to that at 2 weeks. Despite this, collagen fibers on the surface of the tissue were disorganized, directionally ambiguous, and intertwined with each other. Myofilaments within the tissue were also arranged disorderly, with blurry and broken Z-lines. Damage caused by 15J kinetic energy was the most severe and displayed no improvements at 4 weeks compared to 2 weeks. At 4 weeks, IL-1β, IL-6, Collagen I, and Collagen III, MMP2 expressions in the 10J group were lower than those at 2 weeks, showing a tendency towards injury stabilization. Conclusion: After 4 weeks of remodeling and repair, the acute skeletal muscle injury model induced by 10J kinetic energy can stabilize pathological manifestations, inflammatory expression, and extracellular matrix synthesis and catabolism, making it an appropriate model for studying chronic skeletal muscle injuries caused by acute injury.

Diabetes diminishes muscle precursor cell-mediated microvascular angiogenesis.

The skeletal muscles of Type II diabetic (T2D) patients can be characterized by a reduced vessel density, corresponding to deficiencies in microvascular angiogenesis. Interestingly, T2D also inhibits the function of many myogenic cells resident within skeletal muscle, including satellite cells, which are well-known for the role they play in maintaining homeostasis. The current study was undertaken to gain a better understanding of the mechanisms whereby satellite cell progeny, muscle precursor cells (MPCs), influence microvascular angiogenesis. Network growth and the expression of genes associated with angiogenesis were reduced when microvessels were treated with conditioned media generated by proliferating MPCs isolated from diabetic, as compared to control rat skeletal muscle, a phenomenon that was also observed when myoblasts from control or diabetic human skeletal muscle were used. When only exosomes derived from diabetic or control MPCs were used to treat microvessels, no differences in microvascular growth were observed. An evaluation of the angiogenesis factors in control and diabetic MPCs revealed differences in Leptin, vascular endothelial growth factor (VEGF), IL1-β, interleukin 10, and IP-10, and an evaluation of the MPC secretome revealed differences in interleukin 6, MCP-1, VEGF, and interleukin 4 exist. Angiogenesis was also reduced in tissue-engineered skeletal muscles (TE-SkM) containing microvessels when they were generated from MPCs isolated from diabetic as compared to control skeletal muscle. Lastly, the secretome of injured control, but not diabetic, TE-SkM was able to increase VEGF and increase microvascular angiogenesis. This comprehensive analysis of the interaction between MPCs and microvessels in the context of diabetes points to an area for alleviating the deleterious effects of diabetes on skeletal muscle.

Nimbin (N1) and analog N3 from the neem seeds suppress the migration of osteosarcoma MG-63 cells and arrest the cells in a quiescent state mediated via activation of the caspase-modulated apoptotic pathway.

Natural products are considered effective sources for new therapeutic research and development. The numerous therapeutic properties of natural substances in traditional medicine compel us to investigate the anti-cancer properties of Nimbin (N1) and its semi-natural analog Nimbic acid (N3) from Azadirachta indica against MG-63 Osteosarcoma cells. The therapeutic efficacy of N1 and N3 were screened for their toxicity and cytotoxic activity using L6 myotubes, zebrafish larvae and MG-63 osteosarcoma cells. The mitochondrial membrane potential was evaluated using the Rhodamine 123 stain. Further, the nuclear and cellular damage was distinguished using Hoechst and Acridine orange/EtBr stain. The mechanism of cell cycle progression, cellular proliferation and caspase cascade activation was screened using scratch assay, flow cytometry, and mRNA expression analysis. The Nimbin and analogue N3 were found to be non-toxic to normal L6 cells (Rat skeletal muscles), exhibited cytotoxicity in MG-63 cells, and were exposed to be an active inhibitor of cell proliferation and migration. Analogs N1 and N3 induced negative mitochondrial membrane potential when stained with Rhodamine 123, leading to nuclear damage and apoptosis stimulation using AO/EtBr and Hoechst. Further, N1 and N3 induced cell cycle arrest in G0/G1 phase in flow cytometry using PI staining and induced apoptosis by activating the caspase cascade and upregulated Caspase 3 and caspase 9. The study demonstrated cytotoxic activity against MG-63 osteosarcoma cells while being non-toxic to normal L6 cells. These compounds inhibited cell proliferation and migration, induced mitochondrial dysfunction, nuclear damage, and apoptosis stimulation. Furthermore, N1 and N3 caused cell cycle arrest and activated the caspase cascade, ultimately leading to apoptosis. These findings indicate that N1 and N3 hold promise as potential candidates used alone or combined with existing drugs for further investigation and development as anti-cancer agents.

Age-related gene expression signatures from limb skeletal muscles and the diaphragm in mice and rats reveal common and species-specific changes

BackgroundAs a result of aging, skeletal muscle undergoes atrophy and a decrease in function. This age-related skeletal muscle weakness is known as “sarcopenia”. Sarcopenia is part of the frailty observed in humans. In order to discover treatments for sarcopenia, it is necessary to determine appropriate preclinical models and the genes and signaling pathways that change with age in these models.Methods and resultsTo understand the changes in gene expression that occur as a result of aging in skeletal muscles, we generated a multi-time-point gene expression signature throughout the lifespan of mice and rats, as these are the most commonly used species in preclinical research and intervention testing. Gastrocnemius, tibialis anterior, soleus, and diaphragm muscles from male and female C57Bl/6J mice and male Sprague Dawley rats were analyzed at ages 6, 12, 18, 21, 24, and 27 months, plus an additional 9-month group was used for rats. More age-related genes were identified in rat skeletal muscles compared with mice; this was consistent with the finding that rat muscles undergo more robust age-related decline in mass. In both species, pathways associated with innate immunity and inflammation linearly increased with age. Pathways linked with extracellular matrix remodeling were also universally downregulated. Interestingly, late downregulated pathways were exclusively found in the rat limb muscles and these were linked to metabolism and mitochondrial respiration; this was not seen in the mouse.ConclusionsThis extensive, side-by-side transcriptomic profiling shows that the skeletal muscle in rats is impacted more by aging compared with mice, and the pattern of decline in the rat may be more representative of the human. The observed changes point to potential therapeutic interventions to avoid age-related decline in skeletal muscle function.

LCMS Determination and Cytotoxicity of Abrus precatorius on L6 and SK-N-MC Cell Lines.

The present study aimed to investigate the cytotoxic effect of various extracts derived from Abrus precatorius Linn. leaves on rat L6 and human SK-N-MC neuroblastoma cell lines and determine the secondary metabolites responsible for the cytotoxicity of Abrus precatorius. Successive solvent extraction of A.precatorius leaves was carried out using the Soxhlet apparatus with solvents such as petroleum ether, chloroform, ethyl acetate, and ethanol. HPTLC fingerprinting and LC-MS studies were performed to assess the presence of secondary metabolites, such as flavonoids and phenols, in the ethyl acetate extract. Furthermore, the cytotoxic effect of extracts was tested on rat skeletal muscle cell line L6 and human neuroblastoma cell line SK-N-MC using MTT assay. The total phenolic content of ethyl acetate and ethanol extracts of A.precatorius were 72.67 and 60.73 mg, respectively, of GAE/g dry weight of the extract. The total flavonoid content of ethyl acetate and ethanol extract of A.precatorius were 107.33 and 40.66 mg of Quercetin equivalents/g dry weight of the extract. LCMS analysis demonstrated that the flavonoids in specific Naringenin, Diosmetin, Glycitin, and Genistein might play a prominent role in the cytotoxicity of A.precatorius. The cytotoxicity study revealed that the extracts of A.precatorius were non-toxic to rat L6 myotubes, and the IC50 values of the various extracts, such as APPE, APCH, APEA, and APET, were >100 µg/ml. The extracts exhibited cytotoxic activity against human neuroblastoma SK-N-MC cells, and the IC50 values of APPE, APCH, APEA, APET, and the standard drug "Cisplatin" were >100, >100, 64.88, >100, and 3.72 µg/ml, respectively. It was concluded from the study that the extracts of Abrus precatorius were cytotoxic to neuroblastoma cell lines but non-toxic to normal cell lines. HPTLC and LC-MS studies confirmed that flavonoids in the ethyl acetate extract could be responsible for the biological activity.

Effects of voluntary running on the skeletal muscle of rats with pulmonary artery hypertension.

The effects of voluntary running on the skeletal muscle of rats with pulmonary arterial hypertension (PAH) were tested in the present study. PAH was induced in rats by a single injection of monocrotaline (MCT, 60mg/kg). Rats in the sedentary hypertension (HS) group had their tolerance to physical exertion reduced throughout the experiment, while those in the sedentary control (SC), exercise control (EC), exercise hypertension (EH) and median exercise (EM) groups maintained or increased. Despite that, the muscular citrate synthase activity was not different between groups. The survival time was higher in the EH (32days) than in the SH (28days) (p = 0.0032). SH and EH groups showed a lower percentage of muscle fiber and a higher percentage of extracellular matrix compared to control groups (p < 0.0001). However, the EM and EH groups presented higher percentage of muscle fiber and lower percentage of extracellular matrix than SH group (p < 0.0001). Regarding muscular gene expression, the SH and EM groups showed a lower expression of PGC1-α (p = 0.0024) and a higher expression of VEGF (p = 0.0033) compared to SC, while PGC1-α was elevated in the EH. No difference between groups was found for the carbonylated protein levels (p > 0.05), while the TNF-α/IL-10 ratio was augmented in the EH (p = 0.0277). In conclusion, voluntary running augments the proportion of fiber and affects the gene expression of inflammatory and mitochondrial biogenesis' markers in the skeletal muscle of rats with MCT-induced PAH, which benefits their survival and tolerance to physical effort.

A Prochlorperazine-Induced Decrease in Autonomous Muscle Activity during Hindlimb Unloading Is Accompanied by Preserved Slow Myosin mRNA Expression.

Skeletal muscle disuse leads to pathological muscle activity as well as to slow-to-fast fiber-type transformation. Fast-type fibers are more fatigable than slow-type, so this transformation leads to a decline in muscle function. Prochlorperazine injections previously were shown to attenuate autonomous rat soleus muscle electrical activity under unloading conditions. In this study, we found that prochlorperazine blocks slow-to-fast fiber-type transformation in disused skeletal muscles of rats, possibly through affecting calcium and ROS-related signaling.

588-P: Probing the Role of Muscle Glycogen Resynthesis on Postexercise AMPK Activation and Metabolic Protein Abundance Using a Novel Genetic Model

Acute exercise can alter the abundance of key metabolic proteins in skeletal muscle. These effects are influenced by postexercise (PEX) refeeding. Muscle glycogen resynthesis is a putative mediator of these refeeding effects. However, it is challenging to test this idea, because glycogen resynthesis is only one of many outcomes with refeeding. Therefore, we devised a novel genetic model to modify muscle expression of glycogen synthase 1 (GS1) using injection of rat skeletal muscle with adeno-associated viral (AAV) vectors for short hairpin RNA targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Rats were sedentary (SED) or exercised (120min swim-exercise). Muscles were collected immediately postexercise (IPEX) and 9-hours PEX (9hPEX), along with time-matched SED controls. The 9hPEX and their SED-control rats were either refed (RF) or not-refed (NRF). Muscles were analyzed for glycogen and phosphorylation and abundance of key metabolic proteins. Muscles injected with shRNA-GS1 vs shRNA-Scr had lower GS1 abundance in all groups. Regardless of genotype, in IPEX vs SED rats: glycogen fell to similarly low values and phosphorylation of AMPK Thr172 (pAMPK) and its substrate ACC Ser79 (pACC) were comparably increased. Muscle glycogen was increased in all groups of 9h-RF rats, but it was lower for shRNA-GS1 vs shRNA-Scr from RF-9hPEX rats. In the NRF-9hPEX rats, pAMPK and pACC were greater for shRNA-GS1 vs shRNA-Scr muscles. Muscle pyruvate dehydrogenase kinase-4 abundance from 9hPEX rats (both NRF and RF) was greater for shRNA-GS1 vs shRNA-Scr. Hexokinase II (HKII) abundance was greater for 9hPEX vs SED in NRF rats regardless of genotype. In RF-9hPEX rats, HKII abundance was greater for shRNA-GS1 vs shRNA-Scr. These results support the idea that some PEX effects on metabolic protein abundance are influenced by muscle GS1 abundance and the extent of glycogen resynthesis Disclosure S.Kwak: None. A.Zheng: None. E.B.Arias: None. H.Wang: None. X.Pan: None. D.Duan: None. Y.Yue: None. G.D.Cartee: None. Funding National Institutes of Health (R01DK071771); University of Michigan

243-LB: C5a Receptor (C5aR1) Antagonism Ameliorates Saturated Fatty Acid–Induced Mitochondrial Dysfunction in Skeletal Muscle Cells

The complement system is an integral component of the innate immune response and plays a vital role in adaptive immunity. Activation of the system produces peptide fragments, such as C5a, that target G-protein coupled receptors expressed on immune-related cells, such as macrophages, that initiate pro-inflammatory responses. We have discovered that the C5a receptor 1 (C5aR1) is also expressed in skeletal muscle, a primary insulin target tissue. Sustained exposure of muscle cells to palmitate (PA), a saturated fatty acid, or C5aR1 activation is associated with impaired insulin sensitivity. We have studied the role that C5aR1 activation plays in in fatty-acid induced insulin resistance by exploring whether targeted receptor antagonism mitigates PA-induced mitochondrial dysfunction and insulin resistance. Mitochondrial health in rat skeletal muscle cells were examined by assessing mitochondrial morphology and mitophagy via microscopy, and cellular respiration using the XF24 seahorse flux analyser, following overnight incubation with PA in the absence/presence of C5aR1 antagonists. C5aR1, which was detected in mouse gastrocnemius muscle by immunoblot, increased in expression, in parallel with circulating levels of C5a, the peptide agonist for C5aR1, during a high-fat diet (HFD). PA treatment of rat L6 muscle cells suppresses insulin sensitivity as indicated by a reduction in PKB308 phosphorylation and glucose uptake. PA also reduced cellular mitochondrial respiration and promoted mitochondrial fragmentation and mitophagy. C5aR1 antagonism mitigated the PA dependent loss in insulin sensitivity, mitochondrial respiration and restrained mitochondrial fragmentation and the rates of mitophagy. Elevated C5aR1 activity, as might be observed during a HFD, may promote metabolic dysfunction in skeletal muscle, the antagonism of which could have therapeutic implications for mitigating HFD mediated insulin resistance. Disclosure D. S. Shah: None. A. D. Mcneilly: None. R. J. Mccrimmon: Advisory Panel; Sanofi, Speaker's Bureau; Novo Nordisk A/S. H. Hundal: None. Funding Diabetes UK

Chronic repetitive cooling and caffeine-induced intracellular Ca2+ elevation differentially impact adaptations in slow- and fast-twitch rat skeletal muscles.

Intracellular Ca2+ concentration ([Ca2+]i) is considered important in the regulation of skeletal muscle mass. This study tested the hypothesis that chronic repeated cooling and/or caffeine ingestion would acutely increase [Ca2+]i and hypertrophy muscles potentially in a fiber-type-dependent manner. Control rats and those fed caffeine were subjected to repeated bidiurnal treatments of percutaneous icing, under anesthesia, to reduce the muscle temperature below ∼5°C. The predominantly fast-twitch tibialis anterior (TA) and slow-twitch soleus (SOL) muscles were evaluated after 28 days of intervention. The [Ca2+]i elevating response to icing was enhanced by caffeine loading only in the SOL muscle, with the response present across a significantly higher temperature range than in the TA muscle under caffeine-loading conditions. In both the TA and SOL muscles, myofiber cross-sectional area (CSA) was decreased by chronic caffeine treatment (mean reductions of 10.5% and 20.4%, respectively). However, in the TA, but not the SOL, CSA was restored by icing (+15.4 ± 4.3% vs. noniced, P < 0.01). In the SOL, but not TA, icing + caffeine increased myofiber number (20.5 ± 6.7%, P < 0.05) and satellite cell density (2.5 ± 0.3-fold) in cross sections. These contrasting muscle responses to cooling and caffeine may reflect fiber-type-specific [Ca2+]i responses and/or differential responses to elevated [Ca2+]i.

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging.TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found.In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

GLP‑1 receptor agonist protects palmitate-induced insulin resistance in skeletal muscle cells by up-regulating sestrin2 to promote autophagy

In this study, we aimed to determine whether liraglutide could effectively reduce insulin resistance (IR) by regulating Sestrin2 (SESN2) expression in L6 rat skeletal muscle cells by examining its interactions with SESN2, autophagy, and IR. L6 cells were incubated with liraglutide (10–1000 nM) in the presence of palmitate (PA; 0.6 mM), and cell viability was detected using the cell counting kit-8 (CCK-8) assay. IR-related and autophagy-related proteins were detected using western blotting, and IR and autophagy-related genes were analyzed using quantitative real-time polymerase chain reaction. Silencing SESN2 was used to inhibit the activities of SESN2. A reduction in insulin-stimulated glucose uptake was observed in PA-treated L6 cells, confirming IR. Meanwhile, PA decreased the levels of GLUT4 and phosphorylation of Akt and affected SESN2 expression. Further investigation revealed that autophagic activity decreased following PA treatment, but that liraglutide reversed this PA-induced reduction in autophagic activity. Additionally, silencing SESN2 inhibited the ability of liraglutide to up-regulate the expression of IR-related proteins and activate autophagy signals. In summary, the data showed that liraglutide improved PA-induced IR in L6 myotubes by increasing autophagy mediated by SESN2.

The Relationship of p62 Gene Expression with Integrity of Mitochondrial DNA and the Level of Lipid Peroxidation Products in Skeletal Muscles of Rats of Different Ages Exposed to Different Feeding Protocols.

Sequestosome-1 (SQSTM1/p62) is one of the most important multifunctional proteins, which is necessary to maintain mitochondrial stability by eliminating damaged mitochondria through mitophagy. We studied the influence of age and diet on the expression of the p62 gene in the femoral and abdominal muscles of rats, as well as the integrity of some mitochondrial components. In the femoral muscles of 24-month-old rats receiving restricted ration, the expression of the p62 gene increased. We assume that activation of mitophagy contributed to a decrease in the levels of oxidative damage to mitochondrial DNA and LPO intensity in the femoral muscles of 24-month-old rats.

Using Multiscale Simulations as a Tool to Interpret Equatorial X-ray Fiber Diffraction Patterns from Skeletal Muscle.

Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of striated muscle under physiological conditions and on millisecond time scales. The lack of generally applicable computational tools for modeling X-ray diffraction patterns from intact muscles has been a significant barrier to exploiting the full potential of this technique. Here, we report a novel "forward problem" approach using the spatially explicit computational simulation platform MUSICO to predict equatorial small-angle X-ray diffraction patterns and the force output simultaneously from resting and isometrically contracting rat skeletal muscle that can be compared to experimental data. The simulation generates families of thick-thin filament repeating units, each with their individually predicted occupancies of different populations of active and inactive myosin heads that can be used to generate 2D-projected electron density models based on known Protein Data Bank structures. We show how, by adjusting only a few selected parameters, we can achieve a good correspondence between experimental and predicted X-ray intensities. The developments presented here demonstrate the feasibility of combining X-ray diffraction and spatially explicit modeling to form a powerful hypothesis-generating tool that can be used to motivate experiments that can reveal emergent properties of muscle.

Fish oil supplementation during pregnancy attenuates sex-specific changes induced by perinatal maternal high-fat diet in mitochondria of rat skeletal muscle at weaning

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods.

To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods. Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis. The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples. The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

Nano zinc Oxide-Ruthenium (III) complex of novel coumarin derivative: Synthesis, Characterization, DNA Cleavage, anticancer and bioimaging activities

The uniqueness of nanofunctionalized metal complexes would play a prominent role in medicinal chemistry, especially in cancer chemotherapy. Among the metal based chemotherapeutic drugs, ruthenium complexes exhibit different oxidation states, lower toxicity and remarkable antitumor activities than other anticancer drugs. In this investigation bioactive ruthenium(III) complex has been synthesized from novel coumarin derivative and it is functionalized with nanostructured zinc oxide (ZnO) under well defined condition. An octahedral geometry is proposed for the ruthenium complex based on analytical and spectrochemical characterization techniques (UV, FT-IR and NMR). The g|| and g⊥ values obtained from the ESR spectrum indicated high symmetry and octahedral field around ruthenium ion. The XRD patterns of all synthesized compounds explicit average particle sizes are nanometer range. The morphology and particle size of Nano ZnO-Ru(III) complex is also confirmed by using SEM and TEM analysis. The nanofunctionalized material exhibited enhanced fluorescence emissions at 674 nm and 681 nm. The evaluation of DNA cleavage study indicated compounds were effectively cleaved supercoiled pUC18 DNA by gel electrophoresis method. The examinations of in-vitro anticancer activities of compounds were studied against human breast (MCF-7) and leukemia (K-562) cancer cell lines. The ruthenium complex showed greater effect against MCF-7 with < 10 IC50 value. Nowadays, the cell imaging property of nanofunctionalized materials receive more attention. The fluorescence microscopic images of Nano ZnO-Ru(III) complex displayed higher luminescence at 100 μg/ mL against L6 rat skeletal muscle cell line.

Study of changes in the activity of glutathione system enzymes in the blood and liver in experimentally induced dyslipoproteinemia on the background of mechanical damage to the skeletal muscle in rats of different age groups

Currently, there is a steady increase in morbidity and mortality from acute cerebrovascular conditions, which are based on vascular ischemic manifestations, which are systemic in nature. At the heart of the root cause of any ischemic process – heart attack, stroke, hypertension – is atherosclerosis and dyslipoproteinemia. One of the biomarkers of this pathological process is oxidative stress.The purpose of the study: to analyze the dynamics of the activity of glutathione peroxidase and glutathione reductase in the blood and liver of rats of different ages under oxidative stress caused by modeling dyslipoproteinemia followed by mechanical injury to the skeletal muscle.Materials and methods. The study was conducted on outbred healthy male rats, divided into two groups according to age: the first group (c) – 24-month-old (old) rats, weighing 230–250 grams in the amount of 120 pieces and the second group (m) – 6-month-old (young) rats, weighing 190–210 grams in the amount of 120 pieces, kept under standard vivarium conditions. All rats were divided into four subgroups within their group, 30 animals each. Animals of the first subgroup in each group (1s, 1m) are intact (old and young) animals, to which no effects were applied. Animals of the second subgroup (2c, 2m) (regeneration control, old and young) had a mechanical dissection in the region of the middle third of the gastrocnemius muscle of the hind limb. Animals of the first and second subgroups were on a standard vivarium diet and had free access to water and food. Animals of the third (3s, 3m) and fourth (4s, 4m) subgroups of each group were subjected to dyslipoproteinemia modeling for 63 days and received a high-carbohydrate and high-fat diet with an increased fat content of up to 30% by weight of dry matter and replacement of drinking water with a 20% fructose solution. After the specified time, the animals of the fourth subgroups of both groups were injured in the middle third of the gastrocnemius muscle of the hind limb.Conclusions. Against the background of dyslipoproteinemia, oxidative stress occurs in the tissues of young and old rats, which is reflected by a decrease in the activity of enzymes of the glutathione system. Mechanical injury of the calf muscle in combination with dyslipoproteinemia aggravates oxidative processes against the background of inflammation and leads to an even more intense decrease in the activity of glutathione peroxidase and glutathione reductase in the liver and blood tissues of rats, and in old animals these processes are more pronounced than in young ones.

L-carnitine prevents lenvatinib-induced muscle toxicity without impairment of the anti-angiogenic efficacy.

Lenvatinib is an oral tyrosine kinase inhibitor that acts on multiple receptors involved in angiogenesis. Lenvatinib is a standard agent for the treatment of several types of advanced cancers; however, it frequently causes muscle-related adverse reactions. Our previous study revealed that lenvatinib treatment reduced carnitine content and the expression of carnitine-related and oxidative phosphorylation (OXPHOS) proteins in the skeletal muscle of rats. Therefore, this study aimed to evaluate the effects of L-carnitine on myotoxic and anti-angiogenic actions of lenvatinib. Co-administration of L-carnitine in rats treated with lenvatinib for 2weeks completely prevented the decrease in carnitine content and expression levels of carnitine-related and OXPHOS proteins, including carnitine/organic cation transporter 2, in the skeletal muscle. Moreover, L-carnitine counteracted lenvatinib-induced protein synthesis inhibition, mitochondrial dysfunction, and cell toxicity in C2C12 myocytes. In contrast, L-carnitine had no influence on either lenvatinib-induced inhibition of vascular endothelial growth factor receptor 2 phosphorylation in human umbilical vein endothelial cells or angiogenesis in endothelial tube formation and mouse aortic ring assays. These results suggest that L-carnitine supplementation could prevent lenvatinib-induced muscle toxicity without diminishing its antineoplastic activity, although further clinical studies are needed to validate these findings.