Sulforaphane Enhanced Proliferation of Porcine Satellite Cells via Epigenetic Augmentation of SMAD7.

Satellite cells take an indispensable place in skeletal muscle regeneration, maintenance, and growth. However, only limited works have investigated effects of dietary compounds on the proliferation of porcine satellite cells (PSCs) and related mechanisms. Sulforaphane (SFN) at multiple levels was applied to PSCs. The PSCs' viability and HDAC activity were measured with a WST-1 cell proliferation kit and Color-de-Lys® HDAC colorimetric activity assay kit. Gene expression and epigenetics modification were tested with qRT-PCR, Western blot, bisulfite sequencing, and ChIP-qPCR. This study found that SFN enhanced PSC proliferation and altered mRNA expression levels of myogenic regulatory factors. In addition, SFN inhibited histone deacetylase (HDAC) activity, disturbed mRNA levels of HDAC family members, and elevated acetylated histone H3 and H4 abundance in PSCs. Furthermore, both mRNA and protein levels of the Smad family member 7 (SMAD7) in PSCs were upregulated after SFN treatment. Finally, it was found that SFN increased the acetylation level of histone H4 in the SMAD7 promoter, decreased the expression of microRNAs, including ssc-miR-15a, ssc-miR-15b, ssc-miR-92a, ssc-miR-17-5p, ssc-miR-20a-5p, and ssc-miR-106a, targeting SMAD7, but did not impact on the SMAD7 promoter's methylation status in PSCs. In summary, SFN was found to boost PSC proliferation and epigenetically increase porcine SMAD7 expression, which indicates a potential application of SFN in modulation of skeletal muscle growth.

LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages.

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1ß, IL-6, and IFN-ß. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1ß and IFN-ß were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

Comparison of the innate immune responses of porcine monocyte-derived dendritic cells and splenic dendritic cells stimulated with LPS.

Dendritic cell (DC) subsets form a remarkable cellular network that regulate innate and adaptive immune responses. Although pigs are the most approximate model to humans, little is known about the regulation of monocyte-derived DCs (moDCs) and splenic DCs (SDCs) in the initiation of immune responses under inflammatory conditions. We investigated the activation and maturation of porcine moDC and SDC subpopulations following LPS stimulation. Porcine monocytes that would differentiate into moDCs were isolated. SDCs were isolated directly from the porcine spleen. Following LPS stimulation, phagocytosis activity, TLR4/MyD88-dependent gene expression, co-stimulatory molecule, and pro-inflammatory cytokine (TNF-α, IL-1ß) and chemokine (IL-8) expressions were increased in both cell subsets. Furthermore, moDCs showed higher levels of gene and protein expression compared with SDCs. Interestingly, moDCs were found to be more responsive via the TLR4/TRAF-dependent signalling pathway of activation. Only SDCs expressed higher level of IL-12p40 gene and protein, whereas, IFN-γ gene and protein expression were likely to be unchanged after LPS stimulation in both cell subtypes. These data demonstrate that porcine moDCs display a greater ability to initiate innate immune responses, and could be used as a model to investigate immune responses against Ags.