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The overactivation of microglia is known to trigger inflammatory reactions in the central nervous system, which ultimately induce neuroinflammatory disorders including Alzheimer’s disease. However, increasing evidence has shown that menaquinone-4 (MK-4), a subtype of vitamin K2, can attenuate inflammation in the peripheral system. Whereas it was also observed at high levels within the brain, its function in this organ has not been well characterized. Therefore, we investigated the effect of MK-4 on microglial activation and clarified the underlying mechanism. Mouse microglia-derived MG6 cells were exposed to lipopolysaccharide (LPS) either with or without MK-4 pretreatment. Cell responses with respect to inflammatory cytokines (Il-1β, Tnf-α, and Il-6) were measured by qRT-PCR. We further analyzed the phosphorylation of TAK1, IKKα/β, and p65 of the NF-κB subunit by Western blotting. We observed that in LPS-induced MG6 cells, MK-4 dose-dependently suppressed the upregulation of inflammatory cytokines at the mRNA level. It also significantly decreased the phosphorylation of p65, but did not affect that TAK1 and IKKα/β. Furthermore, the nuclear translocation of NF-κB in LPS-induced MG6 cells was inhibited by MK-4. These results indicate that MK-4 attenuates microglial inflammation by inhibiting NF-κB signaling.

The content and composition of dietary supplements is of great interest due to their increasing consumption and variety of available brand offered in the market. Accurate determination of vitamins is important for the improvement of dietary supplement quality and nutrition assessments. In this regard, the simultaneous determination of vitamin D3 (calcitriol—CT and cholecalciferol—CHL) and K2 (menaquinone-4—MK-4 and menaquinone-7—MK-7) in dietary supplements was developed by using ultra-high-pressure liquid chromatography (UHPLC). The overall runtime per sample was above 35 min, with the retention times of 2.40, 6.59, 7.06, and 32.6 min for vitamin D3 (CT and CHL) and vitamin K2 (MK-4 and MK-7), respectively. The limits of detection and limits of quantification for the target nutritional compounds ranged between 0.04–0.05 µg/mL, respectively. The validation results indicated that the method had reasonable linearity (R2 ≥ 0.9990), good recovery (>82%), satisfactory intra-day precision (≤1.9%) and inter-day precision (≤3.5%), and high selectivity and specificity. The validated UHPLC method was demonstrated to be precise, accurate, and robust for the simultaneous determination of vitamins D3 (CT and CHL) and K2 (MK-4 and MK-7) in dietary supplements.

Initially discovered for its role in blood coagulation, there is now convincing evidence that vitamin K (VK) has important actions in the nervous system. In brain, VK is present in the form of menaquinone-4 (MK-4), a byproduct of the main dietary source, phylloquinone. It contributes to the biological activation of various proteins (i.e., Gas6), and participates in the synthesis of sphingolipids, a class of lipids widely present in brain cell membranes with important cell signaling functions. In a previous study, we reported that lifetime consumption of a low VK diet resulted in mild cognitive impairment in aged rats, a finding associated with an alteration of the sphingolipid profile. To confirm the role of VK as it relates to sphingolipids, cognition, and behavior outside the context of aging, we conducted a study of acute VK deficiency using a pharmacological model of VK deficiency in brain. In this procedure, rats (8 weeks) are maintained on a ratio of warfarin (a VK antagonist) to VK whereby coagulation is maintained while inducing VK deficiency in extrahepatic tissues. After 10 weeks of treatment, rats who were subjected to the warfarin plus phylloquinone protocol (WVK) exhibited longer latencies in the Morris water maze test as well as lower locomotor activity and exploratory behavior in the open field test, when compared to control rats. The WVK treatment resulted in a dramatic decrease in MK-4 level in all brain regions despite the presence of high local concentrations of phylloquinone, which suggests an inhibition of the biosynthetic MK-4 pathway in the presence of warfarin. Additionally, WVK treatment affected sphingolipid concentrations in key brain regions, notably those of the ganglioside family. Finally, brain MK-4 was correlated with performances in the open field test. This study confirms the modulatory role of VK in cognition and behavior and the implication of sphingolipids, notably those of the ganglioside family.

The effective delivery of menahydroquinone-4 (MKH), an active form of menaquinone-4 (MK-4, vitamin K2(20)), to the skin is beneficial in the treatment of various skin pathologies. However, its delivery through the application of MK-4 to the skin is hampered due to the photoinstability and phototoxicity of MK-4. This study aimed to evaluate the potential of ester prodrugs of MKH for its delivery into the skin to avoid the abovementioned issues. The ester prodrugs, MKH 1,4-bis-N,N-dimethylglycinate hydrochloride (MKH-DMG) and MKH 1,4-bis-hemisuccinate (MKH-SUC), were prepared using our previously reported methods. Photostability was determined under artificial sunlight and multi-wavelength light irradiation, phototoxicity was determined by intracellular ROS formation and cell viability of UVA-irradiated human epidermal keratinocyte cells (HaCaT), and delivery of MKH into HaCaT cells was assessed by measuring menaquinone-4 epoxide (MKO) levels. MKH prodrugs showed higher photostability than MK-4. Although MK-4 induced cellular ROS and reduced cell viability after UVA irradiation, MKH prodrugs did not affect either ROS generation or cell viability. MKH prodrugs enhanced intracellular MKO, indicating effective delivery of MKH and subsequent carboxylation activity. In conclusion, these MKH prodrugs show potential for the delivery of MKH into the skin without photoinstability and phototoxicity.

Hepatocellular carcinoma (HCC) shows poor prognosis owing to its very frequent recurrence even after curative treatment. Thus, an effective and safe long-term chemopreventive agent is strongly in demand. Menahydroquinone-4 (MKH) is an active form of menaquinone-4 (MK-4, vitamin K2) that is involved in the synthesis of vitamin K-dependent proteins in the liver. We hypothesized that efficient delivery of MKH might be critical to regulate HCC proliferation. The discovery of a suitable prodrug targeting HCC in terms of delivery and activation could reduce the clinical dose of MK-4 and maximize efficacy and safety. We previously showed that MKH dimethylglycinate (MKH-DMG) enables effective delivery of MKH into HCC cells and exhibits strong antitumor effects compared with MK-4. In this study, we prepared anionic MKH hemi-succinate (MKH-SUC) and non-ionic MKH acetate (MKH-ACT), in addition to cationic MKH-DMG, and evaluated MKH delivery profiles and antitumor effects in vitro. MKH-SUC showed the highest uptake and the most efficient release of MKH among the examined compounds and exhibited rapid and strong antitumor effects. These results indicate that MKH-SUC might have a good potential as an MKH delivery system for HCC that overcomes the limitations of MK-4 as a clinical chemopreventive agent.

In this study, we developed a column-switching high-performance liquid chromatography (HPLC) method with fluorescence detection for the analysis of vitamin K. Column-switching is accomplished by changing the direction of flow using a switching valve with a set time program. Using this method, three vitamin K, phylloquinone (PK), menaquinone-4 (MK-4), and menaquinone-7 (MK-7), were separated and identified with high sensitivity, and impurities were eliminated. This method was used to determine the vitamin K content in meat, fish meat, snails, bivalves, sea urchins, seaweeds, vegetables, tea, soy products, milk products, and supplements. The results showed that chicken showed the highest content of MK-4 (15.35 ± 0.35 μg/100 g), matcha showed the highest content of PK (3069.66±80.10 μg/100 g), and dried natto showed the highest content of MK-7 (3997.57±79.42 μg/100 g). This method can also be used to analyze vitamin K in supplements and pharmaceuticals. The results of this study revealed that different manufacturers add different types of vitamin K to their commercial supplements and infant formulas. The developed method provides highly reproducible and quantitative results and allows for the rapid analysis of the three vitamin K types. Thus, the method developed in this study may aid the sequential analysis of vitamin K in different samples to assess food nutrients.

The absence of vitamin E from the diet can lead to cardiovascular disease, cancer, cataracts, and premature aging. Vitamin K deficiency can lead to bleeding disorders. These fat-soluble vitamins are important nutritional factors that can be determined in different methods in vegetables. In this work, the simultaneous determination of α-tocopherol, α-tocopheryl acetate, phylloquinone, and menaquinone-4 by gas chromatography–mass spectrometry (GC–MS) has been optimized using both direct injection and solid phase microextraction (SPME). Three different sample pre-treatment approaches based on: (A) solid–liquid–liquid–liquid extraction (SLE–LLE), (B) SLE, and (C) SPME were then applied to extract the target analytes from vegetables samples using menaquinone as internal standard. All the procedures allowed the determination of the target analytes in onion, carrot, celery, and curly kale samples. Similar results were obtained with the three different approaches, even if the one based on SPME offers the best performance, together with a reduced use of solvent, time consumption, and experimental complexity, which makes it the preferable option for industrial applications.

Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, β-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic β-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects.

Late-onset hypogonadism (LOH) is an age-related condition characterized by a decline in testosterone (Ts) levels and associated symptoms that impair quality of life in older men. Although Ts replacement therapy is available, its clinical use is limited by adverse effects. Vitamin K (VK) is a fat-soluble vitamin that functions as a cofactor for γ-glutamylcarboxylase and plays important roles in blood coagulation and bone homeostasis. Menaquinone-4 (MK-4), a VK homolog predominantly found in animal-derived foods, has been shown to enhance Ts production in healthy male rats. However, whether this effect occurs under low-Ts conditions remains unclear. In this study, we investigated the effects of VK on LOH using a leuprorelin acetate (LA)-induced low-Ts rat model. Male Sprague–Dawley rats were administered sustained-release LA and fed a control diet or diets supplemented with VK1 or MK-4 (75 mg/kg) for 4 weeks. Compared with the control group, MK-4 supplementation significantly ameliorated the reduction in serum Ts levels and seminiferous tubule diameter, whereas VK1 supplementation showed no significant effects. Furthermore, MK-4 supplementation activated the protein kinase A signaling pathway, which is directly involved in testicular Ts production. These findings suggest that MK-4 supplementation may represent a novel nutritional strategy for the management of LOH.

Diabetic osteoporosis (DOP), one of the usual complications in diabetic patients, poses a significant threat to bone health. Type H vessels in metaphysis and medial cortical bone are associated with osteogenesis. As a form of Vitamin K menaquinone-4 (MK-4) is a potential treatment for osteoporosis. We aimed to investigate whether MK-4 ameliorates DOP by promoting bone formation through protecting type H vessels and its associated mechanisms. High fat diet (HDF) feeding and streptozotocin (STZ) injection were applied to establish a mouse model of type 2 diabetic osteoporosis (T2DOP). Micro-CT, Masson staining, HE staining and IHC staining were applied to observe bone mass and the osteoblastic ability of osteoblasts. Tissue immunofluorescence (IF) staining and flow cytometry were employed to assess alteration of type H blood vessels. In vitro, to evaluate the functional level and mitophagy of ECs under high glucose conditions, wound healing assay, tube formation assay, EdU assay and IF were employed. Osteogenic differentiation ability in vitro was evaluated by ALP staining, AR staining, Western blot and RT-qPCR. MK-4 alleviated type H vessel injury and angiogenesis-dependent osteogenesis in DOP mice, thereby maintaining the bone mass. The vitro results showed that MK-4 could mitigate the dysfunction of ECs subjected to HG treatment, and further facilitate the osteogenic differentiation of MC3T3-E1 cells. Moreover, mechanism exploration found that PINK1/Parkin-mediated mitophagy was required for the impact of MK-4 on ECs. Meanwhile, ERK signal pathway is necessary for the improvement of MK-4 in PINK1/Parkin-mediated mitophagy. MK-4 is capable of alleviating the PINK1/Parkin-mediated mitophagy of ECs via the ERK pathway, thereby facilitating angiogenesis-dependent bone formation and further ameliorating DOP.