Objective:The biosynthesis of pilose antler polysaccharides(PAPS)with anti-aging activity relies on the precise regulation of key enzymes;however,the identification of these enzymes is limited by traditional low-throu...Objective:The biosynthesis of pilose antler polysaccharides(PAPS)with anti-aging activity relies on the precise regulation of key enzymes;however,the identification of these enzymes is limited by traditional low-throughput techniques.This study constructed an integrated system combining metabolic engineering and high-throughput screening of fluorescent substrates,aiming to overcome the technical bottlenecks in the identification of key enzymes for PAPS synthesis and the optimization of their catalytic efficiency.Methods:Eighteen candidate genes related to carbohydrate metabolism were screened via transcriptome sequencing of deer antler stem cells.After prokaryotic expression and preliminary screening by high-performance liquid chromatography(HPLC),a self-designed fluorescent substrate(FAM-Glc-β1-3-PNPG)combined with a 96-well plate platform was used to achieve high-throughput optimization and screening of catalytic efficiency.Enzyme function was verified through kinetic analysis and in vitro antioxidant experiments.Results:The screened high-efficiency glycosyltransferase UGT-Wnt3a showed a 2.1-fold increase in catalytic efficiency compared with the wild type,with a maximum reaction rate(Vmax)of12.3μmol·min^(-1)·mg^(-1)and a Michaelis constant(Km)of 0.87 mM.The catalytic product of UGT-Wnt3a increased the superoxide dismutase(SOD)activity by 42%(P<0.01)and decreased the malondialdehyde(MDA)content by 28%(P<0.05)in the oxidative damage system.The detection efficiency of the new platform was 9 times higher than that of HPLC,and the limit of detection was reduced by 50 times.Conclusion:The screening system established in this study provides an innovative technical solution for the directed synthesis of PAPS and the development of anti-aging components,promoting the transformation of active components in traditional Chinese medicine from natural extraction to bioengineering-based production.展开更多
Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not w...Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE’s effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction(Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase(ALP), runt-related transcription factor 2(Runx2), osteocalcin(OCN), bone morphogenetic protein-2(BMP-2), and collagen I(COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200μg·mL^-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.展开更多
Kanglu Brand Pilose Antler Nourishing Cream, developed by the Navy Anti-aging Research Institute and the Hainan Fengmu Deer Breeding Farm, is a kind of high-tech nourishing cream. The cream is produced from the fresh ...Kanglu Brand Pilose Antler Nourishing Cream, developed by the Navy Anti-aging Research Institute and the Hainan Fengmu Deer Breeding Farm, is a kind of high-tech nourishing cream. The cream is produced from the fresh pilose antler of younger deer, and contains the quintessence of pilose antlers——the total fat and SOD. The SOD is regarded as the best anti-aging agent展开更多
应用凝胶过虑和离子交换层析等技术,从鹿茸(pilose antler or Cervus nipponTemminck)中分离纯化出一种多肽(peptide of pilose antler,PAP)。经高效液相色谱和N-末端氨基酸分析鉴定为单一多肽,氨基酸组成分析证明,PAP是由68个氨基酸组...应用凝胶过虑和离子交换层析等技术,从鹿茸(pilose antler or Cervus nipponTemminck)中分离纯化出一种多肽(peptide of pilose antler,PAP)。经高效液相色谱和N-末端氨基酸分析鉴定为单一多肽,氨基酸组成分析证明,PAP是由68个氨基酸组成,分子是为7200。药理研究证明,PAP对大鼠角叉菜胶性足肿胀具有明显的抑制作用并呈现良好的量—效关系。展开更多
文摘Objective:The biosynthesis of pilose antler polysaccharides(PAPS)with anti-aging activity relies on the precise regulation of key enzymes;however,the identification of these enzymes is limited by traditional low-throughput techniques.This study constructed an integrated system combining metabolic engineering and high-throughput screening of fluorescent substrates,aiming to overcome the technical bottlenecks in the identification of key enzymes for PAPS synthesis and the optimization of their catalytic efficiency.Methods:Eighteen candidate genes related to carbohydrate metabolism were screened via transcriptome sequencing of deer antler stem cells.After prokaryotic expression and preliminary screening by high-performance liquid chromatography(HPLC),a self-designed fluorescent substrate(FAM-Glc-β1-3-PNPG)combined with a 96-well plate platform was used to achieve high-throughput optimization and screening of catalytic efficiency.Enzyme function was verified through kinetic analysis and in vitro antioxidant experiments.Results:The screened high-efficiency glycosyltransferase UGT-Wnt3a showed a 2.1-fold increase in catalytic efficiency compared with the wild type,with a maximum reaction rate(Vmax)of12.3μmol·min^(-1)·mg^(-1)and a Michaelis constant(Km)of 0.87 mM.The catalytic product of UGT-Wnt3a increased the superoxide dismutase(SOD)activity by 42%(P<0.01)and decreased the malondialdehyde(MDA)content by 28%(P<0.05)in the oxidative damage system.The detection efficiency of the new platform was 9 times higher than that of HPLC,and the limit of detection was reduced by 50 times.Conclusion:The screening system established in this study provides an innovative technical solution for the directed synthesis of PAPS and the development of anti-aging components,promoting the transformation of active components in traditional Chinese medicine from natural extraction to bioengineering-based production.
基金supported by the National Natural Science Foundation of China(No.81473314)
文摘Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE’s effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction(Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase(ALP), runt-related transcription factor 2(Runx2), osteocalcin(OCN), bone morphogenetic protein-2(BMP-2), and collagen I(COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200μg·mL^-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.
文摘Kanglu Brand Pilose Antler Nourishing Cream, developed by the Navy Anti-aging Research Institute and the Hainan Fengmu Deer Breeding Farm, is a kind of high-tech nourishing cream. The cream is produced from the fresh pilose antler of younger deer, and contains the quintessence of pilose antlers——the total fat and SOD. The SOD is regarded as the best anti-aging agent
文摘应用凝胶过虑和离子交换层析等技术,从鹿茸(pilose antler or Cervus nipponTemminck)中分离纯化出一种多肽(peptide of pilose antler,PAP)。经高效液相色谱和N-末端氨基酸分析鉴定为单一多肽,氨基酸组成分析证明,PAP是由68个氨基酸组成,分子是为7200。药理研究证明,PAP对大鼠角叉菜胶性足肿胀具有明显的抑制作用并呈现良好的量—效关系。
