The biological responses of vitamin K2: A comprehensive review

Abstract Vitamin K1 (VitK1) and Vitamin K2 (VitK2), two important naturally occurring micronutrients in the VitK family, found, respectively, in green leafy plants and algae (VitK1) and animal and fermented foods (VitK2). The present review explores the multiple biological functions of VitK2 from recently published in vitro and in vivo studies, including promotion of osteogenesis, prevention of calcification, relief of menopausal symptoms, enhancement of mitochondrial energy release, hepato‐ and neuro‐protective effects, and possible use in treatment of coronavirus disease. The mechanisms of action associated with these biological effects are also explored. Overall, the findings presented here suggest that VitK, especially VitK2, is an important nutrient family for the normal functioning of human health. It acts on almost all major body systems and directly or indirectly participates in and regulates hundreds of physiological or pathological processes. However, as biological and clinical data are still inconsistent and conflicting, more in‐depth investigations are warranted to elucidate its potential as a therapeutic strategy to prevent and treat a range of disease conditions.

This paper primarily and critically reviews the biological responses of VitK2, with the aim of clarifying the potential mechanistic pathways associated with its observed bioactivities, and of exploring its usefulness as a potential therapeutic strategy for var- clinical trials. The publications were retrieved using the search terms and text words: "vitamin K2" or "menaquinone" in combination with "health" or "diseases". The database search was supplemented by consulting the bibliography of the articles, reviews, and published meta-analyses. The literature research was not limited to a time period, but a particular focus was given to the studies from the past 20 years. Relevant articles were chosen after reviewing through all titles and abstracts, and full texts were obtained if the information contained in the title or abstract was insufficient to exclude the study. As the nature of the research in this area has been largely animal studies and case reports, we did not make a formal assessment of the quality of the research or undertake a formal systematic review with meta-analysis of the quality of evidence for those studies. When the forms of VitK (between VitK1 and VitK2) used were not specified, those studies were excluded unless the outcomes of the studies were considered to be important to report, such as the studies on COVID-19.

| B I OLOG I C AL RE S P ON S E TO VITAMIN K 2: MAIN FIND ING S AND D ISCUSS I ON
To explore the biological activities of VitK2 at multiple levels, extensive in vitro/in vivo studies and hundreds of clinical observations have been performed. Such studies have included, amongst others, morphological, biochemical, immunohistochemical, biometric, biomechanical, and molecular biological approaches. There is accumulating evidence that VitK2 acts on almost every system in the body, and has diverse roles through direct or indirect participation in, and regulation of hundreds of physiological and pathological processes . There is a body of evidence showing that such wide involvement of VitK2 is directly linked to calcium homeostasis, which participates in various physiological processes, and that the main role of VitK2 is to maintain a steady level of calcium in multiple biological pathways (Okano, 2016). Various aspects of these activities of VitK2 are discussed in this section (Figure 2), and Table 1 summarizes the studies discussed in this review linking VitK2 and health. Since its role in maintaining normal blood coagulation has been thoroughly documented as VitK's most well-known function, this bioactivity is not covered in this review.

| Promotion of osteogenesis and bone homeostasis
Bone formation and metabolism involve the nutrient calcium.
Stomach acid converts ingested calcium to calcium ions, which are absorbed into the blood with the help of calcium transport proteins, driven by vitamin D3. Blood calcium ions subsequently enter the bone, regulated by calcitonin, and form the phosphate salt, hydroxyapatite, which gets deposited in the bone. The free calcium in hydroxyapatite is further electrostatically bound to osteocalcin (OC), one of the major noncollagenous VitK-dependent proteins (VKDPs) found in the bones and secreted by osteoblasts, odontoblasts, and hypertrophic cartilage cells (Hauschka et al., 1989).
Such binding must be promoted by VitK to establish the physiological osteogenesis process (bone mineralization). Deficiency in VitK would lead to the movement of free calcium from the bone to the blood again (resorption), resulting in osteoporosis (Maresz, 2015).
However, deficiency of VitK is relatively unusual due to a continually recycled VitK usage process, or redox cycle, in our cells.
2.1.1 | Vitamin K redox cycle and activation of osteocalcin by Vitamin K The major bone constituents are proteins (30%) in the organic part and hydroxyapatite (70%) in the inorganic part. The organic part contains at least two specific VitK-dependent Gla proteins; OC and matrix Gla protein (MGP) (Azuma et al., 2014), which have F I G U R E 2 Biological responses of vitamin K2.
In vitro AD VitK2 possesses antiapoptotic and antioxidant effects and maybe a valuable protective candidate against the progression of AD via inactivating p38 MAP kinase pathway Hadipour et al. (2020) AD VitK2 protects neural cells against Aβ-toxicity probably via regulating PI3K associated-signaling pathway and inhibiting caspase-3mediated apoptosis Huang et al. (2021) AD & mitochondrial dysfunction VitK2 modulates mitochondrial dysfunction induced by 6-hydroxydopamine in SH-SY5Y cells via mitochondrial qualitycontrol loop through the Pink1/Parkin signaling pathway Tang et al. (2022) Arterial calcification VitK2 and pamidronate synergistically inhibit arterial calcification via the increased expression of tropoelastin Saito et al. (2007) Astrocyte dysfunction VitK2 decreases hypoxia-induced damage of astrocytes, provides high astrocyte activity, and reduces the production of ROS and superoxide oxide, with possible involvement of Gas6 and protein S  Tsang and Kamei (2002) Neuron toxicity MK-4 has the potential to protect neurons from methylmercury-induced cell death, without increasing intracellular glutathione levels Sakaue et al. (2011) Osteoblastogenesis & Osteoclastogenesis MK-4 stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture Koshihara et al. (2003) Osteogenesis MK-7 enhances vitamin D3-induced bone development in human mesenchymal stem cells Gigante et al. (2015) Osteoporosis Sulfomenaquinone biosynthesis from MK requires only two genes, cyp128 and stf 3 in Mycobacterium tuberculosis Sogi et al. (2016) In vivo (animal) Abdominal hernia VitK2 can inhibit the expression of MMP-2 and promote the increase of collagen expression to prevent abdominal hernia caused by collagen factors (rat) Chen et al. (2015) AD & Mitochondrial dysfunction Vitk2 is a mitochondrial electron carrier that rescues Pink1 deficiency and severe mitochondrial defects, resulting in more efficient ATP production Vos et al. (2012) Aortic vascular smooth muscle cell calcification MK-4 reduces the mineralization and calcification of rat aortic vascular smooth muscle cells by regulating the BMP-2 signaling pathway in order to attenuate the expression of Runx2. Cui et al. (2018) Behavioral perturbations 25% reduction in the locomotor activity of dietary VitK-deficient rats.
In the radial-arm maze assessment, a similar reduction in locomotor activity in the dietary VitK-deficient rats with no alteration in performance (short-term memory) Cocchetto et al. (1985) Cognition Lifetime consumption of a low-VitK (VitK1 and MK-4) diet resulted in cognitive deficits in the 20-month-old rats but did not affect cognition at 6 and 12 months of age, nor did it affect motor activity or anxiety at any age Carrié et al. (2011) Liver regeneration VitK2 can significantly improve the recovery of liver function after liver regeneration model in rats; when the dose is < 20mg/kg, the recovery of liver function is dose dependent Zhang et al. (2013) Osteoporosis VitK and D may have a synergistic effect on reducing bone loss (rat) Matsunaga et al. (1999) Osteoporosis MK-4 appears to target osteoblasts, consequently inhibiting bone loss induced by ovariectomy (rat) Asawa et al. (2004) Osteoporosis VitK2 promotes bone healing in a rat femoral osteotomy model with or without glucocorticoid treatment Iwamoto et al. (2010) TA B L E 1 (Continued)

Osteoporosis
The combined treatment with PTH1-34 and MK-4 may have a therapeutic advantage on bone healing around hydroxyapatitecoated implants in osteoporotic rats Li et al. (2018) Soft tissue calcification A high dose of VitK2 can suppress experimental calcification of soft tissues induced by vitamin D2 (rat) Seyama et al. (1996) Sphingolipids in brain MK-4 concentration was positively correlated with the concentrations of sulfatides and sphingomyelin, and negatively correlated with ganglioside concentration ( Zaragatski et al. (2016) Bone health in postmenopausal women MK-4 helps improve hip bone geometry and bone strength in postmenopausal women by improving BMC and FNW, but it has little effect on DXA-BMD Knapen et al. (2007) Bone health in postmenopausal women The suppression of serum levels of bone remodeling indices and the positive changes in lumbar spine BMD were observed in postmenopausal women following a 12-month intervention period using a diet enriched with calcium, vitamin D, and VitK1 or MK-7 Kanellakis et al. (2012) Bone health in postmenopausal women Long-term supplementation of MK-7 may help postmenopausal women to prevent bone loss by significantly decreasing the age-related decline in BMD and BMC at the lumbar spine and femoral neck, but not at the total hip Knapen et al. (2013) Bone health in postmenopausal women The use of melatonin, strontium (citrate), vitamin D3, and MK-7 has a positive effect on the prevention or treatment of osteopenia, osteoporosis, or other bone-related diseases Maria et al. (2017) Knapen et al. (2015) CKD High plasma dp-ucMGP level indicating a poor VitK status is a biomarker of kidney damage and cardiovascular risk in CKD patients. VitK2 supplementation may improve the carboxylation status of MGP Kurnatowska et al. (2016) Cognition A clinically and statistically significant association was seen between increased dietary VitK intake and better cognition and behavior among geriatric patients Chouet et al. (2015) Coronary artery calcification Gla protein is associated with coronary artery calcification and VitK status in healthy women. dp-ucMGP may serve as a biomarker of VitK status Dalmeijer et al. (2013) Coronary artery calcification The benefits of MK-4 supplementation were only observed in patients with VitK insufficiencies correlated with high PIVKA-2 baseline levels, reducing brachial-ankle pulse wave velocity, but not coronary artery calcification Treatment with MK-7 as an add-on to calcium and vitamin D increases the carboxylation of osteocalcin. But the treatment of postmenopausal women with osteopenia for 3 years did not affect biochemical markers of bone turnover, BMD, or bone microarchitecture Rønn et al. (2021) Abbreviations: 12-LOX, 12-lipoxygenase; AD, Alzheimer's disease; ATP, adenosine triphosphate; BMC, bone mineral content; BMD, bone mineral density; CKD, chronic kidney disease; Dp-ucMGP, desphospho-uncarboxylated-MGP; FNW, femoral neck width; HCC, hepatocellular carcinoma; HDGF, hepatoma-derived growth factor; HIF-1α, hypoxia-inducible factor-1; MAPK, mitogen-activated protein kinases; MGP, matrix Gla protein; MMP, matrix metalloproteinase; NQO1, NAD(P)H quinone oxidoreductase 1; PD, Parkinson's disease; PI3K, phosphatidylinositol 3-kinase; PIVKA-2, protein induced by VitK absence or antagonist-2; PKC, protein kinase C; PTH, parathyroid hormone; ROS, reactive oxygen species; RXR, retinoid X receptor; TLR, Toll-like receptor; VKDPs, vitamin K-dependent proteins.
TA B L E 1 (Continued) illustrated in Figure 3. Both VitK1 and VitK2 are involved in the activation of VKDPs, but long-chain menaquinones show better bioactivity, suggesting that greater hydrophobicity correlates with higher bioavailability and a longer half-life .
The carboxylation process of VKDPs begins with the conversion of the selective glutamate (Glu) residues on VKDPs to γcarboxyglutamate (Gla) residues by the γ-glutamyl carboxylase (GGCX) enzyme, using as cofactors hydroquinone, CO 2 and H 2 O.
During this process, the reduced form of VitK undergoes a transition from the hydroquinone form to an oxidized state, the 2,3-epoxide.
The Gla residues dissociate, releasing two H + and becoming negatively charged, enhancing their affinity for positively charged Ca 2+ through electrostatic interactions, forming a hydroxyapatite lattice preceding bone mineralization and completing the physiological osteogenesis process. At the same time, after carboxylation, VitK epoxide is further reduced by two separate two-electron reduction steps: to VitK (quinone) by VitK epoxide reductase, and to hydroquinone by a yet-to-be-defined VitK reductase (Tie & Stafford, 2017), possibly a NAD(P)H-dependent reductase (Ingram et al., 2013;Rishavy et al., 2013). Consequently, another Glu residue of VKDPs is carboxylated to Gla; ultimately into active OC being an osteogenic marker (Myneni & Mezey, 2017). Following this process, the majority of OC is accumulated in the bone matrix binding to calcium, while a small proportion of carboxylated-OC (cOC) (~20%) flows into the blood circulation (Wei et al., 2018). So far, it is uncertain if there is a connection between cOC in blood and vascular calcification. It has also been reported that 1,25-dihydroxyvitamin-D [1,25(OH) 2 D 3 ] can upregulate the activity of GGCX for OC production in humans and rats in a dose-dependent manner (Karl et al., 1985;Miyake et al., 2001); conversely, a downregulation effect is seen in mice models (Wen et al., 2018). No clear explanation has been suggested so far for such observed differences in OC between human/rat and mice models.
Depending on the levels of carboxylation on the Glu sites, different forms of OC [cOC, uncarboxylated OC (ucOC) and undercarboxylated OC], with varying calcium-binding affinities, can exist.
However, the latter two forms are often not accurately referred to in most studies, probably due to limitations in their measurement . ucOC or undercarboxylated OC is considered as a clinical indicator of VitK status, due to the dependency on VitK (Shiraki et al., 2010).
While contributing to bone formation by promoting calcium deposition, on the other hand, VitK2 can also prevent bone calcium dissolution and mobilization maintaining calcium homeostasis. Thus, positive effects of VitK2 on degenerative bone conditions, such as osteoporosis, have been suggested (Khalil et al., 2021).

| Regulation of osteoblasts and osteoclasts
Vitamin K2 also regulates the osteoblast function, including modulating the proliferation and differentiation of osteoblasts, inhibiting the induction of osteoblast apoptosis (Koshihara et al., 2003), and increasing the expression of osteogenic genes (Akbari & Rasouli-Ghahroudi, 2018). Increased OC production (Matsunaga et al., 1999) and bone-specific alkaline phosphatase activity (Asawa et al., 2004) were observed after exposure of osteoblasts to VitK2.
Even in the case of glucocorticoid damage, VitK2 still exhibited an F I G U R E 3 Vitamin K (VitK) redox cycle process. VitK-dependent protein (VKDP) carboxylation starts with the reduction of VitK (quinone) to hydroquinone by VitK epoxide reductase (VKOR); during carboxylation, the glutamate (Glu) residue on VKDP is converted to γ-carboxyglutamate (Gla) by γ-glutamyl carboxylase (GGCX) with CO 2 and O 2 as cofactors, while hydroquinone is oxidized to vitamin K epoxide. VitK epoxide is transformed to hydroquinone by two-step reduction by VKOR and yet-to-be-defined VitK reductase (VKR), possibly NAD(P)H-dependent reductase.
osteoprotective effect on osteoblasts (osteocyte density) and promoted bone healing in osteoporotic rat models (Iwamoto et al., 2010;Zhang et al., 2017). Some studies also showed that VitK2 ( In summary, evidence supports the role of VitK2 in maintenance of bone health: (i) increasing bone strength and density, (ii) increasing bone mineral content, (iii) inhibiting bone resorption, (iv) decreasing fracture risk, (v) reducing urinary calcium loss, (vi) lowering serum alkaline phosphatase levels, and (vii) upregulating cOC and carboxylated-MGP levels. This would suggest that VitK2 reduces bone calcium mobilization, increases bone calcium deposition, and strengthens bone construction. However, further investigations are required to support these observations. At the same time, VitK2 limits the occurrence of calcification in other organs due to reduced bone calcium loss (Maresz, 2015;Vermeer, 2012). Figure 4 presents a summary of the various effects of VitK on bone homeostasis.

| Prevention of cardiovascular calcification
Ninety-nine percent of bodily calcium is stored in bone, regulated in part by VitK2, with the remaining 1% circulating in the blood, muscle, and other tissues (Weaver, 2012). Low levels of VitK2 can cause disruption in binding between calcium and OC, leading to loss of calcium from bone and transportation of calcium to other tissues, which results in tissue calcification (Maresz, 2015). Therefore, calcification should be viewed as a standard part of the pathological aging process as the activity of various tissues or organs decreases, leading to functional decline (Roumeliotis et al., 2019). Microcalcifications have been seen in early intimal lesions of atherosclerotic human coronary arteries when considered healthy (Roijers et al., 2011). A notable organ is the pineal gland, which undergoes vigorous secretion in childhood and begins to decline in secretory function during puberty with significant calcification (Tan et al., 2018). Pineal

F I G U R E 4
Biological effects of vitamin K2 on bone homeostasis through promotion of osteogenesis, suppression of bone resorption, and via activity as a carboxylation cofactor; OC: osteocalcin; MGP: matrix Gla protein; cOC: carboxylated OC; cMGP: carboxylated-MGP; OPG: osteoprotegerin.
calcification has long been reported in humans and is seen in nearly all adults leading to reduced melatonin secretion (Goree et al., 1963), which has also been suggested to be associated with Alzheimer's disease (AD) (Song, 2019). In addition, arterial blood vessels are lifelong active tissues, and arterial calcification has been observed in conditions with high atherogenic levels, such as diabetes, oxidative stress, and chronic kidney disease (CKD) (Roumeliotis et al., 2019).
It is commonly seen in the aging population, with 96% of observed aortic and coronary artery calcification seen in people over the age of 70 (Lahtinen et al., 2015), causing arteriosclerosis and myocardial infarction, and accelerating mortality. It has also been recognized that cardiovascular calcification is an active and regulated process.

| Vascular calcification
Vascular calcification is a pathological process and is manifested by the extraosseous deposition of hydroxyapatite in the tunica media or tunica intima of the blood vessel walls (Wasilewski et al., 2019), with a consequent increased risk of hypertension, atheromatosis, ischemia, and myocardial infarction (Roumeliotis et al., 2019).
Emerging evidence from animal and clinical studies has shown that low VitK levels may be associated with vascular calcification and an elevated risk of cardiovascular diseases (CVDs). In women, an association with coronary artery calcification was seen (Dalmeijer et al., 2013).
VitK2 can effectively stabilize mobile calcium, reduce artery calcium levels, inhibit calcium deposition in the blood vessel walls, and prevent the occurrence of CVDs (Beulens et al., 2009;Brandenburg et al., 2015;Kurnatowska et al., 2016). Relevant mechanisms of VitK2 involvement have been suggested to include promotion of bone calcification, prevention of bone calcium loss, and reduction in the source of deposited calcium (Gigante et al., 2015); MGP activation to inhibit the calcification of blood vessels in rat (Cui et al., 2018); growth-arrestspecific gene6 (Gas6) protein modulation to prevent vascular smooth muscle apoptosis and the calcification process; and other pathways to inhibit the calcification of the vascular wall in rat (Qiu et al., 2017).

| Calcification modulated by matrix Gla protein
Matrix Gla protein is the most influential natural inhibitor of all types of calcification in the body and is closely associated with metabolism, Conformational combinations of MGP are possible among various species in the body, depending on its carboxylation and/or phosphorylation state, such as phosphorylated-carboxylated MGP (cMGP, the fully active form), dephosphorylated-uncarboxylated MGP (dp-ucMGP, the fully inactive form), dephosphorylated-carboxylated MGP (dp-cMGP), and phosphorylated-uncarboxylated MGP (ucMGP) . Total uncarboxylated MGP (t-ucMGP) includes dp-ucMGP, but mainly with ucMGP (Dalmeijer et al., 2013). The exact role of MGP phosphorylation remains still unclear but it is believed to be the most crucial step during the MGP activation process. The fully inactive circulating conformation, dp-ucMGP, is independently associated with (peripheral) vascular calcification and carotid femoral/aortic pulse wave velocity, suggesting that it might be a risk biomarker associated with mortality and CVD, allowing early intervention (Dalmeijer et al., 2013;Griffin et al., 2019;Wei et al., 2016). In a study with 40 patients undertaking orthopedic or abdominal surgery, higher plasma dp-ucMGP levels were observed in patients with previous CVD history compared to those without, although no significant difference in dp-cMGP was observed between groups. Postoperatively, the dp-ucMGP concentration was significantly increased in the group with CVD history, and possible causes including nutritional defects were suggested (Dahlberg et al., 2018), thus highlighting the fact that VK intake may have in such situations. Currently, it remains to be elucidated whether OC and MGP would exhibit antagonistic or other interactions.
It has also been found that MGP is capable of downregulating bone morphogenetic protein-2 (BMP-2) and the transforming growth factorβ function which results in vascular smooth muscle cell (VSMC) apoptosis (Li et al., 2008). There is evidence that such
The association of VitK2 involvement in the repression of pathological vascular calcification and the upregulation of Gas6 expression has been shown (Jadhav et al., 2022;Jiang et al., 2016). In a study conducted by Qiu et al. (2017) Western blotting detected that substantial aortic smooth muscle Gas6 expression was restored after VitK2 treatment and that a significant decrease of VSMC calcification and apoptosis induced by CaCl 2 and β-sodium glycerophosphate was also observed. Additionally, it was noted that R428 (an Axl inhibitor) in-

| Cardiac valvular calcification
The atrioventricular and arterial valves are the structural foundations to ensure the unidirectional flow of cardiac blood. Several studies have shown that VKAs (i.e., warfarin) promote coronary artery calcification and valvular calcification (El Asmar et al., 2014;Weijs et al., 2011). It has also been reported that increased plasma dp-ucMGP levels are associated with aortic valve calcification (Brandenburg et al., 2017). Thus, VitK2 supplementation has been suggested as a plausible intervention to inhibit the pathogenetic progression of these conditions (Brandenburg et al., 2015;Marquis-Gravel et al., 2016). However, in a recent study using a hypercholesterolemic mouse model of calcific aortic valve disease, an MK-4 diet did not beneficially impact aortic valve morphology but instead increased plasma levels of total cholesterol, triglycerides, and lowdensity lipoprotein (Weisell et al., 2021).

| Cartilage calcification
Increasing evidence shows that osteoarthritis (OA), the most common form of degenerative joint disease, significantly correlates with progressive loss of articular cartilage due to calcification in articular cartilage, synovial fluid, or synovial membranes (Hawellek et al., 2016;Rafael et al., 2014). A relationship between OA and VitK deficiency has been suggested (Misra et al., 2013).

Gla-rich protein (GRP) is the most recently identified VKDP and
was first identified in sturgeon calcified cartilage (Shea et al., 2015).
It is distributed primarily in bone, cartilage, skin, and vasculature, and possesses a high density of Gla residues (an estimated 15 in human), suggesting a strong calcium-binding affinity (Viegas et al., 2015).
GRP has recently been demonstrated to be directly associated with OA, through observing undercarboxylated GRP accumulation at sites of ectopic calcification in cartilage and synovial membranes of OA patients (Rafael et al., 2014). A similar association between OA and ucMGP/cMGP was also observed by Wallin et al. (2010) where an association between OA and carboxylation deficiency of both GRP and MGP was consistent. Thus, γ-carboxylation modification is also believed to be vital for both GRP and MGP as calcification inhibitors, and a deficiency in carboxylation of these VKDPs may play an important role in causing OA. It has also been suggested that serum ucOC levels could be used as a biomarker of OA, based on a single-arm clinical study of 25 Japanese patients with bilateral knee OA (Naito et al., 2012).
It has been suggested that GRP exhibits its calcification suppres- VitK2 inhibits the expression of MMP-2, thereby promoting collagen expression and normalizing damaged soft tissue (Chen et al., 2015).
In addition, VitK2 significantly suppressed experimental calcification of soft tissues of rats induced by vitamin D2 (Seyama et al., 1996).
Moreover, GRP has been suggested to be involved in the cross talk between inflammation and cartilage calcification in OA, through observing the effects on calcification and inflammation in control and OA cells (Cavaco et al., 2016), which promotes GRP as a potential therapeutic target. However, it is worth noting that there are controversial results regarding the role of VitK2 in the maintenance of bone (and vascular) health, which are summarized in a recent review (Mandatori et al., 2021).
Recommendations regarding VitK intake differ in various countries, but a consistent conclusion arises: there may be certain syner-

| Relief of menopausal symptoms
Hormonal and endocrine changes during menopause can greatly affect the reproductive system, bone density, and nervous and immune system function, among others. Osteoporosis, osteopenia, and increased calcification of abdominal aorta and carotid arteries in postmenopausal women have been noted in many studies (Nike et al., 2016;Wasilewski et al., 2019).
A meta-analysis of 19 randomized, placebo-controlled clinical trials encompassing 6759 participants showed that VitK2 plays an important preventive and therapeutic role for postmenopausal women with osteoporosis (Huang et al., 2015). Intake of MK-4 daily for 3 years improved hip bone geometry and bone strength in postmenopausal women by increasing bone mineral content (BMC), but no increase in bone mineral density (BMD) was observed (Knapen et al., 2007); MK-7 showed comparable effects in terms of improvement in both BMC and BMD (Knapen et al., 2013(Knapen et al., , 2015. Contrarily, no effects

| Hepatoprotection
The protective effects of VitK2 on liver regeneration after partial hepatectomy was studied by Zhang et al. (2013) who used the classical 2-acetamido-fluorene/partial hepatectomy model in Sprague-Dawley rats. In this study, VitK2 significantly increased serum albumin levels with concurrent reduction of the levels of alanine and aspartate aminotransferases, suggesting that VitK2 enhances liver regeneration. Further in vitro experiments suggested that such a liver regeneration effect could be related to the regulation of matrilin-2 and hepatic oval cell proliferation (Lin et al., 2014;Abdelhamid et al., 2019). The combined treatment of VitK2 and an angiotensinconverting enzyme inhibitor was found to clinically ameliorate a hepatic dysplastic nodule in a female patient (aged 66) with liver cirrhosis (Yoshiji et al., 2007). A recent study demonstrated the direct beneficial effect of VitK2 on the control of hyperlipidemia-related hepatic inflammation through activating Gas6 carboxylation via arresting monocyte-hepatocyte adhesion (Bordoloi et al., 2020).
In vitro and in vivo experiments also demonstrated the cytotoxic effect of VitK2 on hepatocellular carcinoma (HCC) cells, with several underpinning mechanisms being reported (Xv et al., 2018;Yamamoto et al., 2009). It was suggested that VitK2 inhibits cell growth by downregulation of cyclin D1 expression through suppressing NF-κB activation (IκB kinase (IKK)/IκB/NF-κB pathway) (Ozaki et al., 2007) or through PKCα/NF-κB and PKCɛ/PKD1/NF-κB pathways (Xia et al., 2012), and suppression of HIF-1α transactivation via inhibiting PKCδ (Xia et al., 2019). It has also been proposed that the inhibitory activity of VitK2 on HCC cell proliferation is associated with upregulation of the cell-cycle regulatory protein p21 transcription (Liu et al., 2007), downregulating hepatoma-derived growth factor expression , as well as activating PKA (Otsuka et al., 2004), causing subsequent G1 cell-cycle arrest (Hitomi et al., 2005). Thus, VitK2 inhibited HCC cell growth by suppressing the expression of cyclin D1 via the IKK/NF-κB pathway and, therefore, could be a useful strategy in treating HCC.
In addition, VitK2 also induces differentiation and apoptosis of HCC cells. For instance, VitK2 suppressed the malignancy of cultured HuH7 cancer cells and promoted a normal hepatocyte phenotype via inhibiting connexin 43 expression and enhancing connexin 32 activity, respectively (Kaneda et al., 2008). The apoptotic effect of VitK2 has also been suggested to be associated with the mitochondrial pathway (Xv et al., 2018) involving inhibition of MAPK system activation (Kanamori et al., 2007), and the extrinsic apoptosis pathway involving activation of p53, a tumor-repressor gene (Li et al., 2010). Furthermore, a preventive effect of VitK2 on tumor invasion was also reported in HCC cell lines via inhibition of MMP expression (Ide et al., 2009). The effects of VitK2 in various (i.e., lung, pancreatic, bladder, and leukemia) cancer cells through multiple signaling pathways are presented in Figure 5.
In summary, supplementation of the recommended dose of VitK2 may provide a positive effect on the prevention and treatment of malignant tumors. Since the mechanisms we have described so far are only part of the complete signaling network regulated by VKDPs, the controversy over the role of some proteins in various cancers would require further investigation. Some studies have yielded promising results, but there was a lack of focus on the specific carboxylated forms of VKDP when carrying out specific measurement and comparison of malignant tumors; therefore, a more comprehensive and unified comparative analysis cannot be performed in this review. In our view, an appropriate strategy or guideline for improving VitK2 status in patients with liver diseases should be established, as current recommendations might not be optimal. in the inner mitochondrial membrane. Coenzyme Q 10 (CoQ 10 ) is an essential VitK-like substance with a naphthoquinone ring and 10 isoprene units ( Figure 6). It is the most common ubiquinone in humans (Kurosu & Begari, 2010). Functions of CoQ 10 as a component of the mitochondrial respiratory chain are well established. Based on the electron transport effects of menaquinones and their structural similarity to CoQ 10 , albeit being less lipophilic, it has been hypothesized that VitK2 treatment of mitochondrial diseases could be advantageous, especially under hypoxic conditions, when the cytochromec-oxidase system cannot function normally. It could potentially be of some significance to ischemic cells in cases of stroke and myocardial infarction.

| Enhancement of mitochondrial energy release
Menaquinones also play an essential part in energy generation in prokaryotes, associated with their role in active electron transport, especially for Gram-positive bacteria under either aerobic or anaerobic respiration, such as Mycobacterium spp. In Gram-negative organisms, ubiquinones are used in aerobic respiration processes, whereas menaquinones are utilized under anaerobic conditions (Suvarna et al., 1998). Even in heliobacteria, VitK2, specifically the quinone unit, plays the role of the secondary electron acceptor in the process of successful photosynthesis (Kondo et al., 2015).
When glucose is broken down in the mitochondria (eukaryotes)/ in the cell membrane (prokaryotes), electrons are released from hydrogen and transported along the membrane by ubiquinones/menaquinones to oxygen, with the help of the enzymes in the respiratory chain, such as the cytochrome-c-oxidase system. Thus, water molecules are formed, causing protons to be pumped across the membrane, with ATP energy released at the same time (Pamplona, 2011).
The tautomerization between the core skeleton naphthoquinone and the naphthol structure in ubiquinones/menaquinones is considered to play a critical role in the electron transport system for various observed protective effects (Ivanova et al., 2018).
One published study attributed VitK2 as serving as a mitochondrial electron carrier and rescuing mitochondrial dysfunction due to Pink1 protein deficiency in the multicellular eukaryote D. melanogaster (Vos et al., 2012). This suggests that VitK2 has the potential to treat mitochondrial-associated diseases or defects in ubiquinone biosynthesis. Pink1 is a protein kinase located in the outer membrane of the mitochondria. It has the function of protecting mitochondria when the cell has abnormally high energy requirements. When mitochondria are damaged, the cell recognizes and clears the dysfunctional mitochondria through the Pink1 protein (Vos et al., 2012) and/or mitochondrial quality-control loop (Tang et al., 2022). It is now known that this steady-state imbalance is related to the occurrence of Parkinson's disease (PD) (Opdebeeck et al., 2019). Such a result indicates a molecular mechanism of VitK2 in the treatment of PD. However, in a 2019 study using human CoQ 10 -deficient cell lines and yeast carrying mutations in genes required for CoQ 6 biosynthesis, Cerqua and coauthors claimed that VitK2, despite reaching mitochondria, restored neither electron flow in the respiratory chain nor ATP synthesis (Cerqua et al., 2019).
It was considered that the role of VitK2 as electron carrier (if confirmed) might probably be restricted to Drosophila, rather than being a general phenomenon in eukaryotic cells.
In recent years, because of the lack of VitK2 biosynthesis enzymes in humans and the vital role played by VitK2 in bacteria, the design of inhibitors targeting the VitK2 biosynthetic pathway has received considerable attention. This is especially so for the design of novel and selective antimicrobial agents targeting multidrugresistant Gram-positive pathogens including Mycobacterium tuberculosis (Sogi et al., 2016).

| Neuroprotection
Oxidative and neuroinflammatory mechanisms of cellular damage are associated with many neurological disorders, including neurodegenerative conditions such as Alzheimer's disease (AD) and Parkinson's disease (PD). An increasing body of evidence suggests the possible role of VitK supplementation as a novel neuroprotective strategy in the maintenance of nerve integrity and normal brain function, including cognition and behavior (Carrié et al., 2011;Chouet et al., 2015;Cocchetto et al., 1985;Ferland, 2013;Josey et al., 2013).
Modulatory roles of VitK in cognition and behavior and in sphingolipid homeostasis have been supported by a growing body of preclinical evidence. Following a 6-month treatment with a VitK1 diet, rats had increased MK-4 levels in various brain regions, with significantly higher MK-4 levels in myelinated regions (the pons medulla and midbrain; Carrié et al., 2004). The same research group also revealed that long-term VitK1 and MK-2 depletion correlated with increased cognitive impairment, as measured by the Morris Water Maze (MWM) test in rat models, especially in older animals (20 months), who took longer to perform the task (Carrié et al., 2011). In addition, this observation was further supported by human studies (Chouet et al., 2015;Soutif-Veillon et al., 2016).
In a behavioral-perturbations study, Cocchetto et al. (1985) observed a 25% reduction in locomotor activity in a rat group fed a VitK-deficient diet compared with that of the control group in the open-field paradigm assessment. Significantly less exploratory behaviors were also seen in the group receiving warfarin treatment.
The radial-arm maze assessment provided similar results in terms of F I G U R E 6 Chemical structure of CoQ 10 (ubiquinone, n = 9). reduction in locomotor activity by VitK dietary depletion, but no alteration in short-term memory was evident.
Several studies have provided evidence that MK-4 is the predominant form of VitK in both rat and human brain tissue Nakagawa et al., 2010), even though the majority of extrahepatic tissues have VitK1 and MK-4 (Ferland, 2012). MK-4 was reported to account for >98% of the total VK content in the rat brain between 6 and 21 months (Carrié et al., 2004). Sex, age, and diet also influence the concentration of MK-4 in brain .
Higher MK-4 levels in the cortex and cerebellum of female Brown Norway rats were observed compared to males on a similar diet.
It is well known that VitK is a necessary factor for the biosynthesis and metabolism of sphingolipids, by modulating certain key enzyme activities involved (Lev, 1979). Sphingolipids represent an important class of lipids, which exist in high concentrations in neuronal and glial cell membranes and function in brain cell events, including signaling, proliferation, differentiation, survival, synaptic transmission, neuronal-glial interaction, and myelin stability (Olsen & Faergeman, 2017). The major sphingolipids, including ceramide, sphingomyelin, cerebroside, sulfatides, and gangliosides (Ferland, 2012), are associated with neuroinflammation and neurodegeneration because of their effects on microglial activation and accumulation of amyloid precursor protein (Alisi et al., 2019). In rat experiments conducted by Carrié et al. (2004), MK-4 levels were found to be positively correlated with concentrations of sphingomyelin and sulfatides, and negatively correlated with ganglioside concentration in both low (80 μg/kg) and adequate (500 μg/kg) VitK1 diet groups. Crivello et al. (2010) also showed significant positive correlations between sulfatide and MK-4 levels in the hippocampus and cortex of 12-and 24-month male Fisher rats (n = 344) receiving VitK1 dietary supplementation, but no significant correlations were found in the striatum.
Due to the wide distribution of Gas6 in the central and peripheral nervous systems, the neurofunctions of Gas6 to maintain adequate cerebral homeostasis have been studied, including antiapoptotic, cell growth, mitogenic, myelinating activity in neuronal and glial cells (especially oligodendrocytes, Schwann cells, and microglia) (Ferland, 2012). Since Gas6 is a VKDP, it has been suggested that VitK2 must play an important protective role in the nervous system by regulating Gas6. Huang et al. (2021) suggested that VitK2's protective effect against amyloid β-protein (Aβ) cytotoxicity in neural cells might be through Gas6 activation, leading to upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt/Axl pathway and inhibition of caspase-3-mediated apoptosis. Aβ plaque accumulation in F I G U R E 7 The protective effect of vitamin K2 on neuroprotection. It is considered to be associated with modulating neurodegeneration, inflammation, and oxidative stress via with (green route) or without (red) the involvement of VKDPs (Gas6, Protein S). VKDP: vitamin Kdependent protein; Gas6: growth-arrest-specific gene 6; Aβ: amyloid β-protein; ROS: reactive oxygen species; MAPK: mitogen-activated protein kinase; PI3K: phosphatidylinositol 3-kinase; Akt: protein kinase B; Axl: Axl receptor tyrosine kinase; PKA: protein kinase A; TAM: Tyro3, Axl, and MERTK. the brain is clinically considered as a biocharacteristic feature of AD, being responsible for the massive neuronal death seen. Specifically, Hadipour et al. (2020) recently demonstrated that VitK2 protected PC12 cells against Aβ  and H 2 O 2 -induced apoptosis in a model of AD cell damage, via inactivating the p38 MAPK pathway. Similarly, Tsang and Kamei (2002) showed that both MK-4 and VitK1 en- In both primary cultured neurons and human neuroblastoma IMR-32 cells, MK-4 and VitK1 inhibited methylmercury-induced neuronal death without affecting glutathione levels (Sakaue et al., 2011), suggesting the potential of VitK treatment for neural disease conditions involving glutathione depletion. Evidence also indicates that VitK2 directly suppresses rotenone-induced activation of cultured microglial BV2 cells by inhibiting ROS production of reactive oxygen species and p38 MAPK activation (Yu et al., 2016), and by inhibiting the activation of 12-lipoxygenase in developing oligodendrocytes to prevent oxidative cell death (Li et al., 2009).

| Coronavirus disease
A recent review has collected evidence exploring the potential beneficial role of VitK in the pathogenesis of COVID-19 through proposed effects on coagulation and/or immuno-regulation ( Figure 8; Kudelko et al., 2021). By using a causal loop diagram, Goddek proposed a potential synergistic effect between VitK and vitamin D against COVID-19, to prevent long-term health risks (Goddek, 2020).
This was demonstrated by a prospective observational study involving 100 COVID-19 patients and 50 controls (median age 55), where worse disease severity was found to be positively and independently correlated with both VitK and vitamin D deficiency using the dp-ucMGP level as a biomarker (Desai et al., 2021), but the clinical study is lacking in order to prove whether optimization of VitK and vitamin D status could have such positive impact clinically.

F I G U R E 8
The potential modulating role of vitamin K (VitK) in thromboembolism and respiratory impairment related to COVID-19 pathogenesis. Following the entry of SARS-CoV-2 into alveolar type II cells, the virus causes lung epithelial cell and endothelial cell infection. The former leads to respiratory impairment, associated with (i) the production of pro-inflammatory cytokines [i.e., Interleukin 6 (IL-6), Tumor Necrosis Factor-alpha (TNFα), IL-1, and C-reactive protein (CRP)], causing acute respiratory distress syndrome (ARDS), and (ii) increased levels of metalloproteinases (MMPs) 8 and 9, inducing pulmonary fibrosis (grey route). The latter affects normal coagulation processes, resulting in hemostatic abnormalities including intravascular coagulopathy, thrombosis, pulmonary microthrombi, heart attacks, and stroke (red route). This pathogenesis is associated with insufficient carboxylation of Matrix Gla protein (MGP), Protein S, and Protein C; therefore, a modulating role of VitK is proposed (green route). Chiodini et al. (2021) very recently published a meta-analysis based on 54 studies, showing that COVID-19 patients requiring hospital admission and with low vitamin D levels present an increased risk of respiratory distress and mortality due to respiratory failure or other complications. This supports the protective role of administration of vitamin D against acute respiratory tract infection based on its anti-inflammatory properties as previously suggested (Martineau et al., 2019). However, pro-calcification effects of vitamin D in COVID-19 patients have been hypothesized, based on a recent study with 135 hospitalized patients, where vitamin D sufficient patients (25(OH)D >50 nmol/L) had increased degradation of elastic fiber in lung compared to those with mild deficiency (25(OH) D 25-50 nmol/L); whereas no difference in vitamin D level was seen between patients in good and bad (intubation and/or death) condition (Walk et al., 2020). This suggests that VitK might be considered when administration of calcium is needed for COVID-19 patients, in order to compensate for the potential negative consequence of fiber degradation caused by calcification. Similar findings have been further demonstrated in human trials with measurement of VitK alone, including a trial with 138 COVID-19 patients and 138 controls, where it was found that an elevated dp-ucMGP level was associated with mortality in patients, after adjusting for gender and age factors (Linneberg et al., 2021). These data so far suggest that there might be a pathway of pneumonia-induced extrahepatic VitK depletion in COVID-19 patients resulting in accelerated elastic fiber degradation and thrombosis formation, due to impaired activation of VKDPs (MGP and Protein S; Dofferhoff et al., 2021). A randomized, controlled phase-2 trial has recently been set up to investigate effects of VitK2 (MK-7) in COVID-19 (NCT04770740) (Hospital, 2021).
More immediate observational studies and larger randomized clinical trials are warranted to further evaluate the effect of VitK on the prognosis of COVID-19 patients and the beneficial impact of VitK supplementation on clinical severity.
In the meanwhile, we further suggest that monitoring and supplementation of VitK2 (or even VitK in general), based on recommended adequate daily doses of 90 mg (female >19 years old)/120 mg (male >19 years old) (NIH, 2021), could also be considered as a part of the COVID-19 treatment strategy in counteracting COVID-19 infection and reducing complications; thus, reinforcing the health maintenance and therapeutic potential of optimal circulatory levels of VitK. It is also recommended that more investigations using other biomarkers (rather than dp-ucMGP) to measure the VitK2 level are needed to provide more robust scientific evidence.

| CON CLUS ION
Vitamin K2, together with VitK1, is one of the important and essential naturally occurring vitamins for human health. This study provides a comprehensive overview of available data to add to the understanding of its biological 'fingerprint', and maps the effects of VitK2, extending from its well-known role in blood coagulation to promotion of osteogenesis, prevention of calcification, relief of menopausal symptoms, enhancement of mitochondrial energy release, and its protective effects on the liver and nerves. It is clear that VKDPs are a primary target for VitK2, such as OC, MGP, Gas6, and Protein S, thus controlling the body's various physiological functions by regulation of Ca 2+ distribution. Mitochondrial respiratory chains have been considered to be the second target of VitK2 in energy conversion and release, as well as cell survival through the electron transfer process. But there is currently limited proof for this claim, especially for mammalian cells.
Taken together, according to current studies, VitK, especially VitK2, is an important bioactive nutrient family for human health. The beneficial effects of VitK2 in various conditions emphasize the importance of VitK2 in the global diet, as evidenced by the results presented in this review. However, biological and clinical data are still inconsistent and conflicting; therefore, more in-depth investigations, including larger and longer duration trials with better design are warranted to optimize its use as a potentially useful strategy for the prevention and treatment of a range of disease conditions.

ACK N OWLED G M ENT
The authors would like to thank Kin Aik Kok for reviewing the references presented in Table 1.

FU N D I N G I N FO R M ATI O N
This research received no external funding.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
There is no primary data associated with this manuscript.