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C. elegans numr-1/2 ( nu clear-localized m etal- r esponsive) is an identical gene pair encoding a nuclear protein previously shown to be activated by cadmium and disruption of the integrator RNA metabolism complex. We took a chemical genetic approach to further characterize regulation of this novel metal response by screening 41,716 compounds and extracts for numr-1p::GFP activation. The most potent activator was chaetocin, a fungal 3,6-epidithiodiketopiperazine (ETP) with promising anticancer activity. Chaetocin activates numr-1/2 strongly in the alimentary canal but is distinct from metal exposure, because it represses canonical cadmium-responsive metallothionine genes. Chaetocin has diverse targets in cancer cells including thioredoxin reductase, histone lysine methyltransferase, and acetyltransferase p300/CBP; further work is needed to identify the mechanism in C. elegans as genetic disruption and RNAi screening of homologues did not induce numr-1/2 in the alimentary canal and chaetocin did not affect markers of integrator dysfunction. We demonstrate that disulfides in chaetocin and chetomin, a dimeric ETP analog, are required to induce numr-1/2. ETP monomer gliotoxin, despite possessing a disulfide linkage, had almost no effect on numr-1/2 , suggesting a dimer requirement. Chetomin inhibits C. elegans growth at low micromolar levels, and loss of numr-1/2 increases sensitivity; C. elegans and Chaetomiaceae fungi inhabit similar environments raising the possibility that numr-1/2 functions as a defense mechanism. There is no direct orthologue of numr-1/2 in humans, but RNaseq suggests that chaetocin affects expression of cellular processes linked to stress response and metal homeostasis in colorectal cancer cells. Our results reveal interactions between metal response gene regulation and ETPs and identify a potential mechanism of resistance to this versatile class of preclinical compounds.

Pinostrobin is a dietary flavonoid found in several plants that possesses pharmacological properties, such as anti-cancer, anti-virus, antioxidant, anti-ulcer, and anti-aromatase effects. However, it is unclear if pinostrobin exerts anti-melanogenic properties and, if so, what the underlying molecular mechanisms comprise. Therefore, we, in this study, investigated whether pinostrobin inhibits melanin biosynthesis in vitro and in vivo, as well as the potential associated mechanism. Pinostrobin reduced mushroom tyrosinase activity in vitro in a concentration-dependent manner, with an IC 50 of 700 μM. Molecular docking simulations further revealed that pinostrobin forms a hydrogen bond, as well as other non-covalent interactions, between the C-type lectin-like fold and polyphenol oxidase chain, rather than the previously known copper-containing catalytic center. Additionally, pinostrobin significantly decreased α-melanocyte-stimulating hormone (α-MSH)-induced extracellular and intracellular melanin production, as well as tyrosinase activity, in B16F10 melanoma cells. More specifically, pinostrobin inhibited the α-MSH-induced melanin biosynthesis signaling pathway by suppressing the cAMP-CREB-MITF axis. In fact, pinostrobin also attenuated pigmentation in α-MSH-stimulated zebrafish larvae without causing cardiotoxicity. The findings suggest that pinostrobin effectively inhibits melanogenesis in vitro and in vivo via regulation of the cAMP-CREB-MITF axis.

Pinostrobin is a natural flavonoid with valuable pharmacological properties, including anti-cancer, anti-viral, and anti-oxidant activities. However, the anti-inflammatory effects of pinostrobin have not been well studied. In this study, we investigated whether pinostrobin attenuates lipopolysaccharide (LPS)-induced inflammation and endotoxemia. Additionally, the target molecule of pinostrobin was identified through molecular docking simulation. Pinostrobin decreased LPS-induced nitric oxide (NO) and prostaglandin E 2 production, and reduced the expression of inducible NO synthase and cyclooxygenase-2. Furthermore, pinostrobin inhibited the production of proinflammatory cytokines, including interleukin-12 and tumor necrosis factor-α in LPS-stimulated RAW 264.7 macrophages accompanied by inhibiting nuclear translocation of nuclear factor-κB. The anti-inflammatory effect of pinostrobin was further confirmed in LPS-microinjected zebrafish larvae by diminishing the recruitment of macrophages and neutrophils, and proinflammatory gene expression. Moreover, LPS-microinjected zebrafish larvae showed a decrease in heart rate and an increase in mortality and abnormalities. However, pinostrobin significantly attenuated these adverse effects. Molecular docking showed that pinostrobin fits into myeloid differentiation factor (MD2) and Toll-like receptor 4 (TLR4) with no traditional hydrogen bonds (pose 1). The 2D ligand interaction diagram showed that pinostrobin forms a carbon hydrogen bond with LYS89 in MD2 and many non-covalent interactions, including π-alkyl or alkyl and van der Waals interactions, indicating that pinostrobin hinders LPS binding between MD2 and TLR4 and consequently inhibits TLR4/MD2-mediated inflammatory responses. These data suggest that pinostrobin attenuates LPS-induced inflammation and endotoxemia by binding to the TLR4/MD2 complex., Competing Interests: Conflict of interest statement The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Osteoporosis is a bone resorptive disease characterized by the loss of bone density, causing an increase in bone fragility. In our previous study, we demonstrated that gamma aminobutyric acid-enriched fermented oyster ( Crassostrea gigas ) extract (FO) stimulated osteogenesis in MC3T3-E1 preosteoblast cells and vertebral formation in zebrafish. However, the efficacy of FO in prednisolone (PDS)-induced bone resorption remains unclear. In this study, we evaluated the osteogenic potential of FO in MC3T3-E1 preosteoblast cells and zebrafish larvae under both PDS-pretreated and PDS-post-treated conditions. We found that FO recovered osteogenic activity by upregulating osteoblast markers, such as alkaline phosphatase ( ALP ), runt-related transcription factor 2, and osterix, in both PDS-pretreated and post-treated MC3T3-E1 osteoblast cells and zebrafish larvae. In both conditions, PDS-induced decrease in calcification and ALP activity was recovered in the presence of FO. Furthermore, vertebral resorption in zebrafish larvae induced by pretreatment and post-treatment with PDS was restored by treatment with FO, along with the recovery of osteogenic markers and downregulation of osteoclastogenic markers. Finally, whether FO disturbs the endocrine system was confirmed according to the Organization for Economic Cooperation and Development guideline 455. We found that FO did not stimulate estrogen response element-luciferase activity or proliferation in MCF7 cells. Additionally, in ovariectomized mice, no change in uterine weight was observed during FO feeding. These results indicate that FO effectively prevents and treats PDS-induced osteoporosis without endocrine disturbances.

Insulin-like growth factor-1 (IGF-1) primarily increases the release of gamma-aminobutyric acid (GABA) in neurons; moreover, it is responsible for the promotion of longitudinal growth in children and adolescents. Therefore, in this study, we investigated whether exogenous GABA supplementation activates IGF-mediated growth performance. Zebrafish larvae treated with GABA at three days post fertilization (dpf) showed a significant increase in the total body length from 6 to 12 dpf through upregulation of growth-stimulating genes, including IGF-1 , growth hormone-1 ( GH-1 ), growth hormone receptor-1 ( GHR-1 ), and cholecystokinin A ( CCKA ). In particular, at 9 dpf, GABA increased total body length from 3.60 ± 0.02 to 3.79 ± 0.03, 3.89 ± 0.02, and 3.92 ± 0.04 mm at concentrations of 6.25, 12.5, and 25 mM, and the effect of GABA at 25 mM was comparable to 4 mM β-glycerophosphate (GP)-treated larvae (3.98 ± 0.02 mm). Additionally, the highest concentration of GABA (50 mM) -induced death in 50% zebrafish larvae at 12 dpf. GABA also enhanced IGF-1 expression and secretion in preosteoblast MC3T3-E1 cells, concomitant with high levels of the IGF-1 receptor gene ( IGF-1R ). In zebrafish larvae, the GABA-induced growth rate was remarkably decreased in the presence of an IGF-1R inhibitor, picropodophyllin (PPP), which indicates that GABA-induced IGF-1 enhances growth rate via IGF-1R. Furthermore, we investigated the effect of GABA receptors on growth performance along with IGF-1 activation. Inhibitors of GABA A and GABA B receptors, namely bicuculline and CGP 46381, respectively, considerably inhibited GABA-induced growth rate in zebrafish larvae accompanied by a marked decrease in the expression of growth-stimulating genes, including IGF-1 , GH-1 , GHR-1 , and CCKA , but not with an inhibitor of GABA C receptor, TPMPA. Additionally, IGF-1 and IGF-1R expression was impaired in bicuculline and CGP 46381-treated MC3T3-E1 cells, but not in the cells treated with TPMPA. Furthermore, treatment with bicuculline and CGP 46381 significantly downregulated GABA-induced IGF-1 release in MC3T3-E1 cells. These data indicate that GABA stimulates IGF-1 release via GABA A and GABA B receptors and leads to growth promotion performance via IGF-1R.

Fisetin has numerous therapeutic properties, such as anti-inflammatory, antioxidative, and anticancer effects. However, the mechanism by which fisetin inhibits NLRP3 inflammasome remains unclear. In this study, we observed that fisetin bound to TLR4 and occluded the hydrophobic pocket of MD2, which in turn inhibited the binding of LPS to the TLR4/MD2 complex. This prevented the initiation of scaffold formation by the inhibition of MyD88/IRAK4 and subsequently downregulated the NF-κB signaling pathway. The result also demonstrated that fisetin downregulated the activation of the NLRP3 inflammasome induced by LPS and ATP (LPS/ATP) and the subsequent maturation of IL-1β. Fisetin also activated mitophagy and prevented the accumulation of damaged mitochondria and the excessive production of mitochondrial reactive oxygen species. The transient knockdown of p62 reversed the inhibitory activity of fisetin on the LPS/ATP-induced formation of the NLRP3 inflammasome. This indicated that fisetin induces p62-mediated mitophagy for eliminating damaged mitochondria. Recently, the existence of inflammasomes in non-mammalian species including zebrafish have been identified. Treatment of an LPS/ATP-stimulated zebrafish model with fisetin aided the recovery of the impaired heart rate, decreased the recruitment of macrophage to the brain, and gradually downregulated the expression of inflammasome-related genes. These results indicated that fisetin inhibited the TLR4/MD2-mediated activation of NLRP3 inflammasome by eliminating damaged mitochondria in a p62-dependent manner.

Ziziphus jujuba extracts possess a broad spectrum of biological activities, such as antioxidant and anticancer activities in melanoma cancers. Nevertheless, the compounds contain high antioxidant capacities and anticancer activities in melanoma cells, shown to be effective in hyperpigmentation disorders, but whether flavonoid glycosides from Z . jujuba regulate anti-melanogenesis remains unclear. In this study, we evaluated the anti-melanogenic activity of five flavonoid glycosides from Z. jujuba var. inermis (Bunge) Rehder seeds, including jujuboside A (JUA), jujuboside B (JUB), epiceanothic acid (EPA), betulin (BTL), and 6'''-feruloylspinosin (FRS), in B16F10 melanoma cells and zebrafish larvae. According to our results, JUB, EPA, and FRS potently inhibited α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis and prevented hyperpigmentation in zebrafish larvae. In particular, under α-MSH-stimulated conditions, FRS most significantly inhibited α-MSH-induced intracellular and extracellular melanin content in B16F10 melanoma cells. Additionally, JUB, EPS, and FRS remarkably downregulated melanogenesis in α-MSH-treated zebrafish larvae, with no significant change in heart rate. Neither JUA nor BTA were effective in downregulating melanogenesis in B16F10 melanoma cells and zebrafish larvae. Furthermore, JUB, EPA, and FRS directly inhibited in vitro mushroom tyrosinase enzyme activity. JUB, EPA, and FRS also downregulated cyclic adenosine monophosphate (cAMP) levels and the phosphorylation of cAMP-response element-binding protein (CREB), and subsequent microphthalmia transcription factor (MITF) and tyrosinase expression. In conclusion, this study demonstrated that JUB, EPA, and FRS isolated from Z. jujuba var. inermis (Bunge) Rehder seeds exhibit potent anti-melanogenic properties by inhibition of the cAMP-CERB-MITF axis and consequent tyrosinase activity.