OBJECTIVES: To investigate the protective effects of dexmedetomidine (DEX) against heat stress (HS)-induced oncosis in human skeletal muscle cells (HSKMCs) and its underlying mechanisms. METHODS: A HSKMC model of HS-induced oncosis were established by 43 ℃ water bath for 4 h, and the effects of treatments with 30 μmol/L DEX, ML385 (a Nrf2 inhibitor) +DEX, si-Nrf2+HS, and si-Nrf2+DEX prior to modeling on cell viability was assessed using CCK-8 assay. Oncosis characteristics were evaluated using transmission electron microscopy and Annexin V-FITC/PI flow cytometry. The oxidative stress markers (GSH, GSH-Px, MDA, SOD and ROS), mitochondrial membrane potential, energy metabolism, and inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the cells were quantified using standard kits, and the expressions of porimin, caspase-3 and Nrf2 pathway proteins were analyzed using Western blotting and qRT-PCR. RESULTS: HS induced typical oncotic features in HSKMCs including organelle swelling and cytoplasmic vacuolization. DEX pretreatment significantly attenuated these changes, reduced Annexin V⁺/PI⁺ cell ratio and cellular porimin expression, and lowered the levels of ROS and MDA while restoring GSH and SOD levels. DEX pretreatment also significantly increased the mitochondrial membrane potential and ATP level, upregulated the expressions of Nrf2, p-Nrf2, HO-1 and NQO1, and suppressed the expressions of TNF-α, IL-6 and IL-1β. The protective effects of DEX were obviously attenuated by interventions with ML385 or si-Nrf2. CONCLUSIONS DEX mitigates HS-induced HSKMC oncosis by activating the Nrf2/HO-1 pathway to relieve oxidative stress, mitochondrial dysfunction, and inflammatory responses.
Humans ; Dexmedetomidine/pharmacology* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress/drug effects* ; Heat-Shock Response/drug effects* ; Signal Transduction/drug effects* ; Membrane Potential, Mitochondrial ; Muscle, Skeletal/cytology* ; Heme Oxygenase-1/metabolism* ; Apoptosis/drug effects*

The purpose of this study is to construct a muscle-specific synthetic promoter library, screen out muscle-specific promoters with high activity, analyze the relationship between element composition and activity of highly active promoters, and provide a theoretical basis for artificial synthesis of promoters. In this study, 19 promoter fragments derived from muscle-specific elements, conserved elements, and viral regulatory sequences were selected and randomLy connected to construct a muscle-specific synthetic promoter library. The luciferase plasmids pCMV-Luc and pSPs-Luc were constructed and transfected into the myoblast cell line C2C12. The activities of the synthesized promoters were evaluated by the luciferase activity assay. Two non-muscle-derived cell lines HeLa and 3T3 were used to verify the muscle specificity of the highly active promoters. The sequences of promoters with high activity, good muscle specificity, and correct sequences were analyzed to explore the relationship between the element composition and activity of promoters. We successfully constructed a muscle-specific promoter library and screened out 321 effective synthetic promoter plasmids. Among them, the activity of SP-301 promoter was 5.63 times that of CMV. The 15 promoters with high activity were muscle-specific. In the promoters with high activity and correct sequences, there was a relationship between their element composition and activity. Muscle-specific elements accounted for a high proportion in the promoters, while they had weak correlations with the promoter activity, being tissue-specific determinants. Viral elements accounted for no less than 20% in highly active promoters, which may be the key elements for the promoter activity. The content of conserved elements was proportional to the promoter activity. This study lays a theoretical foundation for the synthesis of tissue-specific efficient promoters and provides a new idea for the construction and application of in-situ gene delivery systems.
Promoter Regions, Genetic ; Humans ; Animals ; Mice ; Gene Library ; Cell Line ; Transfection ; HeLa Cells ; Luciferases/metabolism* ; Muscle, Skeletal/metabolism* ; Plasmids/genetics* ; Myoblasts/cytology*

We examined the effect of a combination of astaxanthin (AX) supplementation, repeated heat stress, and intermittent reloading (IR) on satellite cells in unloaded rat soleus muscles. Forty-nine male Wistar rats (8-week-old) were divided into control, hind-limb unweighting (HU), IR during HU, IR with AX supplementation, IR with repeated heat stress (41.0-41.5 °C for 30 min), and IR with AX supplementation and repeated heat stress groups. After the experimental period, the antigravitational soleus muscle was analyzed using an immunohistochemical technique. Our results revealed that the combination of dietary AX supplementation and heat stress resulted in protection against disuse muscle atrophy in the soleus muscle. This protective effect may be partially due to a higher satellite cell number in the atrophied soleus muscle in the IR/AX/heat stress group compared with the numbers found in the other groups. We concluded that the combination treatment with dietary AX supplementation and repeated heat stress attenuates soleus muscle atrophy, in part by increasing the number of satellite cells.
Animals ; Body Weight ; Dietary Supplements ; Fibrinolytic Agents/pharmacology* ; Heat-Shock Response ; Hindlimb ; Hot Temperature ; Immunohistochemistry ; Male ; Muscle, Skeletal ; Muscular Atrophy/drug therapy* ; Oxidative Stress ; Rats ; Rats, Wistar ; Satellite Cells, Skeletal Muscle/cytology* ; Xanthophylls/pharmacology*

Muscle-derived stem cells (MDSC) are defined as myogenic stem cells endowed with their ability to self-renew and differentiate into multiple cell types of their derivative tissue, and are proved to be over 10 times more efficient in hematopoiesis than hematopoietic stem cells (HSC). Although the mechanism which MDSC differentiate into blood cells is still unclear, MDSC were considered to replace HSC to treat the patients suffering from bone marrow diseases such as aplastic anemia and tumor. MDSC are different from HSC in a variety aspects like biological characteristics, protein expression and cell proliferation. On the other hand, MDSC contain multiple distinct stem cell populations. Among these, there is only a small part with the ability to repopulate hematopoietic cells, and it is still uncertain whether their origin is same as HSC. This review summarizes the difference between MDSC and HSC, the ability of MDSC to repopulate hematopoietic cells, and the prospect of MDSCs' transplantation.
Anemia, Aplastic ; Cell Differentiation ; Cell Proliferation ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Humans ; Muscle, Skeletal ; cytology

It is now well established that inflammation plays an important role in the development of numerous chronic metabolic diseases including insulin resistance (IR) and type 2 diabetes (T2DM). Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Our pre- vious study has shown that rutaecarpine (Rut) can benefit blood lipid profile, mitigate inflammation, and improve kidney, liver, pan- creas pathology status of T2DM rats. However, the effects of Rut on inflammatory cytokines in the development of IR-skeletal muscle cells have not been studied. Thus, our objective was to investigate effects of Rut on inflammatory cytokines interleukiri (IL)-1, IL-6 and tumor necrosis factor (TNF)-α in insulin resistant primary skeletal muscle cells (IR-PSMC). Primary cultures of skeletal muscle cells were prepared from 5 neonate SD rats, and the primary rat skeletal muscle cells were identified by cell morphology, effect of ru- taecarpine on cell proliferation by MTT assay. IR-PSMC cells were induced by palmitic acid (PA), the glucose concentration was measured by glucose oxidase and peroxidase (GOD-POD) method. The effects of Rut on inflammatory cytokines IL-1, IL-6 and TNF-α in IR-PSMC cells were tested by enzyme-linked immunosorbent assay (ELISA) kit. The results show that the primary skeletal muscle cells from neonatal rat cultured for 2-4 days, parallel alignment regularly, and cultured for 7 days, cells fused and myotube formed. It was shown that Rut in concentration 0-180. 0 μmol x L(-1) possessed no cytotoxic effect towards cultured primary skeletal muscle cells. However, after 24 h exposure to 0.6 mmol x L(-1) PA, primary skeletal muscle cells were able to induce a state of insulin resistance. The results obtained indicated significant decrease (P < 0.05 to P < 0.001) IL-1, IL-6 and TNF-α production by cultured IR-PSMC cells when incubating 24 hours with Rut, beginning from 20 to 180.0 μmol x L(-1). IL-1, IL-6 and TNF-α in the Rut treated groups were dose-dependently decreased compared with that in the IR-PSMC control group. Our results demonstrated that the Rut promoted glucose consumption and improved insulin resistance possibly through suppression of inflammatory cytokines in the IR-PSMC cells.
Animals ; Cell Proliferation ; drug effects ; Cytokines ; metabolism ; Female ; Glucose ; metabolism ; Indole Alkaloids ; pharmacology ; Inflammation ; metabolism ; Insulin Resistance ; Male ; Muscle, Skeletal ; cytology ; drug effects ; metabolism ; Quinazolines ; pharmacology ; Rats

Skeletal muscle possesses a remarkable ability for its regeneration and injured tissue repair. This ability depends on the activity and contributions of muscle satellite cells. Proliferating satellite cells, termed myogenic precursor cells or myoblasts, are activated and driven out of their quiescent state upon muscle injury. In this summary, we present a review to summarize the molecular regulation in skeletal satellite cells to light on the satellite cells' self-renewal mechanism.
Cell Proliferation ; Humans ; Muscle, Skeletal ; Regeneration ; Satellite Cells, Skeletal Muscle ; cytology ; Soft Tissue Injuries

OBJECTIVETo explore the effect of different functional groups on self-assembled monolayers on the biological characteristics of rabbit skeletal muscle cells in vitro.

METHODSRabbit skeletal muscle cells were cultured on self-assembled monolayers of gold on which different terminal chemical groups including methyl groups (-CH(3)), amino(-NH(2)), hydroxyl(-OH) and carboxyl (-COOH ) were anchored with self-assembled methods. Contact angle measurements and atomic force microscopy were employed to confirm the similar density of different functional groups occupation. Fluorescence microscopy, MTT assay, flow cytometry, and scanning electron microscopy (SEM) were used to analyze the morphological and biological alterations of the cells.

RESULTSSEM results revealed that the chemical groups on the surface of the monolayer modulated the structure of skeletal muscle cells and the cell morphology. Skeletal muscle cells cultured on the monolayer with -CH3 exhibited the smallest contact area with a spherical morphology, while the cells on the monolayers with -NH(2), -OH and -COOH showed much larger contact area and flatter morphology. The functional groups -NH(2) and -COOH obviously promoted cell adhesion and proliferation, while -CH(3) group produced significantly greater toxicity than -NH(2), -OH and -COOH groups to inhibit the cell growth and adhesion and promote cell death. Cell attachment and growth was enhanced, in the order the magnitude of the effect, by -NH(2)>-COOH>-OH>-CH(3), and the toxicity decreased in the order of -NH(2)>-COOH>-OH>-CH(3).

CONCLUSIONThe terminal chemical groups can obviously affect the phenotype of skeletal muscle cells in vitro, and this finding provides a theoretical basis for surface design of biomaterials.

Animals ; Cell Adhesion ; Cell Proliferation ; Cells, Cultured ; Microscopy, Atomic Force ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Muscle Fibers, Skeletal ; cytology ; Rabbits

OBJECTIVESTo evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.

METHODSThe expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.

RESULTSThe expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.

CONCLUSIONMyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Gene Expression Regulation ; drug effects ; physiology ; Glucose ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Insulin ; pharmacology ; Mice ; MicroRNAs ; biosynthesis ; genetics ; Multigene Family ; physiology ; Muscle Fibers, Skeletal ; cytology ; metabolism ; MyoD Protein ; genetics ; metabolism ; Myoblasts ; cytology ; metabolism ; Sweetening Agents ; pharmacology

Regeneration of periodontal tissue is the most promising method for restoring periodontal structures. To find a suitable bioactive three-dimensional scaffold promoting cell proliferation and differentiation is critical in periodontal tissue engineering. The objective of this study was to evaluate the biocompatibility of a novel porcine acellular dermal matrix as periodontal tissue scaffolds both in vitro and in vivo. The scaffolds in this study were purified porcine acellular dermal matrix (PADM) and hydroxyapatite-treated PADM (HA-PADM). The biodegradation patterns of the scaffolds were evaluated in vitro. The biocompatibility of the scaffolds in vivo was assessed by implanting them into the sacrospinal muscle of 20 New Zealand white rabbits. The hPDL cells were cultured with PADM or HA-PADM scaffolds for 3, 7, 14, 21 and 28 days. Cell viability assay, scanning electron microscopy (SEM), hematoxylin and eosin (H&E) staining, immunohistochemistry and confocal microscopy were used to evaluate the biocompatibility of the scaffolds. In vitro, both PADM and HA-PADM scaffolds displayed appropriate biodegradation pattern, and also, demonstrated favorable tissue compatibility without tissue necrosis, fibrosis and other abnormal response. The absorbance readings of the WST-1 assay were increased with the time course, suggesting the cell proliferation in the scaffolds. The hPDL cells attaching, spreading and morphology on the surface of the scaffold were visualized by SEM, H&E staining, immnuohistochemistry and confocal microscopy, demonstrated that hPDL cells were able to grow into the HA-PADM scaffolds and the amount of cells were growing up in the course of time. This study proved that HA-PADM scaffold had good biocompatibility in animals in vivo and appropriate biodegrading characteristics in vitro. The hPDL cells were able to proliferate and migrate into the scaffold. These observations may suggest that HA-PADM scaffold is a potential cell carrier for periodontal tissue regeneration.
Absorbable Implants ; Acellular Dermis ; Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; physiology ; Cell Culture Techniques ; Cell Movement ; physiology ; Cell Proliferation ; Cell Shape ; physiology ; Cell Survival ; physiology ; Durapatite ; chemistry ; Fibrosis ; Humans ; Microscopy, Electron, Scanning ; Muscle, Skeletal ; surgery ; Necrosis ; Periodontal Ligament ; cytology ; surgery ; Rabbits ; Regeneration ; physiology ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVETo reveal the effects of three days' repeated exhausted eccentric exercise on the skeletal muscle apoptosis and proliferation in rats.

METHODSFifty male SD rats aged at 8 week old were randomly divided into control group (C) and training groups (B1, B2, B3, B4) (n = 10), the training groups ran on a treadmill every day till exhausted. After they had been trained repeatedly for three days, their medial head of triceps brachii muscle cell apoptosis was detected in paraffin section by the TUNEL, expression of proliferating cell nuclear antigen (PCNA) protein was examined by immunohistochemistry.

RESULTS(1) The apoptosis appeared sequential change, and it was consistent with the exercise-induced skeletal muscle micro-injury (EIMmI). The apoptosis index in the training group after exercise was much greater than that in the control group (P < 0.05), and it reached the peak at 24 h after exercise, then it reduced at 48 h after exercise. (2) The express of PCNA exhibited a sequential change after exercise, the proliferation index in the training group after exercise was greater than that in the control group (P < 0.05), it increased after exercise immediately, but it reduced at 3 h after exercise, then was reached the peak at 24 h after exercise, the proliferation index was moderately correlated with the apoptosis index (P < 0.05).

CONCLUSION(1) Cell apoptosis can induce the delayed skeletal muscle damage. (2) Apoptosis may be a start factor of skeletal muscle regeneration.

Animals ; Apoptosis ; Cell Proliferation ; Male ; Muscle, Skeletal ; cytology ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley