The capacity for quantification of active metabolites of vitamin D (VitD) is highly valuable to evaluate the risks and therapies for numerous diseases such as multiple sclerosis. However, the extremely low circulating levels and poor detectability of some dihydroxyl metabolites such as the 1alpha,25-dihydroxy-VitD(3) constitute a daunting challenge. Based on the combination of a selective solid-phase extraction (SPE) and a microflow liquid chromatography tandem mass spectrometry (microLC-MS/MS), we developed an ultrasensitive method for the robust, selective, and accurate quantification of four key VitD metabolites, including 25-hydroxy-VitD(2), 25-hydroxy-VitD(3), 24(R),25-dihydroxy-VitD(3), and 1alpha,25-dihydroxy-VitD(3), in serum samples. A one-step derivatization was employed to improve the ionization efficiency of the metabolites. The SPE procedure was optimized so that the analytes were selectively extracted from serum, while the sample matrix was substantially simplified. By eliminating majority of undesirable compounds from the matrix, the selective SPE enabled a high sample loading volume on the microLC column without causing overcapacity of the microLC column and thus helped to achieve ultralow detect limits in serum. An on-column sample focusing approach was employed to prevent band-broadening, and a sufficient microLC separation was achieved to eliminate endogenous interferences and to minimize ion suppression effect. Detect limits of the four metabolites ranged from 0.5-1 pg/mL, and the linearity was excellent for all compounds. The method showed high quantitative accuracy (error < 13.8%) and precision (CV < 14.1%). For 1alpha,25-dihydroxy-VitD(3), a lower limit of quantification (LLOQ) of 5 pg/mL was validated. This high level of sensitivity, for the first time, enabled the robust and consistent LC/MS/MS-based analysis of the four metabolites in a large-scale clinical investigation. Serum samples from 281 multiple sclerosis patients and 22 healthy subjects were analyzed, and it was discovered that the levels of both 24(R),25-dihydroxy-VitD(3) and 1alpha,25-dihydroxy-VitD(3) were significantly lower in patients than healthy subjects (P < 0.05). This novel observation may imply that the incidence of multiple sclerosis is inversely associated with the levels of the two metabolites. Moreover, the method was highly robust and reproducible as evaluated extensively in the clinical analysis; therefore, it could serve as a more selective and accurate alternative to immunoassay for large-scale clinical studies.

Vitamin D metabolites have protective associations with disability and brain atrophy in MS. In particular, the results indicate strong associations for the 24, 25(OH)(2)VD(3) metabolite, which has not been extensively investigated in MS patients.

Long-term exposure to ultraviolet irradiation to skin leads to deleterious intracellular effects, including reactive oxygen species (ROS) production and inflammatory responses, causing accelerated skin aging. Previous studies have demonstrated that increased expression and activation of protease-activated receptor 2 (PAR2) and Akt is observed in keratinocyte proliferation, suggesting their potential regulatory role in skin photoaging. However, the specific underlying molecular mechanism of PAR2 and the Akt/NF-κB/FoxO6-mediated signaling pathway is not clearly defined. In this study, we first used the UVB-irradiated photoaged skin of hairless mice and observed an increase in PAR2 and Gαq expression and PI3-kinase/Akt, NF-κB, and suppressed FoxO6. Consequently, increased levels of proinflammatory cytokines and decreased levels of antioxidant MnSOD was observed. Next, to investigate PAR2-specific roles in inflammation and oxidative stress, we used photoaged hairless mice topically applied with PAR2 antagonist GB83 and photoaged PAR2 knockout mice. PAR2 inhibition and deletion significantly suppressed inflammatory and oxidative stress levels, which were associated with decreased IL-6 and IL-1β levels and increased MnSOD levels, respectively. Furthermore, NF-κB phosphorylation and decreased FoxO6 was reduced by PAR2 inhibition and deletion in vivo . To confirm the in vivo results, we conducted PAR2 knockdown and overexpression in UVB-irradiated HaCaT cells. In PAR2 knockdown cells by si-PAR2 treatment, it suppressed Akt/NF-κB and increased FoxO6, whereas PAR2 overexpression reversed these effects and subsequently modulated proinflammatory target genes. Collectively, our data define that PAR2 induces oxidative stress and inflammation through Akt-mediated phosphorylation of NF-κB (Ser536) and FoxO6 (Ser184), which could be a critical upstream regulatory mechanism in ROS-mediated inflammatory response.

Chronic inflammation, a pervasive feature of the aging process, is defined by a continuous, multifarious, low-grade inflammatory response. It is a sustained and systemic phenomenon that aggravates aging and can lead to age-related chronic diseases. In recent years, our understanding of age-related chronic inflammation has advanced through a large number of investigations on aging and calorie restriction (CR). A broader view of age-related inflammation is the concept of senoinflammation, which has an outlook beyond the traditional view, as proposed in our previous work. In this review, we discuss the effects of CR on multiple phases of proinflammatory networks and inflammatory signaling pathways to elucidate the basic mechanism underlying aging. Based on studies on senoinflammation and CR, we recognized that senescence-associated secretory phenotype (SASP), which mainly comprises cytokines and chemokines, was significantly increased during aging, whereas it was suppressed during CR. Further, we recognized that cellular metabolic pathways were also dysregulated in aging; however, CR mimetics reversed these effects. These results further support and enhance our understanding of the novel concept of senoinflammation, which is related to the metabolic changes that occur in the aging process. Furthermore, a thorough elucidation of the effect of CR on senoinflammation will reveal key insights and allow possible interventions in aging mechanisms, thus contributing to the development of new therapies focused on improving health and longevity.

FSH is an essential regulator of mammalian reproduction. Its synthesis by pituitary gonadotrope cells is regulated by multiple endocrine and paracrine factors, including TGFβ superfamily ligands, such as the activins and inhibins. Activins stimulate FSH synthesis via transcriptional regulation of its β-subunit gene (Fshb). More recently, bone morphogenetic proteins (BMPs) were shown to stimulate murine Fshb transcription alone and in synergy with activins. BMP2 signals via its canonical type I receptor, BMPR1A (or activin receptor-like kinase 3 [ALK3]), and SMAD1 and SMAD5 to stimulate transcription of inhibitor of DNA binding proteins. Inhibitor of DNA binding proteins then potentiate the actions of activin-stimulated SMAD3 to regulate the Fshb gene in the gonadotrope-like LβT2 cell line. Here, we report the unexpected observation that BMP2 also stimulates the SMAD2/3 pathway in these cells and that it does so directly via ALK3. Indeed, this novel, noncanonical ALK3 activity is completely independent of ALK4, ALK5, and ALK7, the type I receptors most often associated with SMAD2/3 pathway activation. Induction of the SMAD2/3 pathway by ALK3 is dependent upon its own previous activation by associated type II receptors, which phosphorylate conserved serine and threonine residues in the ALK3 juxtamembrane glycine-serine-rich domain. ALK3 signaling via SMAD3 is necessary for the receptor to stimulate Fshb transcription, whereas its activation of the SMAD1/5/8 pathway alone is insufficient. These data challenge current dogma that ALK3 and other BMP type I receptors signal via SMAD1, SMAD5, and SMAD8 and not SMAD2 or SMAD3. Moreover, they suggest that BMPs and activins may use similar intracellular signaling mechanisms to activate the murine Fshb promoter in immortalized gonadotrope-like cells.

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Dietary interventions such as prolonged calorie restriction (CR) and intermittent fasting provide health benefits including a reduction in the inflammatory burden and regulation of energy metabolism. During CR, β-hydroxybutyrate (BHB) level is elevated in the serum. BHB is a ligand of GPR109A, which inhibits lipolysis and exerts anti-inflammatory effects on cells. During aging, comorbidities related to dyslipidemia are significantly associated with fatty liver. However, the underlying mechanisms of BHB in hepatic ER stress and dyslipidemia are unclear and remain to be elucidated. Here, we used aged rats that were administered with BHB and compared the modulatory effects of BHB through the GPR109A/AMPK pathway on the hepatic endoplasmic reticulum (ER) stress and lipid accumulation to CR rats. BHB caused suppression of hepatic ER stress and lipid accumulation through GPR109A/AMPK pathway in the aged rats. Aged rats of both treatment groups showed reduced cAMP level and PKA phosphorylation. Furthermore, AMPK-Ser173 phosphorylation via PKA was decreased, whereas AMPK-Thr172 phosphorylation was increased by BHB and CR. Further supporting evidence was provided in HepG2 cells that BHB inhibited ER stress and lipid accumulation induced by palmitate. These results suggest that BHB activates GPR109A and regulates the activation of AMPK. These findings were further confirmed by GPR109A-siRNA transfection in vitro. In addition, BHB treatment elevated the protein levels of AMPK leading to significant inhibition of hepatic steatosis, whereas AMPK-siRNA treatment abolished these effects. Taken together, these findings suggest that BHB could be a effective molecule that mimics CR in ameliorating age-related hepatic lipid accumulation via GPR109A signaling pathway.

Tyrosinase is a key enzyme in melanin synthesis, catalyzing the initial rate-limiting steps of melanin synthesis. Abnormal and excessive melanin synthesis is the primary cause of serious skin disorders including melasma, senile lentigo, freckles, and age spots. In attempts to find potent and safe tyrosinase inhibitors, we designed and synthesized a novel compound, (Z)-3-(3-bromo-4-hydroxybenzylidene)thiochroman-4-one (MHY1498), and evaluated its tyrosinase inhibitory activity in vitro and in silico. The chemical structures of (Z)-3-benzylidenethiochroman-4-one analogues, including the novel compound MHY1498, were rationally designed and synthesized as hybrid structures of reported potent tyrosinase inhibitors, which were confirmed both in vitro and in vivo: (Z)-5-(substituted benzylidene)thiazolidine-2,4-diones (Compound A) and 2-(substituted phenyl)benzo[d]thiazoles (Compound B). During screening, MHY1498 showed a strong dose-dependent inhibitory effect on mushroom tyrosinase. The IC50 value of MHY1498 (4.1 ± 0.6 μM) was significantly lower than that of the positive control, kojic acid (22.0 ± 4.7 μM). In silico molecular multi-docking simulation and inhibition mechanism studies indicated that MHY1498 interacts competitively with the tyrosinase enzyme, with greater affinity for the active site of tyrosinase than the positive control. Furthermore, in B16F10 melanoma cells treated with α-melanocyte-stimulating hormone, MHY1498 suppressed both melanin production and tyrosinase activity. In conclusion, our data demonstrate that MHY1498, a synthesized novel compound, effectively inhibits tyrosinase activity and has potential for treating hyperpigmentation and related disorders.