Propolis Ethanol Extract Stimulates Cytokine and Chemokine Production through NF-κB Activation in C2C12 Myoblasts

Myoblast activation is a triggering event for muscle remodeling. We assessed the stimulatory effects of propolis, a beehive product, on myoblasts. After an 8 h treatment with 100 μg/mL of Brazilian propolis ethanol extract, expression of various chemokines, including CCL-2 and CCL-5, and cytokines, such as IL-6, increased. This propolis-induced cytokine production appears to depend on NF-κB activation, because the IKK inhibitor BMS-345541 repressed mRNA levels of CCL-2 by ~66%, CCL-5 by ~81%, and IL-6 by ~69% after propolis treatment. Supernatant from propolis-conditioned C2C12 cells upregulated RAW264 macrophage migration. The supernatant also stimulated RAW264 cells to produce angiogenic factors, including VEGF-A and MMP-12. Brazilian green propolis therefore causes myoblasts to secrete cytokines and chemokines, which might contribute to tissue remodeling of skeletal muscle

Propolis Ethanol Extract Stimulates Cytokine and Chemokine Production through NF-κB Activation in C2C12 Myoblasts

Myoblast activation is a triggering event for muscle remodeling. We assessed the stimulatory effects of propolis, a beehive product, on myoblasts. After an 8 h treatment with 100 μg/mL of Brazilian propolis ethanol extract, expression of various chemokines, including CCL-2 and CCL-5, and cytokines, such as IL-6, increased. This propolis-induced cytokine production appears to depend on NF-κB activation, because the IKK inhibitor BMS-345541 repressed mRNA levels of CCL-2 by ~66%, CCL-5 by ~81%, and IL-6 by ~69% after propolis treatment. Supernatant from propolis-conditioned C2C12 cells upregulated RAW264 macrophage migration. The supernatant also stimulated RAW264 cells to produce angiogenic factors, including VEGF-A and MMP-12. Brazilian green propolis therefore causes myoblasts to secrete cytokines and chemokines, which might contribute to tissue remodeling of skeletal muscle

Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco’s modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique

Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism