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.

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.

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.

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 IC₅₀ 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.

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.

This study assesses the relationship between arthropod and vegetation diversity in four ecosystems with different types of vegetation, during a post-monsoonal season. We determined the arthropod diversity in vegetation surrounding an aquatic environment (AQ), a broad-leaved wet, evergreen forest ecosystem (BL), a Pinus caribaea monoculture plantation (PN), and a Pinus plantation artificially enriched with indigenous broad-leaved tree species (PNEN) located in the Hanthana mountain range, Sri Lanka. Arthropods randomly sampled from three randomly selected sites (5 m × 5 m) of each ecosystem were identified up to the highest possible taxa using standard identification keys. Woody and herbal vegetation was identified via a plant census. Arthropod and vegetation diversities were computed separately for each site using the Shannon–Wiener Index (H). Arthropods of 68 species and 43 families were found. AQ had the greatest arthropod diversity (H = 2.642), dominated by Olios spp., followed by BL (H = 2.444), dominated by a tettigonid species, Oxytate spp. and Psechrus spp. PN was third (H = 1.411), dominated by Dicaldispa spp. PNEN had the lowest (H = 1.3500), dominated by an ant species. Contrastingly, PNEN had the highest plant diversity (H = 2.614) and PN, the lowest (H = 0.879). In AQ, BL, and PN, the arthropod diversity was linearly dependent on plant diversity (R² = 0.423, ≤ 0.001), whereas it was not so when PNEN was also included (R² = 0.008, ≤ 0.001). This shows that higher plant diversity contributes to greater arthropod diversity in ecosystems where human intervention is minimal. But this pattern was not visible in PNEN, which is an artificially created ecosystem.