GeneChem Inc.

Abstract

The safety of 3′-sialyllactose (3′-SL) sodium salt was evaluated by testing for gene mutations, in vivo and in vitro clastogenic activity, and animal toxicity in beagle dogs and rats. The results of all mutagenicity and genotoxicity tests were negative, indicating that 3′-SL does not have any mutagenic or clastogenic potential. The mean lethal dose (LD50) of 3′-SL sodium salt was well above 20 g/kg body weight (bw) in rats. A dose escalation acute toxicity study in Beagle dogs also indicated no treatment-related abnormalities. Subsequent 28-day and 90-day toxicity studies in Sprague- Dawley (SD) rats involved dietary exposure to 500, 1,000, and 2000 mg/kg bw of 3′-SL sodium salt and a water (vehicle) control. There were no treatment-related abnormalities on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, organ weights, relative organ weights, urinalysis parameters, or necropsy and histopathological findings. The No Observed Adverse Effect Level (NOAEL) of 3′-SL sodium salt was determined to be higher than 2000 mg/kg bw/day in an oral subchronic toxicity study in rats, indicating that the substance is an ordinary carbohydrate with the lowest toxicity rating. Results confirm that 3′-SL sodium salt has a toxicity profile similar to other non-digestible carbohydrates and naturally occurring human milk oligosaccharides (HMOs) and support its safety for human consumption in foods.

Abstract

We performed a series of toxicity studies on the safety of 6′-sialyllactose (6′-SL) sodium salt as a food ingredient. 6′-SL sodium salt, up to a maximum dose of 5000 μg/plate, did not increase the number of revertant colonies in five strains of Salmonella typhimurium in the presence or absence of S9 metabolic activation. A chromosomal aberration assay (using Chinese hamster lung cells) found no clastogenic effects at any concentration of 6′-SL sodium salt in the presence or absence of S9 metabolic activation. An in vivo bone marrow micronucleus test in Kunming mice showed no clastogenic activities with 6′-SL sodium salt doses up to 2000 mg/kg body weight (bw). In an acute toxicity study, the mean lethal dose of 6′-SL sodium salt was greater than 20 g/kg bw in rats. In a 13-week subchronic toxicity investigation, no effects were found at doses up to 5.0 g/kg bw of 6′-SL sodium salt in food consumption, body weight, clinical signs, blood biochemistry and hematology, urinalysis, or ophthalmic and histological macroscopic examination of organs. The no-observed-adverse-effect level (NOAEL) was 5.0 g/kg bw/day in rats.

Abstract

Sialyllactose (SL) is a representative human milk oligosaccharide (HMO) of human breast milk. The roles of SL in infant brain development and immunity have been reported in previous studies. In this study, we identified the impact of SL on innate immunity. Our results showed that the administration of SL had significant efficacy on bacterial clearance in Pseudomonas aeruginosa K-infected mice. We also examined the role of SL in the human THP-1 macrophage-like cell line. SL effectively promoted receptor-mediated endocytosis and phagocytosis. Furthermore, SL accelerated the recruitment of Rac1 to the cell membrane, leading to the generation of reactive oxygen species for the elimination of phagocytosed bacteria. Our findings provide a new perspective on the role of SL in breast milk and suggest its application as a therapeutic agent to treat bacterial and viral infections.

Abstract

BackgroundThis study assessed the gastrointestinal tolerance and safety of the human milk oligosaccharide 3’-sialyllactose sodium salt in subjects positive for Helicobacter pylori.

Methods

In a randomized, double-blind, placebo-controlled study, 48 adults positive for H. pylori were randomized to receive 12g of 3’-sialyllactose sodium salt or placebo (4g after breakfast, lunch, and dinner) for 4 weeks. We used one-way analysis of variance repeated measures analysis and a two-sample t-test to evaluate pre-and post-dose safety and efficacy. Physical examinations and clinical laboratory tests were performed to assess the primary endpoints of gastrointestinal symptoms, safety, and tolerability. The secondary endpoint was 13C-urea breath test values at week 4 compared with baseline.

Results

There were no clinically significant differences between pre- and post-dose gastrointestinal tolerance and clinical chemistry (serum biochemistry, hematology, and urine analysis) outcomes. Pre- and post-dose urea breath test values were not significantly different within or between the treatment and placebo groups. Differences in physical examination and vital signs in the treatment and placebo groups were not statistically significant. Compliance and adverse events were similar between the two groups.

Conclusions

3’-sialyllactose sodium salt was well tolerated, with no major side effects, but it did not effectively attenuate H. pylori positive status.

Abstract

3′-Sialyllactose has specific physiological functions in a variety of tissues; however, its effects on osteoarthritic development remain unknown. Here, we demonstrated the function of 3′-sialyllactose on osteoarthritic cartilage destruction. In vitro and ex vivo, biochemical and histological analysis demonstrated that 3′-sialyllactose was sufficient to restore the synthesis of Col2a1 and accumulation of sulphated proteoglycan, a critical factor for cartilage regeneration in osteoarthritic development, and blocked the expression of Mmp3, Mmp13 and Cox2 induced by IL-1β, IL-6, IL-17 and TNF-α, which mediates cartilage degradation. Further, reporter gene assays revealed that the activity of Sox9 as a transcription factor for Col2a1 expression was accelerated by 3′-sialyllactose, whereas the direct binding of NF-κB to the Mmp3, Mmp13 and Cox2 promoters was reduced by 3′-sialyllactose in IL-1β-treated chondrocytes. Additionally, IL-1β induction of Erk phosphorylation and IκB degradation, representing a critical signal pathway for osteoarthritic development, was totally blocked by 3′-sialyllactose in a dose-dependent manner. In vivo, 3′-sialyllactose protected against osteoarthritic cartilage destruction in an osteoarthritis mouse model induced by destabilization of the medial meniscus, as demonstrated by histopathological analysis. Our results strongly suggest that 3′-sialyllactose may ameliorate osteoarthritic cartilage destruction by cartilage regeneration via promoting Col2a1 production and may inhibit cartilage degradation and inflammation by suppressing Mmp3, Mmp13 and Cox2 expression. The effects of 3′-sialyllactose could be attributed in part to its regulation of Sox9 or NF-κB and inhibition of Erk phosphorylation and IκB degradation. Taken together, these effects indicate that 3′-sialyllactose merits consideration as a natural therapeutic agent for protecting against osteoarthritis.

Abstract

Sialyllactose (SL) is an abundant oligosaccharide in human milk with health benefits that include intestinal maturation, gut microbiota modulation, and cognitive development. Recent technological advances support large scale production of different forms of sialyllactose, which will enable their use as a food ingredient. The objective of the study was to investigate the dose-dependent effects of novel enzymatically-synthesized 3′-sialyllactose (3′SL) sodium salt supplemented to swine milk replacer on growth, hematological parameters and tissue histology in a pre-clinical neonatal pig model. Forty-five two-day-old male and female pigs were provided one of four experimental diets for 21 days. Diets were formulated to contain 0 (CON), 140 (LOW), 200 (MOD) or 500 (HIGH) mg/L of 3′SL sodium salt. Samples were collected on days 8 and 22 of the study for hematological and histological analyses. The addition of 3′SL sodium salt to formula at all doses was well-tolerated by neonatal piglets and supported growth and development comparable to those observed in the CON group. In addition, serum chemistries as well as hematology and organ microscopic structure were unaffected by 3′SL (p > 0.05). These data provide supportive evidence for the safety of supplementation of this enzymatically-synthesized 3′SL sodium salt to human infant formula.

Abstract

Sialylated milk oligosaccharides (SMOs) have a multifunctional health benefit, yet the molecular details underlying their potential role in modulating intestinal maturation remains unknown. To test the hypothesis that sialyllactose (SL) may mediate intestinal maturation and function through controlling neuronal function, studies were carried out where the diet of postnatal piglets was supplemented with a mixture of 3′- and 6′-sialyllactose from postnatal day 3 to 38. Gene transcription pathways regulating enteric nervous system function, polysialic acid (polySia) synthesis, and cell proliferation were quantified. Our new findings show that SL intervention: (1) upregulated the level of gene and protein expression of the glial-derived neurotrophic factor (GDNF) in the ileum; (2) upregulated phosphorylation of the cAMP responsive element-binding protein (CREB), the downstream target of GDNF signaling pathway; (3) promoted cell proliferation based on an increase in the number and density of Ki-67 positive cells in the crypts; (4) increased the crypt width in the ileum by 10%, while gene markers for the functional cells were not affected; (5) upregulated mRNA expression level of ST8Sia IV, a key polysialyltransferase responsible for synthesis of polySia-NCAM; (6) reduced the incidence and severity of diarrhea. These results show that SL promotes intestinal maturation in neonatal piglets by upregulating GDNF, synthesis of polySia and CREB-interactive pathway.

Abstract

Oligosaccharides are complex, non-digestible glycans found in large abundance in human milk. The abundance and the profile of bovine milk oligosaccharides and bovine milk based in infant formula differ from those in human milk. Recently, some human milk oligosaccharides (HMOs) have been supplemented to infant formula, however, not all forms have been available in large scale. The objective of the study was to investigate the dose-dependent effects of an enzymatically-synthesized 6′-sialyllactose (6′-SL) sodium salt supplemented to swine milk replacer on growth, hematological parameters, and organ microscopic assessment in our pre-clinical neonatal pig model. Two-day-old male and female pigs (n = 47) were provided one of four experimental diets for 21 days. Diets were formulated to contain 0 (CON), 300 (LOW), 600 (MOD), or 1200 (HIGH) mg/L of 6′-SL sodium salt. On days 8 and 22, samples were collected for hematological and histological analyses. Supplemental 6′-SL sodium salt at all doses supported growth and development comparable to those observed in control animals. In addition, serum chemistries, hematology, and organ microscopic structure were unaffected by 6′-SL (p > 0.05). Thus, addition of enzymatically-synthesized 6′-SL to a milk replacer formula supported growth and clinical outcomes similar to the control formula in the neonatal piglet.

Abstract

Breast milk contains human milk oligosaccharides (HMOs), including sialyllactose (SL). SL is composed of sialic acid and lactose, and is divided into 3′-SL and 6′-SL according to the binding position. SL has immunoprotective effects against bacteria and viruses, and acts as a probiotic in the gastrointestinal tract. In this study, we developed a bioanalytical method for simultaneous analysis of 3′-SL and 6′-SL in liver and kidney tissues of Yucatan minipigs using liquid chromatography–tandem mass spectrometry (LC-MS/MS) under conditions optimized in our previous study. LC-MS/MS was performed using a hydrophilic interaction liquid chromatography (HILIC) column (50 mm × 2.1 mm, 3 μm) with a mobile phase consisting of 10 mM ammonium acetate in water (pH 4.5) and acetonitrile with gradient elution at a flow rate of 0.3 mL/min. A surrogate matrix method using water was applied for analysis of endogenous SL. The developed method was validated with regard to selectivity, linearity, precision, accuracy, the matrix effect, recovery, parallelism, dilution integrity, carryover, and stability according to the US Food and Drug Administration guidelines. We performed a tissue distribution study of minipigs, and analyzed liver and kidney tissues using the developed method to determine the tissue distribution of 3′-SL and 6′-SL. The tissue concentrations of 3′-SL and 6′-SL were readily measurable, suggesting that the method would be useful for evaluating the tissue distributions of these compounds in minipigs.

Abstract

Sialyllactose (SL) is an acidic oligosaccharide, consisting of a combination of sialic acid and lactose. It is found in human milk. It has immune-protective effects against pathogens in newborns and helps with the development of the immune system and intestinal microorganisms. We developed and validated a method by which 3′-SL and 6′-SL levels were simultaneously analyzed via liquid chromatography–tandem mass spectrometry (LC–MS/MS), and evaluated the pharmacokinetics of the materials after systemic delivery to minipigs.

To improve chromatographic selectivity, several types of columns (C18, amide, and HILIC phase) were used to separate the peaks of 3′-SL and 6′-SL. Ultimately the HILIC phase column was selected, as it had a good peak shape and quick resolution. The mobile phase comprised ammonium acetate buffer and acetonitrile with gradient elution. MS was performed in the negative ion and multiple reaction monitoring modes. Plasma samples were prepared using the protein precipitation method with methanol. A surrogate matrix was used for quantification because SLs are endogenous plasma compounds.

The method developed was validated according to U.S. Food and Drug Administration guidance. A pharmacokinetic study was performed with intravenous administration of 3′-SL and 6′-SL in minipigs (Sus scrofa/Yucatan). The concentrations of 3′-SL and 6′-SL were readily measurable in the plasma samples, which suggests that the method adequately determined systemic exposure in minipigs.

Abstract

Sialyllactose (SL), an acidic oligosaccharide, has immune-protective effects against pathogens and helps with the development of the immune system and intestinal microorganisms. To elucidate the pharmacokinetic characterization after oral administration to rats, the simultaneous quantification method for 3′-SL and 6′-SL in rat plasma was validated, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an electrospray ionization (ESI) mode. Several types of columns [C18, amide, and hydrophilic interaction liquid chromatography (HILIC) phase] were used to separate the peaks of 3′-SL and 6′-SL, which improved chromatographic selectivity. Ultimately, the HILIC phase column had a good peak shape and quick resolution, with a mobile phase comprising ammonium acetate buffer and acetonitrile obtained by gradient elution. In addition, the simultaneous quantification of 3′-SL and 6′-SL in rat plasma samples were adequately applied to pharmacokinetic study.

Abstract

Osteoarthritis (OA) is a major degenerative joint disease. Oxidative stress and inflammation play key roles in the pathogenesis of OA. 3′-Sialyllactose (3′-SL) is derived from human milk and is known to regulate a variety of biological functions related to immune homeostasis. This study aimed to investigate the therapeutic mechanisms of 3′-SL in interleukin-1β (IL-1β)-treated SW1353 chondrocytic cells. 3′-SL potently suppressed IL-1β-induced oxidative stress by increasing the levels of enzymatic antioxidants. 3′-SL significantly reversed the IL-1β mediated expression levels of reactive oxygen species in IL-1β-stimulated chondrocytic cells. In addition, 3′-SL could reverse the increased levels of inflammatory markers such as nitrite, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2, IL-1β, and IL-6 in IL-1β-stimulated chondrocytic cells. Moreover, 3′-SL significantly inhibited the apoptotic process, as indicated by the downregulation of the pro-apoptotic protein Bax, upregulation of the anti-apoptotic protein Bcl-2 expression, and significant reduction in the number of TUNEL-positive cells in the IL-1β-treated chondrocytic cells. Furthermore, 3′-SL reversed cartilage destruction by decreasing the release of matrix metalloproteinases (MMP), such as MMP1, MMP3, and MMP13. In contrast, 3′-SL significantly increased the expression levels of matrix synthesis proteins, such as collagen II and aggrecan, in IL-1β-treated chondrocytic cells. 3′-SL dramatically suppressed the activation of mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways, which are related to the pathogenesis of OA. Taken together, our data suggest that 3′-SL alleviates IL-1β-induced OA pathogenesis via inhibition of activated MAPK and PI3K/AKT/NF-κB signaling cascades with the downregulation of oxidative stress and inflammation. Therefore, 3′-SL has the potential to be used as a natural compound for OA therapy owing to its ability to activate the antioxidant defense system and suppress inflammatory responses.

Abstract

Sialyllactoses (SL) are an abundant component of human milk. There have been many studies on the biological effects of SL in humans. SL can be produced using an economical method of enzyme synthesis. Although the European Food Safety Authority has published the human safety and appropriate intake dose of 6′-SL sodium salt as a novel food, it has suggested that the appropriate dose for particular medical purposes be judged on a case-by-case basis. Also, as revealed in the same report, there are no data on toxicity when 6′-SL is used in human intervention. However, clinical studies have only confirmed the safety of 3′-SL for therapeutic intervention in humans, and the safety for therapeutic use of 6′-SL, which is more abundant than 3′-SL in human milk, has not been confirmed. In this study, to determine the safety of 6′-SL use in humans, participants were randomly assigned to the placebo (maltodextrin) and 6′-SL groups, and then 3 g of powder was orally administered twice a day for 12 weeks. There were no serious adverse reactions, such as life-threatening complications requiring hospitalization, causing disability, or causing deformity during the use of 6′-SL. There were no clinically significant differences among the baseline, sixth, and twelfth week clinical chemistry tests, such as aspartate aminotransferase, alanine aminotransferase, and creatinine. Most of the adverse reactions were gastrointestinal problems such as diarrhea, abdominal discomfort, and bloating, with no significant difference in the proportions between the placebo and 6′-SL groups. These results support the safety of the 6′-SL for human use.

Abstract

Sialyllactose, known to be abundant in human breast milk, has anti-inflammatory properties, but its preventive effect on osteoarthritis remainunclear. Here, we demonstrated the efficacy of 3’ sialyllactose (3’ SL) and 6’ sialyllactose (6’ SL) in preventing osteoarthritis in Yucatan mini-pigs. Twelve female Yucatan mini-pigs were administered 0, 200, 400 mg 3’ SL or a combination of 200 mg 3’ SL + 200 mg 6’ SL for 12 weeks (4weeks before and 8 weeks after surgery); then, osteoarthritis was induced in the left knee by anterior cruciate ligament transection surgery. Kinematic variables were used to quantify gait analysis on the treadmill, and the degree of osteoarthritis was analyzed in the femur and tibia cartilage. It was confirmed that lameness of the left hind limb was reduced in all treated groups compared to the control group. Cartilage disruption was alleviated through macroscopic and microscopic observation of the knee joint. In addition, the expression of pro-inflammatory cytokines (IL-1β, TNF-α) and anti-inflammatory cytokines (IL-10, TGF-β) was decreased in human macrophages (THP-1) by 3’ SL. This reduction in cytokine expression was due to the maintenance of M0 macrophages, which did not differentiate into M1 or M2 macrophages. Thus, we suggest that 3’ SL and 6’ SL have the potential to act as natural therapeutic agents for the prevention of osteoarthritis.

Abstract

Background

Rheumatoid arthritis (RA) is a chronic inflammatory disease of joint, but there is no known cure.

3′-sialyllactose (3′-SL) is an oligosaccharide that is abundant in breast milk of mammals, and has anti-inflammatory properties. However, the efficacy of 3′-SL on RA remains unclear. The objective of the present study was to evaluate the therapeutic effect of 3′-SL after it was directly injected into the knee joint cavity of a RA minipig model.

Results

Minipig RA model was induced by intra-articular injection of bovine type II collagen emulsified with complete or incomplete Freund’s adjuvant into left knee joint. In clinical assessment, lameness and swelling of the hindlimb and increased knee joint width were observed in all animals. After the onset of arthritis, 3′-SL (0, 2, 10, and 50 mg/kg) was directly administered to the left knee joint cavity once a week for 4 weeks. Compared to the vehicle control group, no significant difference in macroscopic observation of the synovial pathology or the expression of inflammation-related genes (IL-1β, TNF-α, and COX2) in the synovial membrane of the knee joint was found. In microscopic observation, cell cloning of the articular cartilage was significantly reduced in proportion to the concentration of 3′-SL administered.

Conclusions

Our results suggest that intra-articular injected 3′-SL had a therapeutic effect on collagen-induced arthritis at the cellular level with potential as a medication for RA.

Abstract

Excessive production and migration of vascular smooth muscle cells (VSMCs) are associated with vascular remodeling that causes vascular diseases, such as restenosis and hypertension. Angiotensin II (Ang II) stimulation is a key factor in inducing abnormal VSMC function. This study aimed to investigate the effects of 6′-sialyllactose (6′SL), a human milk oligosaccharide, on Ang II-stimulated cell proliferation, migration and osteogenic switching in rat aortic smooth muscle cells (RASMCs) and human aortic smooth muscle cells (HASMCs). Compared with the control group, Ang II increased cell proliferation by activating MAPKs, including ERK1/2/p90RSK/Akt/mTOR and JNK pathways. However, 6′SL reversed Ang II-stimulated cell proliferation and the ERK1/2/p90RSK/Akt/mTOR pathways in RASMCs and HASMCs. Moreover, 6′SL suppressed Ang II-stimulated cell cycle progression from G0/G1 to S and G2/M phases in RASMCs. Furthermore, 6′SL effectively inhibited cell migration by downregulating NF-κB-mediated MMP2/9 and VCAM-1 expression levels. Interestingly, in RASMCs, 6′SL attenuated Ang II-induced osteogenic switching by reducing the production of p90RSK-mediated c-fos and JNK-mediated c-jun, leading to the downregulation of AP-1-mediated osteopontin production. Taken together, our data suggest that 6′SL inhibits Ang II-induced VSMC proliferation and migration by abolishing the ERK1/2/p90RSK-mediated Akt and NF-κB signaling pathways, respectively, and osteogenic switching by suppressing p90RSK- and JNK-mediated AP-1 activity.

Abstract

Disruption of the endothelial barrier function and reduction in cell migration leads to endothelial dysfunction. One of the most abundant human milk oligosaccharides, 6′-sialylactose (6′-SL), is reported to exert various biological functions related to inflammatory responses. In this study, we evaluated the effects of 6′-SL on lipopolysaccharide (LPS)-induced inflammation caused by endothelial barrier damage. Our results showed that LPS at 500 ng/mL strongly not only abolished cell migration but also hyperactivated MAPK and NF-κB pathways. 6′-SL suppressed LPS-induced endothelial inflammation via ERK1/2, p38, and JNK MAPK pathways. 6′-SL supported endothelial junctions by upregulating PECAM-1 expression and mRNA levels of tight junctions, such as ZO-1 and occludin, which were downregulated by LPS stimulation. It significantly inhibited the nuclear translocation of NF-κB, along with the downregulation of inflammatory cytokines, including TNF-α, IL-1β, MCP-1, VCAM-1, and ICAM-1. Furthermore, 6′-SL abolished NF-κB-mediated STAT3 in controlling endothelial migration and hyperpermeability via downregulating STAT3 activation and nuclear translocation. Finally, LPS induced over-expression of VCAM-1 and ZO-1 disassembly in both atheroprone and atheroprotective areas of mouse aorta, which were reversed by 6′-SL treatment. Altogether, our findings suggest that 6′-SL is a potent therapeutic agent for modulating inflammatory responses and endothelial hyperpermeability.

Abstract

Sialyllactose (SL) is the most abundant acidic oligosaccharide in human breast milk and plays a primary role in various biological processes. Recently, SL has attracted attention as an excellent dietary supplement for arthritis because it is effective in cartilage protection and treatment. Despite the superior function of SL, there are few pharmacological studies of SL according to blood concentrations in arthritis models. In this study, we investigated quantitative changes in SL and sialic acids in the plasma obtained from mini-pigs with osteoarthritis throughout exogenous administration of SL using liquid chromatography-multiple reaction monitoring mass spectrometry. Plasma concentrations of SL and sialic acids in the SL-fed group showed a significant difference compared to the control group. Mini pigs were fed only Neu5Ac bound to SL, but the concentration patterns of the two types of sialic acid, Neu5Ac and Neu5Gc, were similar. In addition, the relative mRNA expression level of matrix metalloproteinases (MMPs), which is known as a critical factor in cartilage matrix degradation, was remarkably decreased in the synovial membrane of the SL-fed group. Consequently, the temporal quantitative profiling suggests that dietary SL can be metabolized and utilized in the body and may protect against cartilage degradation by suppressing MMP expression during osteoarthritis progression.

Abstract

GNE myopathy, caused by biallelic mutations in the GNE gene, is characterized by initial ankle dorsiflexor weakness and rimmed vacuoles in the muscle histopathology, resulting in reduced sialic acid production. Sialyllactose is a source of sialic acid. We performed a pilot clinical trial to analyze the pharmacokinetic properties of 6′-sialyllactose (6SL) and evaluated the safety, and efficacy of oral 6SL in patients with GNE myopathy. Ten participants were in the pharmacokinetic study, and 20 in the subsequent clinical trial. For the pharmacokinetic study, participants were administered either 3 g (low-dose) or 6 g (high-dose) of 6SL in a single dose. Plasma concentrations of 6SL, sialic acid, and sialic acid levels on the surface of red blood cells were periodically assessed in blood samples. Patients were randomly allocated to test (low- and high-dose groups) or placebo groups for the trial. Motor function, ambulation, plasma 6SL and sialic acid concentrations, GNE myopathy-functional activity scale scores, and MRI findings were assessed. 6SL was well tolerated, except for self-limited gastrointestinal discomfort. Free sialic acid in both low- and high-dose groups significantly increased at 6 and 12 weeks, but not in the placebo group. In the high-dose group, proximal limb powers improved with daily 6SL. Considering the fat fraction on muscle MRI, results in the high-dose group were superior to those in the low-dose group. 6SL may be a good candidate for GNE myopathy therapeutics as it induces an increase or reduces the decrease in limb muscle power, attenuates muscle degeneration, and improves the biochemical properties of sialic acid.

Abstract

Acute lung injury (ALI) is the leading cause of respiratory diseases induced by uncontrolled inflammation and cell death. Lipopolysaccharide (LPS) is a major trigger of ALI in the progression through macrophage differentiation and the accelerated release of pro-inflammatory cytokines. The present study aimed to investigate the protective effects of human milk oligosaccharides, specifically 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL), on LPS-induced ALI and elucidate their underlying signaling pathways. The inhibitory effects of 3′-SL and 6′-SL on inflammation were evaluated using LPS-treated RAW 264.7 macrophages. To establish the ALI model, mice were treated with 10 mg/kg LPS for 24 h. Histological changes in the lung tissues were assessed using hematoxylin and eosin staining and immunofluorescence. LPS causes thickening of the alveolar wall infiltration of immune cells in lung tissues and increased serum levels of TNF-α, IL-1β, and GM-CSF. However, these effects were significantly alleviated by 100 mg/kg of 3′-SL and 6′-SL. Consistent with the inhibitory effects of 3′-SL and 6′-SL on LPS-induced pro-inflammatory cytokine secretion in serum, 3′-SL and 6′-SL suppressed mRNA expression of TNF-α, IL-1β, MCP-1, iNOS, and COX2 in LPS-induced RAW 264.7 cells. Mechanistically, 3′-SL and 6′-SL abolished LPS-mediated phosphorylation of NF-κB and STAT1. Interestingly, fludarabine treatment, a STAT1 inhibitor, did not affect LPS-mediated NF-κB phosphorylation. In summary, 3′-SL and 6′-SL protect LPS-induced macrophage activation and ALI through the STAT1 and NF-κB signaling pathways.

Abstract

Osteoporosis is a common skeletal disease that results in an increased risk of fractures. However, there is no definitive cure, warranting the development of potential therapeutic agents. 3′-Sialyllactose (3′-SL) in human milk regulates many biological functions. However, its effect on bone metabolism remains unknown. This study aimed to investigate the molecular mechanisms underlying the effect of 3′-SL on bone homeostasis. Treatment of human bone marrow stromal cells (hBMSCs) with 3′-SL enhanced osteogenic differentiation and inhibited adipogenic differentiation of hBMSCs. RNA sequencing showed that 3′-SL enhanced laminin subunit gamma-2 expression and promoted osteogenic differentiation via the phosphatidylinositol 3‑kinase/protein kinase B signaling pathway. Furthermore, 3′-SL inhibited the receptor activator of nuclear factor κB ligand-induced osteoclast differentiation of bone marrow-derived macrophages through the nuclear factor κB and mitogen‑activated protein kinase signaling pathway, ameliorated osteoporosis in ovariectomized mice, and positively regulated bone remodeling. Our findings suggest 3′-SL as a potential drug for osteoporosis.

Abstract

Aim

Endothelial hyperpermeability is an early stage of endothelial dysfunction associated with the progression and development of atherosclerosis. 3′-Sialyllactose (3′-SL) is the most abundant compound in human milk oligosaccharides, and it has the potential to regulate endothelial dysfunction. This study investigated the beneficial effects of 3′-SL on lipopolysaccharide (LPS)-induced endothelial dysfunction in vitro and in vivo.

Main methods

We established LPS-induced endothelial dysfunction models in both cultured bovine aortic endothelial cells (BAECs) and mouse models to determine the effects of 3′-SL. Western blotting, qRT-PCR analysis, immunofluorescence staining, and en face staining were employed to clarify underlying mechanisms. Superoxide production was measured by 2′,7′-dichlorofluorescin diacetate, and dihydroethidium staining.

Key findings

LPS significantly decreased cell viability, whereas 3′-SL treatment mitigated these effects via inhibiting ERK1/2 activation. Mechanistically, 3′-SL ameliorated LPS-induced ROS accumulation leading to ERK1/2 activation-mediated STAT1 phosphorylation and subsequent inhibition of downstream transcriptional target genes, including VCAM-1, TNF-α, IL-1β, and MCP-1. Interestingly, LPS-induced ERK1/2/STAT1 activation leads to the HMGB1 release from the nucleus into the extracellular space, where it binds to RAGE, while 3′-SL suppressed EC hyperpermeability by suppressing the HMGB1/RAGE axis. This interaction also led to VE-cadherin endothelial junction disassembly and endothelial cell monolayer disruption through ERK1/2/STAT1 modulation. In mouse endothelium, en face staining revealed that 3′-SL abolished LPS-stimulated ROS production and VCAM-1 overexpression.

Significance

Our findings suggest that 3′-SL inhibits LPS-induced endothelial hyperpermeability by suppressing superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis. Therefore, 3′-SL may be a potential therapeutic agent for preventing the progression of atherosclerosis.

Abstract

Sialyllactose (SL) is a functional human milk oligosaccharide essential for immune support, brain development, intestinal maturation, and antiviral defense. However, despite its established health benefits, the effect of SL on exercise performance and muscle mass in mice remains unknown. Here, we aimed to investigate, for the first time, the effects of 6′-SL on muscle functions. Seven-week-old male C57BL/6J mice were administered 100 mg/kg 6′-SL for 12 weeks, after which exhaustive treadmill performance was conducted. Moreover, muscle strength was examined by grip strength, and muscle phenotype characteristics such as muscle mass, muscle fiber size, and muscle protein expression were also examined. The administration of 6′-SL significantly improved exhaustive treadmill performance metrics, including distance and exhaustion time. Grip strength was also increased by 6′-SL administration. Additionally, 6′-SL increased muscle mass in both the gastrocnemius (GAS) and soleus. 6′-SL administration led to an increase in the minimum Feret’s diameter and the protein expression of total myosin heavy chain in the GAS muscle. In conclusion, 6′-SL administration in vivo led to increased running distance and time by increasing muscle mass and strength. These findings collectively indicate that 6′-SL is a potential agent for improving muscle health and exercise performance.

Abstract

6′-Sialyllactose (6′-SL), found in human breast milk, exhibits anti-inflammatory, immune function-enhancing, brain development-promoting, and gut health-improving effects. However, its effects on muscle fatigue remain unknown. Here, we aimed to investigate the effects of 6′-SL on blood lactate level, muscle fiber type, and oxidative phosphorylation protein complexes (OXPHOS) in muscle after exercise using C57BL/6J male mice. C57BL/6J mice were randomly assigned to control or 100 mg/kg 6′-SL. After 12 weeks of 6′-SL administration, the mice were made to perform treadmill exercise; their blood lactate and glucose levels were measured at the basal level (rest) and 0, 5, and 10 min after treadmill exercise. Results showed that 6′-SL treatment in C57BL/6J mice significantly reduced blood lactate level and improved blood glucose level. Moreover, 6′-SL increased the expression of slow-myosin heavy chain (MHC) and OXPHOS in gastrocnemius muscle. In addition, 6′-SL treatment for 12 weeks did not affect food intake, serum biomarkers of tissue injury, and lipid profiles compared with those of the controls. These findings indicate that non-toxic 6′-SL suppressed muscle fatigue during exercise by promoting protein expression of muscle fibers, especially slow-twitch muscle fibers characterized by abundant OXPHOS complexes and decreased blood lactate level. This study suggests that 6′-SL holds promise as a nutritional supplement in exercise and clinical settings, subject to further validation.

Abstract

Background/Objectives

3′-Sialyllactose (3′-SL), a human milk oligosaccharide, has anti-inflammatory effects and is demonstrated to have protective effects against osteoarthritis (OA) in vitro and in vivo. However, this hypothesis remains to be investigated in a clinical setting. Herein, we investigated the effects of 3′-SL on pain and physical function in patients with knee OA.

Methods

Sixty patients with knee OA with Kellgren and Lawrence grades (KL-grades) 1–4 and Korean Western Ontario and McMaster Universities Osteoarthritis Index (KWOMAC) scores ≥30 were randomly assigned to the placebo (n = 20), 3′-SL 200 mg (n = 20), and 3′-SL 600 mg (n = 20) groups. For 12 weeks, 3′-SL or placebo was administered to patients once a day. Clinical efficacy was evaluated using a visual analog scale (VAS) for pain and KWOMAC for physical function at baseline and at 6 and 12 weeks. Adverse effects were assessed for 12 weeks.

Results

Significant reductions in VAS and KWOMAC scores were observed at 12 weeks compared with the baseline in the 3′-SL group. No severe adverse effects were observed over 12 weeks.

Conclusions

3′-SL reduced pain in patients with knee OA, improved daily life movements, and was safe, suggesting that 3′-SL might be an effective treatment for knee OA without severe side effects.

Abstract

Macrophages play a central role in cardiovascular diseases, like atherosclerosis, by accumulating in vessel walls and inducing sustained local inflammation marked by the release of chemokines, cytokines, and matrix-degrading enzymes. Recent studies indicate that 6'-sialyllactose (6'-SL) may mitigate inflammation by modulating the immune system. Here, we examined the impact of 6'-SL on lipopolysaccharide (LPS)-induced acute inflammation using RAW 264.7 cells and a mouse model. In vivo, ICR mice received pretreatment with 100 mg/kg 6'-SL for 2 h, followed by intraperitoneal LPS injection (10 mg/kg) for 6 h. In vitro, RAW 264.7 cells were preincubated with 6'-SL before LPS stimulation. Mechanistic insights were gained though Western blotting, qRT-PCR, and immunofluorescence analysis, while reactive oxygen species (ROS) production was assessed via DHE assay. 6'-SL effectively attenuated LPS-induced p38 MAPK and Akt phosphorylation, as well as p65 nuclear translocation. Additionally, 6'-SL inhibited LPS-induced expression of tissue damage marker MMP9, IL-1β, and MCP-1 by modulating NF-κB activation. It also reduced ROS levels, mediated by p38 MAPK and Akt pathways. Moreover, 6'-SL restored LPS-suppressed Nrf2 and HO-1 akin to specific inhibitors SB203580 and LY294002. Consistent with in vitro results, 6'-SL decreased oxidative stress, MMP9, and MCP-1 expression in mouse endothelium following LPS-induced macrophage activation. In summary, our findings suggest that 6'-SL holds promise in mitigating atherosclerosis by dampening LPS-induced acute macrophage inflammation.

Abstract

Sarcopenia is associated with various geriatric diseases, such as gait disorders, falls, malnutrition, and osteoporosis. Accordingly, interest in the prevention and treatment of sarcopenia has grown over the years. The human milk oligosaccharide (HMO) 6′-sialyllactose (6′-SL) is known to improve exercise performance, reduce muscle fatigue, and improve GNE myopathy; however, its effect on sarcopenia has not yet been reported. In this study, we aimed to investigate the efficacy of 6′-SL in dexamethasone-induced muscle atrophy, which is a widely used model for the study of sarcopenia. The effects of 6′-SL on differentiated C2C12 skeletal muscle cells and on mice were examined by treatment with 6′-SL in the presence or absence of dexamethasone. 6′-SL was found to inhibit the dexamethasone-induced decrease of MHC expression, as well as to prevent reduction in the number, length, and width of myotubes. Furthermore, the dexamethasone-induced upregulation of myostatin, muscle RING-finger protein-1 (MuRF1), and atrogin-1 were also inhibited by 6′-SL treatment. In mice, intraperitoneal administration of dexamethasone caused decreases in muscle fiber diameter, muscle weight, and exercise performance, most of which were significantly inhibited by oral treatment with 6′-SL. Therefore, utilization of 6′-SL could contribute to the prevention and treatment of muscle atrophy and sarcopenia.

Abstract

GNE myopathy is a rare genetic muscle disorder characterized by initial ankle dorsiflexor weakness and the presence of rimmed vacuoles in muscle histopathology. Biallelic mutations in the GNE gene are causative, leading to reduced production of sialic acid. In our previous clinical trial, we used 6′-sialyllactose (6SL) as a supplement to increase sialic acid levels and compared the effects of 6SL at doses of 3 g and 6 g. The findings from the trial revealed superior outcomes in muscle strength, attenuation of muscle degeneration, and bioavailability in the 6 g group.

This trial was planned to complement the lack of placebo arm from the previous trial and to provide more conclusive evidence for therapeutic value of 6SL in GNE myopathy. Of the 11 participants, five were allocated to the 6SL and six to the placebo group after undergoing 12 weeks of pre-study observation and stratified randomization. At every visit with an interval of 12 weeks for 48 weeks, all participants underwent muscle strength measurement, muscle MRI, biochemical evaluations, 6-min-walk test, and completed a questionnaire.

No safety concerns arose during the trial period. Muscle strength, excluding hand grip power, did not show a significant difference between the two groups, which is attributed to the lack of pronounced muscle strength decline in both groups. Hand grip power tended to decrease in both groups, and this decline was statistically significant in the placebo group (p = 0.0004). The fat fraction measured by MRI showed the most significant results in the posterior thigh. The increase in fat fraction, indicating muscle degeneration, was statistically significant between the two groups (p = 0.0004). Although no statistically significant differences were observed between the groups in anterior thigh and both anterior and posterior lower leg, a trend of slowed increase in fat fraction was noted in the 6SL group compared to the placebo group starting from 24 or 36 weeks. Resialylation of cell surface glycoconjugate was demonstrated in 6SL group by measuring lectin bindings on peripheral blood monocytes.

The GNEM-FAS, used to assess patient-reported outcomes, did not show statistical significance in the total score or any of the three domains. However, the tendency for scores in the self-care and upper extremity domains to rebound after 24 weeks in the 6SL group suggests the potential for long-term benefits.

The effect of 6SL on muscle strength appeared to be minimal compared to our previous clinical trial, likely due to the short duration of the study and the inclusion of relatively early-stage patients. However, the changes in fat fraction measured by muscle MRI and the results of biochemical assays are more promising, suggesting potential benefits with long-term administration in the future.

Abstract

6′-Sialyllactose (6′-SL) sodium salt is the sodium salt of a naturally occurring trisaccharide found in dairy products and human milk. 6′-SL sodium salt formulations produced by different methods have been the subject of other safety studies and are approved for use in food in the United States and other countries. Because the manufacturing process can influence the toxicological profile of a substance, studies were undertaken to assess the safety of 6′-SL sodium salt produced by a novel manufacturing process. The results of genetic toxicity tests (bacterial reverse mutation assay, an in vitro mammalian chromosome aberration test, and an in vivo micronucleus test in mice), and acute and 26-week repeated dose oral toxicity studies in rats for this novel 6′-SL sodium salt are reported herein. All studies were conducted according to Good Laboratory Practice (GLP) and the respective guidelines from the Korean Ministry of Food and Drug Safety (MFDS). The substance showed no evidence of genotoxicity in all three assays that were conducted to address this endpoint and was not acutely toxic to rats at doses up to 6000 mg/kg body weight (bw). The no observed adverse effect level (NOAEL) for toxicity in the 26-week study in both male and female rats was 6000 mg/kg bw/day, the highest dose administered. This new 6′-SL sodium salt is safe in rats at a higher level and for a longer period of time than previously studied in either rats or pigs, demonstrating a more robust safety profile.

Filing Date	\|	2007-04-13	Registration Date	\|	2009-02-25
Application Number	\|	10-2007-0036276	Registration Number	\|	10-0886650

Filing Date	\|	2007-06-11	Registration Date	\|	2009-03-05
Application Number	\|	10-2007-0056919	Registration Number	\|	10-0888513

Filing Date	\|	2008-09-09	Registration Date	\|	2009-08-21
Application Number	\|	10-2008-0088762	Registration Number	\|	10-0914525

Filing Date	\|	2007-12-07	Registration Date	\|	2010-04-30
Application Number	\|	21541215	Registration Number	\|	05209639

Filing Date	\|	2010-04-02	Registration Date	\|	2012-03-02
Application Number	\|	10-2010-0030154	Registration Number	\|	10-1125211

Journal

Date

Reference

DOI

Regulatory Toxicology and Pharmacology

2018.01.28

Regul Toxicol Pharmacol. 2018 Apr;94:83-90.

Abstract

Journal

Date

Reference

DOI

Regulatory Toxicology and Pharmacology

2018.03.27

Regul Toxicol Pharmacol. 2018 Jun;95:182-189.

Abstract

Journal

Date

Reference

DOI

Infection and Immunity

2018.12.19

Infect Immun. 2018 Dec 19;87(1):e00694-18

Abstract

Journal

Date

Reference

DOI

EC Nutrition

2018.09.10

EC Nutrition 13.9 (2018): 600-608

(원본 : https://ecronicon.net/assets/ecnu/pdf/ECNU-13-00499.pdf)

Abstract

Journal

Date

Reference

DOI

Journal of Cellular and Molecular Medicine

2017.08.07

J. Cell. Mol. Med. Vol 22, No 1, 2018 pp. 57-66

Abstract

Journal

Date

Reference

DOI

Regulatory Toxicology and Pharmacology

2018.11.16

Regul Toxicol Pharmacol. 2019 Feb:101:57-64

Abstract

Journal

Date

Reference

DOI

Molecular Neurobiology

2019.06.03

Mol Neurobiol. 2019 Dec;56(12):7994-8007

Abstract

Journal

Date

Reference

DOI

Nutrients

2020.04.09

Nutrients. 2020 Apr 9;12(4):1030

Abstract

Journal

Date

Reference

DOI

Molecules

2020.12.03

Molecules 2020, 25, 5721

Abstract

Journal

Date

Reference

DOI

Journal of Pharmaceutical and Biomedical Analysis

2020.12.07

J Pharm Biomed Anal. 2021 Feb 20:195:113827