Interleukin-1ß induces apoptosis in annulus fibrosus cells through the extracellular signal-regulated kinase pathway.

PURPOSE: The loss of intervertebral disc (IVD) cells due to excessive apoptosis induced by inflammatory cytokines is a major cause of IVD degeneration. This study aims to explore the mechanism of interleukin-1ß (IL-1ß)-induced apoptosis of annulus fibrosus cells (AFCs). It's hypothesized that IL-1ß induces apoptosis through the extracellular signal-regulated kinase (ERK) pathway in AFCs. METHODS: The mRNA and protein expression levels of apoptosis-associated genes were analyzed by quantitative real-time PCR and Western blotting. The apoptotic rate was measured by flow cytometry. Three experimental groups were established, including Control, IL-1ß, and IL-1ß+U0126 groups, respectively. RESULTS: Increase in the expression of apoptosis-associated genes including B-cell lymphoma-2 associated X (Bax), caspase-3, and caspase-9, and meanwhile, decrease in the expression of B-cell lymphoma-2 (Bcl-2) gene were found in patients with degenerative IVDs. In in vitro tests, both apoptosis and phosphorylated ERK expression in rat AFCs decreased in the IL-1ß+U0126 group compared with the IL-1ß group. The expression levels of Bax, caspase-3, and caspase-9 in AFCs decreased significantly in the IL-1ß+U0126 group compared with those in the IL-1ß group. The expression level of Bcl-2, on the other hand, significantly increased. CONCLUSIONS: Findings from this study suggest that IL-1ß induces apoptosis in AFCs through the ERK pathway, and therefore, ERK inhibition may provide certain protection against the adverse effects of IL-1ß.

The novel gene pFAM134B positively regulates fat deposition in the subcutaneous fat of Sus scrofa.

In this study, we analyzed the global gene expression profiles in the subcutaneous fat (SAT) of Jinhua pigs and Landrace pigs at 90d. Several genes were significantly highly expressed in Jinhua pigs, including genes encoding the rate limiting enzymes in the TCA cycle, fatty acid activation, fatty acid synthesis and triglyceride synthesis. We identified a novel gene tagged by the EST sequences as public No. BF702245.1, which was named porcine FAM134B (pFAM134B) and the pFAM134B mRNA levels of SAT was significantly higher in Jinhua pigs than that in Landrace pigs at 90d (P<0.01). Then the effects of pFAM134B on lipid accumulation were investigated by using RNAi and gene overexpression in the subcutaneous adipocytes. The results showed that pFAM134B played a significant positive role in regulating lipid deposition by increasing the mRNA levels of PPARγ, lipogenic genes fatty acid synthetase (FAS) and acetyl-CoA carboxylase (ACC) (P<0.01) and reducing the mRNA levels of adipose triglyceride lipase (ATGL) and lipase, hormone-sensitive (HSL) (P<0.01). This study implied that pFAM134B might be a positive factor in lipid deposition, providing insight into the control of fat accumulation and lipid-related disorders.

Correction: Preparation and characterizations of an injectable and biodegradable high-strength iron-bearing brushite cement for bone repair and vertebral augmentation applications.

Correction for 'Preparation and characterizations of an injectable and biodegradable high-strength iron-bearing brushite cement for bone repair and vertebral augmentation applications' by Luguang Ding et al., Biomater. Sci., 2023, 11, 96-107, https://doi.org/10.1039/D2BM01535H.

Moderate mechanical stimulation antagonizes inflammation of annulus fibrosus cells through YAP-mediated suppression of NF-κB signaling.

Intervertebral disc degeneration (IDD) is a leading cause of low back pain. The inflammatory responses caused by aberrant mechanical loading are one of the major factors leading to annulus fibrosus (AF) degeneration and IDD. Previous studies have suggested that moderate cyclic tensile strain (CTS) can regulate anti-inflammatory activities of AF cells (AFCs), and Yes-associated protein (YAP) as a mechanosensitive coactivator senses diverse types of biomechanical stimuli and translates them into biochemical signals controlling cell behaviors. However, it remains poorly understood whether and how YAP mediates the effect of mechanical stimuli on AFCs. In this study, we aimed to investigate the exact effects of different CTS on AFCs as well as the role of YAP signaling involving in it. Our results found that 5% CTS inhibited the inflammatory response and promoted cell growth through inhibiting the phosphorylation of YAP and nuclear localization of NF-κB, while 12% CTS had a significant proinflammatory effect with the inactivation of YAP activity and the activation of NF-κB signaling in AFCs. Furthermore, moderate mechanical stimulation may alleviate the inflammatory reaction of intervertebral discs through YAP-mediated suppression of NF-κB signaling in vivo. Therefore, moderate mechanical stimulation may serve as a promising therapeutic approach for the prevention and treatment of IDD.

Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration.

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades. However, efficient harvest and handling of cell sheets remain challenging, including insufficient extracellular matrix content and poor mechanical strength. Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types. However, currently, there are no effective ways to apply mechanical loading to cell sheets. In this study, we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide) (PNIPAAm) to poly(dimethylsiloxane) (PDMS) surfaces. The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting. Subsequently, MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates. Upon maturation, the cell sheets were harvested by lowering the temperature. We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning. Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated. After implantation into the critical-sized calvarial defects of mice, the mechanically conditioned cell sheets significantly promoted new bone formation. Findings from this study reveal that thermo-responsive elastomer, together with mechanical conditioning, can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.

Engineering the viscoelasticity of gelatin methacryloyl (GelMA) hydrogels via small "dynamic bridges" to regulate BMSC behaviors for osteochondral regeneration.

The dynamic extracellular matrix (ECM) constantly affects the behaviors of cells. To mimic the dynamics of ECM with controllable stiffness and energy dissipation, this study proposes a strategy in which a small molecule, 3,4-dihydroxybenzaldehyde (DB), was used as fast "dynamic bridges'' to construct viscoelastic gelatin methacryloyl (GelMA)-based hydrogels. The storage modulus and loss modulus of hydrogels were independently adjusted by the covalent crosslinking density and by the number of dynamic bonds. The hydrogels exhibited self-healing property, injectability, excellent adhesion and mechanical properties. Moreover, the in vitro results revealed that the viscous dissipation of hydrogels favored the spreading, proliferation, osteogenesis and chondrogenesis of bone marrow mesenchymal stem cells (BMSCs), but suppressed their adipogenesis. RNA-sequencing and immunofluorescence suggested that the viscous dissipation of hydrogels activated Yes-associated protein (YAP) by stabilizing integrin ß1, and further promoted nuclear translocation of smad2/3 and ß-catenin to enhance chondrogenesis and osteogenesis. As a result, the viscoelastic GelMA hydrogels with highest loss modulus showed best effect in cartilage and subchondral bone repair. Taken together, findings from this study reveal an effective strategy to fabricate viscoelastic hydrogels for modulating the interactions between cells and dynamic ECM to promote tissue regeneration.

Preparation and characterizations of an injectable and biodegradable high-strength iron-bearing brushite cement for bone repair and vertebral augmentation applications.

Brushite cements have good osteoconductive and resorbable properties, but the low mechanical strength and poor injectability limit their clinical applications in load-bearing conditions and minimally invasive surgery. In this study, an injectable brushite cement that contains monocalcium phosphate monohydrate (MCPM) and ß-tricalcium phosphate (ß-TCP) as its solid phase and ammonium ferric citrate (AFC) solution as the aqueous medium was designed to have high mechanical strength. The optimized formulation achieved a compressive strength of 62.8 ± 7.2 MPa, which is above the previously reported values of hand-mixing brushite cements. The incorporation of AFC prolonged the setting times and greatly enhanced the injectability and degradation properties of the cements. In vitro and in vivo experiments demonstrated that the brushite cements exhibited good biocompatibility and bone regeneration capacity. The novel brushite cement is promising for bone healing in load-bearing applications.

Fucoidan-loaded nanofibrous scaffolds promote annulus fibrosus repair by ameliorating the inflammatory and oxidative microenvironments in degenerative intervertebral discs.

Tissue engineering holds potential in the treatment of intervertebral disc degeneration (IDD). However, implantation of tissue engineered constructs may cause foreign body reaction and aggravate the inflammatory and oxidative microenvironment of the degenerative intervertebral disc (IVD). In order to ameliorate the adverse microenvironment of IDD, in this study, we prepared a biocompatible poly (ether carbonate urethane) urea (PECUU) nanofibrous scaffold loaded with fucoidan, a natural marine bioactive polysaccharide which has great anti-inflammatory and antioxidative functions. Compared with pure PECUU scaffold, the fucoidan-loaded PECUU nanofibrous scaffold (F-PECUU) decreased the gene and protein expression related to inflammation and the oxidative stress in the lipopolysaccharide (LPS) induced annulus fibrosus cells (AFCs) significantly (p<0.05). Especially, gene expression of Il 6 and Ptgs2 was decreased more than 50% in F-PECUU with 3.0 wt% fucoidan (HF-PECUU). Moreover, the gene and protein expression related to the degradation of extracellular matrix (ECM) were reduced in a fucoidan concentration-dependent manner significantly, with increased almost 3 times gene expression of Col1a1 and Acan in HF-PECUU. Further, in a 'box' defect model, HF-PECUU decreased the expression of COX-2 and deposited more ECM between scaffold layers when compared with pure PECUU. The disc height and nucleus pulposus hydration of repaired IVD reached up to 75% and 85% of those in the sham group. In addition, F-PECUU helped to maintain an integrate tissue structure with a similar compression modulus to that in sham group. Taken together, the F-PECUU nanofibrous scaffolds showed promising potential to promote AF repair in IDD treatment by ameliorating the harsh degenerative microenvironment. STATEMENT OF SIGNIFICANCE: Annulus fibrosus (AF) tissue engineering holds potential in the treatment of intervertebral disc degeneration (IDD), but is restricted by the inflammatory and oxidative microenvironment of degenerative disc. This study developed a biocompatible polyurethane scaffold (F-PECUU) loaded with fucoidan, a marine bioactive polysaccharide, for ameliorating IDD microenvironment and promoting disc regeneration. F-PECUU alleviated the inflammation and oxidative stress caused by lipopolysaccharide and prevented extracellular matrix (ECM) degradation in AF cells. In vivo, it promoted ECM deposition to maintain the height, water content and mechanical property of disc. This work has shown the potential of marine polysaccharides-containing functional scaffolds in IDD treatment by ameliorating the harsh microenvironment accompanied with disc degeneration.

High-resolution 3D printing of angle-ply annulus fibrosus scaffolds for intervertebral disc regeneration.

Intervertebral disc (IVD) degeneration is one of the leading causes of disability, and current therapies are mainly unsatisfactory. The key pathological feature during IVD degeneration is the dysfunction of annulus fibrosus (AF). Although tissue-engineered AF has shown great promise for IVD regeneration, the design and fabrication of biomimetic AF scaffold remains a challenge due to the complexity of its structure. Nowadays, 3D printing technology has drawn great attention due to its customizable processes and ability to produce complex tissue architecture. However, few existing 3D printing methods can accurately replicate the fine angle-ply architecture of native AF, which is one of the most critical steps for IVD regeneration, due to the limited printing resolution. In this study, we aimed to fabricate high-resolution polycaprolactone (PCL) scaffolds using a newly developed electrohydrodynamic 3D printing technique. The structural advantages of such scaffolds were verified by finite element analysis (FEA). The PCL scaffolds were further assembled into AF construct to replicate the angle-ply architecture of AF. The optimal assembling method was confirmed by FEA and mechanical tests. Thein vitroexperiments showed that the 3D printed AF scaffolds presented favorable biocompatibility and supported the adhesion and growth of AF cells. Thein vivoperformance of tissue-engineered IVDs (TE-IVDs), which consisted of 3D printed AF scaffold and GelMA hydrogel that simulated nucleus pulposus (NP), were evaluated using a rat total disc replacement model. We found that the implantation of TE-IVDs helped maintain the disc height, reduced the loss of NP water content, and partially restored the biomechanical function of IVD. In addition, the TE-IVDs achieved well integration with adjacent tissues and promoted new tissue formation. In summary, being able to accurately simulate the structural characteristics of native AF, the 3D printed angle-ply AF scaffolds hold potential for future applications in IVD regeneration.

Regulation of differentiation of annulus fibrosus-derived stem cells using heterogeneous electrospun fibrous scaffolds.

BACKGROUND: Tissue engineering of the annulus fibrosus (AF) shows promise as a treatment for patients with degenerative disc disease (DDD). However, it remains challenging due to the intrinsic heterogeneity of AF tissue. Fabrication of scaffolds recapitulating the specific cellular, componential, and microstructural features of AF, therefore, is critical to successful AF tissue regeneration. METHODS: Poly-L-lactic acid (PLLA) fibrous scaffolds with various fiber diameters and orientation were prepared to mimic the microstructural characteristics of AF tissue using electrospinning technique. AF-derived stem cells (AFSCs) were cultured on the PLLA fibrous scaffolds for 7 days. RESULTS: The morphology of AFSCs significantly varied when cultured on the scaffolds with various fiber diameters and orientation. AFSCs were nearly round on scaffolds with small fibers. However, they became spindle-shaped on scaffolds with large fibers. Meanwhile, upregulated expression of collagen-I gene happened in cells cultured on scaffolds with large fibers, while enhanced expression of collagen-II and aggrecan genes was seen on scaffolds with small fibers. The production of related proteins also showed similar trends. Further, culturing AFSCs on a heterogeneous scaffold by overlaying membranes with different fiber sizes led to the formation of a hierarchical structure approximating native AF tissue. CONCLUSION: Findings from this study demonstrate that fibrous scaffolds with different fiber sizes effectively promoted the differentiation of AFSCs into specific cells similar to the types of cells at various AF zones. It also provides a valuable reference for regulation of cell differentiation and fabrication of engineered tissues with complex hierarchical structures using the physical cues of scaffolds. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Effective AF repair is an essential need for treating degenerative disc disease. Tissue engineering is a promising approach to achieving tissue regeneration and restoring normal functions of tissues. By mimicking the key structural features of native AF tissue, including fiber size and alignment, this study deciphered the effect of scaffold materials on the cell differentiation and extracellular matrix deposition, which provides a solid basis for designing new strategies toward more effective AF repair and regeneration.

Substrate Topography Regulates Differentiation of Annulus Fibrosus-Derived Stem Cells via CAV1-YAP-Mediated Mechanotransduction.

Regeneration of annulus fibrosus (AF) through tissue engineering techniques shows promise as a treatment for patients with degenerative disc disease (DDD). Yet, it remains challenging because of the intrinsic heterogeneity of AF tissue and shortage of in-depth knowledge of its structure-function correlation. In the current study, we fabricated fibrous poly(ether carbonate urethane)urea (PECUU) scaffolds with various fiber sizes to mimic the microstructural feature of native AF and aimed to regulate the differentiation of AF-derived stem cells (AFSCs) by controlling the topographical cues of the scaffold. We found that the morphology of AFSCs varied significantly on scaffolds with various fiber sizes. Meanwhile, the expression of the phenotypic marker genes of outer AF was up-regulated on scaffolds with large fibers. Meanwhile, enhanced expression of the phenotypic marker genes of inner AF was seen on scaffolds with small fibers. Such topography-dependent gene expression in AFSCs approximated the biochemical profile of AF tissue in various zones. Moreover, cell spreading and nucleus translocation of Yes-associated protein (YAP) were facilitated with increased fiber size. Formation and maturation of focal adhesions of AFSCs were also promoted. We also found that Caveolin-1 (CAV1) positively modulated the mechano-responses of YAP in response to substrate topography. In conclusion, depending on the activation of the CAV1-YAP mechanotransduction axis, tuning the fiber size of scaffolds can effectively induce changes in cell shape, adhesions, and extracellular matrix expression. This work may therefore provide new insights in the design of novel materials toward AF tissue regeneration.

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway.

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1ß stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin ß1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin ß1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.

Tissue Engineering and Regenerative Medicine: Achievements, Future, and Sustainability in Asia.

Exploring innovative solutions to improve the healthcare of the aging and diseased population continues to be a global challenge. Among a number of strategies toward this goal, tissue engineering and regenerative medicine (TERM) has gradually evolved into a promising approach to meet future needs of patients. TERM has recently received increasing attention in Asia, as evidenced by the markedly increased number of researchers, publications, clinical trials, and translational products. This review aims to give a brief overview of TERM development in Asia over the last decade by highlighting some of the important advances in this field and featuring major achievements of representative research groups. The development of novel biomaterials and enabling technologies, identification of new cell sources, and applications of TERM in various tissues are briefly introduced. Finally, the achievement of TERM in Asia, including important publications, representative discoveries, clinical trials, and examples of commercial products will be introduced. Discussion on current limitations and future directions in this hot topic will also be provided.

Substrate stiffness- and topography-dependent differentiation of annulus fibrosus-derived stem cells is regulated by Yes-associated protein.

Annulus fibrosus (AF) tissue engineering has attracted increasing attention as a promising therapy for degenerative disc disease (DDD). However, regeneration of AF still faces many challenges due to the tremendous complexity of this tissue and lack of in-depth understanding of the structure-function relationship at cellular level within AF is highly required. In light of the fact that AF is composed of various types of cells and has gradient mechanical, topographical and biochemical features along the radial direction. In this study, we aimed to achieve directed differentiation of AF-derived stem cells (AFSCs) by mimicking the mechanical and topographical features of native AF tissue. AFSCs were cultured on four types of electrospun poly(ether carbonate urethane)urea (PECUU) scaffolds with various stiffness and fiber size (soft, small size; stiff, small size; soft, large size and stiff, large size). The results show that with constant fiber size, the expression level of the outer AF (oAF) phenotypic marker genes in AFSCs increased with the scaffold stiffness, while that of inner AF (iAF) phenotypic marker genes showed an opposite trend. When scaffold stiffness was fixed, the expression of oAF phenotypic marker genes in AFSCs increased with fiber size. While the expression of iAF phenotypic marker genes decreased. Such substrate stiffness- and topography-dependent changes of AFSCs was in accordance with the genetic and biochemical distribution of AF tissue from the inner to outer regions. Further, we found that the Yes-associated protein (YAP) was translocated to the nucleus in AFSCs cultured with increasing stiffness and fiber size of scaffolds, yet it remained mostly phosphorylated and cytosolic in cells on soft scaffolds with small fiber size. Inhibition of YAP down-regulated the expression of tendon/ligament-related genes, whereas expression of the cartilage-related genes was upregulated. The results illustrate that matrix stiffness is a potent regulator of AFSC differentiation. Moreover, we reveal that fiber size of scaffolds induced changes in cell adhesions and determined cell shape, spreading area, and extracellular matrix expression. In all, both mechanical property and topography features of scaffolds regulate AFSC differentiation, possibly through a YAP-dependent mechanotransduction mechanism. STATEMENT OF SIGNIFICANCE: Physical cues such as mechanical properties, topographical and geometrical features were shown to profoundly impact the growth and differentiation of cultured stem cells. Previously, we have found that the differentiation of annulus fibrosus-derived stem cells (AFSCs) could be regulated by the stiffness of scaffold. In this study, we fabricated four types of poly(ether carbonate urethane)urea (PECUU) scaffolds with controlled stiffness and fiber size to explore the potential of induced differentiation of AFSCs. We found that AFSCs are able to present different gene expression patterns simply as a result of the stiffness and fiber size of scaffold material. This work has, for the first time, demonstrated that larger-sized and higher-stiffness substrates increase the amount of vinculin assembly and activate YAP signaling in pre-differentiated AFSCs. The present study affords an in-depth comprehension of materiobiology, and be helpful for explain the mechanism of YAP mechanosensing in AF in response to biophysical effects of materials.

Antibacterial activity and osseointegration of silver-coated poly(ether ether ketone) prepared using the polydopamine-assisted deposition technique.

Poly(ether ether ketone) (PEEK) is a popular orthopaedic implant material due to the outstanding biocompatibility and mechanical properties. However, bacterial infections and aseptic loosening during implantation can cause many clinical problems that may eventually lead to implant failure. Therefore, endowing implants with antibacterial functions plays an important role in promoting integration between implants and bone tissue and ultimately, in successful implantation. This study aimed to develop a biocompatible and antibacterial coating for PEEK implants using polydopamine (PDA)-based surface modification technology and subsequent deposition of silver (Ag) nanoparticles (PEEK-PDA-Ag). Formation of Ag nanoparticles was clearly observed on the surface of PEEK-PDA-Ag using scanning electron microscopy. PEEK-PDA-Ag showed low toxicity to MC3T3-E1 cells. It exhibited outstanding antibacterial properties against both S. aureus and E. coli in vitro as well as decent antibacterial performance in vivo. In addition, in vivo studies demonstrated good osseointegration of PEEK-PDA-Ag implants, as shown by micro-CT evaluation and push-out tests. Together, the findings from this study indicate that the facilely prepared PEEK-PDA-Ag substrates possess considerable biocompatibility and antibacterial properties, permitting their potential use as a promising orthopaedic implant material.

Selenium­enriched exopolysaccharides produced by Enterobacter cloacae Z0206 alleviate adipose inflammation in diabetic KKAy mice through the AMPK/SirT1 pathway.

Polysaccharides belong to a structurally diverse class of macromolecules, with the necessary flexibility for the precise regulatory mechanisms and high capacity for carrying biological information. On the basis of a previous study regarding the administration of selenium-enriched exopolysaccharides (Se-ECZ-EPS) produced by Enterobacter cloacae (E. cloacae) Z0206 which resulted in a reduction of blood glucose levels and showed significant anti-inflammatory and anti-diabetic effects, the present study was conducted to evaluate the effects and mechanism of EPS on the alleviation of fat inflammation in high-fat-diet (HFD) induced-diabetic KKAy mice. The HFD induced-diabetic KKAy mice were gavaged once daily with EPS (0.2 mg/g body weight) or distilled water, while the C57BL/6J mice were gavaged with distilled water. Six weeks later visceral adipose tissue (VAT) was collected for quantified polymerase chain reaction (qPCR) and western blot (WB) analysis. The results showed that following supplementation with EPS, interleukin (IL) 6, IL1ß and tumor necrosis factor (TNF) α mRNA expression in VAT were significantly reduced, while Glut4, pAMPK and SirT1 protein expression were markedly increased when compared with KKAy mice gavaged with water. Furthermore, ATGL and HSL mRNA were also significantly decreased. Subsequently, 3T3-L1 adipocytes were treated with insulin to induce insulin resistance to determine the mechanism by which EPS affects inflammation. Following the treatment of adipocytes with 100 nM insulin for 8 h, IL6 and TNFα mRNA expression were significantly increased, while the content of glucose uptake and Glut4 protein expression were significantly decreased. When treated with 100 nM insulin and 0.1 mg/ml EPS, no significant change in IL6 and TNFα mRNA expression or glucose uptake were observed. However, when SirT1­siRNA or AMPKα1-siRNA was transfected into the 3T3-L1 adipocytes prior to treatment with insulin and EPS, there was a significant increase in IL6 and TNFα mRNA abundance. In conclusion, VAT inflammation and lipolysis in HFD-induced KKAy mice were significantly decreased following EPS usage. Moreover, EPS may alleviate VAT inflammation primarily through the AMPK/SirT1 pathway.

Distinctive genes determine different intramuscular fat and muscle fiber ratios of the longissimus dorsi muscles in Jinhua and landrace pigs.

Meat quality is determined by properties such as carcass color, tenderness and drip loss. These properties are closely associated with meat composition, which includes the types of muscle fiber and content of intramuscular fat (IMF). Muscle fibers are the main contributors to meat mass, while IMF not only contributes to the sensory properties but also to the plethora of physical, chemical and technological properties of meat. However, little is known about the molecular mechanisms that determine meat composition in different pig breeds. In this report we show that Jinhua pigs, a Chinese breed, contains much higher levels of IMF than do Landrace pigs, a Danish breed. We analyzed global gene expression profiles in the longissimus dorsi muscles in Jinhua and Landrace breeds at the ages of 30, 90 and 150 days. Cross-comparison analysis revealed that genes that regulate fatty acid biosynthesis (e.g., fatty acid synthase and stearoyl-CoA desaturase) are expressed at higher levels in Jinhua pigs whereas those that regulate myogenesis (e.g., myogenic factor 6 and forkhead box O1) are expressed at higher levels in Landrace pigs. Among those genes which are highly expressed in Jinhua pigs at 90 days (d90), we identified a novel gene porcine FLJ36031 (pFLJ), which functions as a positive regulator of fat deposition in cultured intramuscular adipocytes. In summary, our data showed that the up-regulation of fatty acid biosynthesis regulatory genes such as pFLJ and myogenesis inhibitory genes such as myostatin in the longissimus dorsi muscles of Jinhua pigs could explain why this local breed produces meat with high levels of IMF.