• Bovine milk fat globule epidermal growth factor Ⅷ activates PI3K/Akt signaling pathway and attenuates sarcopenia in rat model induced by D-galactose

      Li, He; Wang, Rongchun; Wang, Lifeng; Li, Lin; Ma, Ying; Zhou, Shaobo; Jiangsu Normal University; Harbin Institute of Technology; Northeast Agriculture University; University of Bedfordshire (Elsevier, 2020-12-17)
      To develop a more effective and safer treatment for sarcopenia, this research investigated the anti-sarcopenia mechanism of Milk Fat Globule Epidermal Growth Factor Ⅷ (MFG-E8) from the liver function and metabolism in sarcopenic model rat. After 4 weeks nutritional intervention experiment, MFG-E8 can significantly increase the gastrocnemius mass in rat. The mechanism of MFG-E8 in improving sarcopenia was related to its promotional capacity to the activities of superoxide dismutase (SOD) activity in serum, Glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) in liver. Meanwhile, MFG-E8 could also down-regulate obesity-related indicators, such as triglyceride (TG) and Non-esterified fatty acid (NEFA). The analysis of liver and gastrocnemius histopathology found that MFG-E8 could reduce the accumulation of fatty vesicles, improve liver function, thereby alleviating gastrocnemius tissue inflammation. In vitro experiments, myoblasts obtained from gastrocnemius tissue showed that MFG-E8 could reduce mitochondrial autophagy and inhibit cell apoptosis. In addition, MFG-E8 could up-regulate the phosphorylation level of PI3K via activating PI3K/Akt signaling pathway in gastrocnemius tissue, and promote the formation of muscle fibers, thereby increasing muscle mass. Moreover, MFG-E8 could also promote the formation of neuromuscular junctions by up-regulating the mRNA and protein expression of MusK in gastrocnemius.
    • Encapsulation of α-tocopherol in whey protein isolate/chitosan particles using oil-in-water emulsion with optimal stability and bioaccessibility

      Xu, Weili; Lv, Kangxing; Mu, Wei; Zhou, Shaobo; Yang, Yang; University of Bedfordshire; Harbin Institute of Technology (Elsevier, 2021-05-23)
      The aim of this study was to develop an oil-in-water (O/W) emulsion using whey protein isolate (WPI)-chitosan (CN) complex to encapsulate α-tocopherol and to characterize their stability and bioaccessibility in vitro. The O/W emulsions prepared under the optimal conditions (mass ratio of WPI:CN = 1: 1, corn oil containing 5 g/100 g of α-tocopherol) exhibited a monomodal distribution (d = 803.3 ± 6.9 nm) with encapsulation rate of 86.3 ± 2.3%. The emulsions were stable under NaCl (0–150 mmol/L), sugar (0–5 g/100 g), 55 °C for 30 min, pH 5–6.5, even storage for 20 d at 4 °C and 25 °C. During gastric digestion, WPI situated at the surface of emulsion particles can be digested into small molecular peptides by pepsin, but the structure of the core-shell particles remained due to the cross-linking with CN. During intestinal digestion, the structure of the particles disintegrated over the digestion time, and the inner-oil phase was released. Release profiles of the α-tocopherol and free fatty acids showed a burst effect followed by slow release. These results suggest that the WPI-CN complex could be used to achieve a controlled and sustainable release of liposoluble bioactive compounds from O/W emulsions.
    • Establishment of a stable complex formed from whey protein isolate and chitosan and its stability under environmental stresses

      Xu, Weili; Tang, Yinzhao; Yang, Yang; Wang, Guijie; Zhou, Shaobo; Harbin Institute of Technology; University of Bedfordshire (Elsevier, 2020-10-22)
      This study aimed to investigate the stability of a complex formed with whey protein isolate (WPI) and chitosan under environmental stress. The optical density, particle size, zeta potential, chemical characteristics, electrostatic interactions, and surface morphology were evaluated for the stable complexes; the optimum conditions for the generation of the stable complex were 0.2% (wt/wt) whey protein with 0.05% (wt/wt) chitosan at pH 5.7. Under these conditions, the complex particle size was 217.8 ± 11.3 nm and the zeta potential was 16.7 ± 0.92 mV. The complex was formed through electrostatic interactions between the amine groups of chitosan (-NH3+) and carboxyl groups of whey protein (-COO−), and contained a porous network interspaced by heterogeneously sized vacuoles. The complex displayed stable physiochemical characteristics under environmental stresses including NaCl (0–75 mM) or sugar (0–5%) at ambient temperature and upon heating for 15 min at 25–65 °C, up to 65 °C for 30 min. Moreover, the complex could be stably stored for 30 d at 4 °C and for 20 d at 25 °C. The present results provide theoretical insights into the industrial production of chitosan-protein complexes and for microencapsulation of sensitive food or medicinal ingredients to increase their intestinal absorption.
    • Gamma-tocotrienol stimulates the proliferation, differentiation, and mineralization in osteoblastic MC3T3-E1 cells

      Xu, Weili; He, Pan; He, Shenghua; Cui, Pengju; Mi, Yaqing; Yang, Yang; Li, Yang; Zhou, Shaobo; Harbin Institute of Technology; Harbin Medical University; et al. (Hindawi Publishing Corporation, 2018-01-15)
      Gamma-tocotrienol, a major component of tocotrienol-rich fraction of palm oil, has been suggested to exhibit bone protective effects in vivo. However, the effects of γ-tocotrienol on osteoblast cells are still unclear. In this study, the effects of γ-tocotrienol on the proliferation, differentiation, and mineralization in osteoblastic MC3T3-E1 cells were investigated. Our results showed that γ-tocotrienol (2–8 μmol/L) significantly improved the cell proliferation (), but it did not affect cell cycle progression. γ-Tocotrienol significantly increased alkaline phosphatase (ALP) activity (), secretion levels of osteocalcin (OC) and osteonectin (ON), and mRNA levels of collagen type I (Col I) of MC3T3-E1 cells. Meanwhile, we found that γ-tocotrienol is promoted in differentiation MC3T3-E1 cells by upregulation of the expression of Runx2 protein. Moreover, the number of bone nodules increased over 2.5-fold in cells treated with γ-tocotrienol (2–8 μmol/L) for 24 d compared to control group. These results indicated that γ-tocotrienol at low dose levels, especially 4 μmol/L, could markedly enhance the osteoblastic function by increasing the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Moreover, our data also indicated that Runx2 protein may be involved in these effects. Further studies are needed to determine the potential of γ-tocotrienol as an antiosteoporotic agent.
    • In vitro bioaccessibility and physicochemical properties of phytosterol linoleic ester synthesized from soybean sterol and linoleic acid

      Yang, Fuming; Oyeyinka, Samson A.; Xu, Weili; Ma, Ying; Zhou, Shaobo; Harbin Institute of Technology; University of Ilorin; University of Bedfordshire (Elsevier, 2018-02-14)
      Phytosterols are bioactive components capable of reducing cholesterol level in serum and reducing risk of arteriosclerosis. In this study, conditions for the synthesis of maximum yield of phytosterol linoleic ester (PLE) was optimized and the physicochemical properties and in vitro bioaccessibility of the PLE were assessed. Under the optimized condition of 1:1.1 mol ratio of phytosterol and linoleoyl chloride at 80 °C for 1.5 h, the conversion rate of phytosterol reached 96.1%. Its solubility in oil increased 20 times, up to 33.8%. Also, peroxide value of PLE was much lower than linoleic acid (32.9 and 47.0 mmol/kg), which means better oxidative stability. Bioaccessibility of PLE was affected by time, concentration of bile extract, and dissolved medium. It was 4.93% alone, increased by 2.5 times compare to phytosterol; or 53.46% in oil, under the condition of 40 mg/mL bile extract for 120 min. In conclusion, under the tested condition, phytosterol conversion rate, its solubility in oil and bioaccessibility were improved significantly. The method showed great potential in manufacture high quality and quantity of PLE.
    • MFG-E8 induced differences in proteomic profiles in mouse C2C12 cells and its effect on PI3K/Akt and ERK signal pathways

      Li, He; Guan, Kaifang; Li, Xu; Ma, Ying; Zhou, Shaobo; Harbin Institute of Technology; Jilin University; University of Bedfordshire (Elsevier, 2018-11-28)
      Milk fat globule-EGF factor 8 (MFG-E8) is one of the major proteins in milk fat globule membrane. In this study, mouse-derived C2C12 myoblast cells were served as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocytes formation comprehensively, a liquid chromatography coupled with tandem mass spectrometry label-free semi-quantitative  approach was used to analyse the myogenic C2C12 proliferation program. Over 1987  proteins were identified in C2C12 cells. The MFG-E8 (200 mg/mL) and MFG-E8 (500 26 mg/mL) with significant differences were compared based on the relative abundance. The result profiles of regulation of MFG-E8 to the expression of proteins in C2C12 cells revealed that differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across during the C2C12 cell proliferation process. Based on the analysis of  KEGG and STRING database, further to verification the expression of PI3K and ERK phosphorylation levels by Western blot. This study found that the data of proteomic was complementary to recent MFG-E8 studies of protein expression patterns in developing myotubes and provided a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development.
    • Milk fat globule membrane protein promotes C2C12 cell proliferation through the PI3K/Akt signaling pathway

      Li, He; Xu, Weili; Ma, Ying; Zhou, Shaobo; Xiao, Ran; Harbin Institute of Technology; University of Bedfordshire (Elsevier, 2018-04-07)
      Milk fat globule membrane (MFGM) protein is known to have several health benefits, including an anti-sarcopenia effect; however, its mechanism is unclear. The aim of this study was to investigate the potential mechanism of action of the MFGM protein. The MFGM protein was extracted and separated into 4 fractions, and Fraction 2 (57 % of total MFGM) demonstrated the greatest effect on C2C12 cell proliferation. Milk fat globule-EGF factor 8 (MFG-E8) accounted for 82.35 % of the MFGM protein. The effects of whole Fraction 2 (100 μg/mL, 200 μg/mL and 300 μg/mL) on cell proliferation and morphology were measured. Using qRT-PCR or a Western blot assay, several regulatory factors, e.g., PI3K P85α, p-pI3K p85α (Tyr 508), Akt, p-Akt (Ser 473), mTOR and p-mTOR (Ser 2448), were measured in cells incubated with 200 μg/mL of Fraction 2 with or without wortmannin. The results demonstrated that Fraction 2 induced C2C12 cell proliferation in a dose-dependent manner, upregulated the mRNA expression of mTOR and p70S6K, and activated PI3K, Akt, mTOR and P70S6K phosphorylation; however, Fraction 2 inhibited FOXO3a and 4E-BP. The results demonstrate that the MFGM protein, predominantly MFG-E8, promotes cell proliferation through the PI3K/Akt/mTOR signaling pathway. This study elucidated the molecular mechanism of the MFGM protein, primarily MFG-E8, in promoting C2C12 cell proliferation via the PI3K/Akt/mTOR/P70S6K signal pathway.
    • Separation and purification of the bovine milk fat globule membrane protein and its effect on improvement of C2C12 mouse skeletal muscle cell proliferation

      Li, He; Xu, Weili; Ma, Ying; Zhou, Shaobo; Harbin Institute of Technology; University of Bedfordshire (Royal Society of Chemistry, 2017-06-07)
      A novel method to improve the proliferation activity of C2C12 cells by the bovine milk fat globule membrane (MFGM) protein was established in this study. The MFGM protein was extracted and isolated into 4 fractions using an electric cream separator, and purified by a cellulose DEAE-52 column. Fraction 2 accounted for 57.8% of the total MFGM protein, and was used in the following study. The MTT assay showed that it induced cell proliferation activity, increased the cell survival rate and the cell number using flow cytometry and fluorescence microscopy analysis. There were only subtle changes in the morphology as observed using confocal scanning laser microscopy, but the number of mitochondria was significantly increased as observed using transmission electron microscopy analysis. Furthermore, the mRNA expression of MyoD, cyclin D1, p70S6K and mTOR was up-regulated as determined utilizing the quantitative real-time PCR assay, and the activation of Akt and mTOR phosphorylation was up regulated as determined using the Western blot assay. The main protein in fraction 2, assayed by 1-D gel electrophoresis and MALDI TOF-TOF, was identified as milk fat globule-EGF factor 8, the content was 65.6% of the total protein in fraction 2. The results elucidate a new molecular mechanism of the MFGM protein fraction 2: the activation of the Akt signal pathway in promoting cell proliferation.