Huang Wen, Lao Linhui, Deng Yuliang, Li Ziwei, Liao Wanwen, Duan Shan, Xiao Suyao, Cao Yong, Miao Jianyin
Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of Food Science, South China Agricultural University, Guangzhou, China.
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China.
Front Nutr. 2022 Aug 2;9:960228. doi: 10.3389/fnut.2022.960228. eCollection 2022.
Casein phosphopeptides (CPPs) are good at calcium-binding and intestinal calcium absorption, but there are few studies on the osteogenic activity of CPPs. In this study, the preparation of casein phosphopeptide calcium chelate (CPP-Ca) was optimized on the basis of previous studies, and its peptide-calcium chelating activity was characterized. Subsequently, the effects of CPP-Ca on the proliferation, differentiation, and mineralization of MC3T3-E1 cells were studied, and the differentiation mechanism of CPP-Ca on MC3T3-E1 cells was further elucidated by RNA sequencing (RNA-seq). The results showed that the calcium chelation rate of CPPs was 23.37%, and the calcium content of CPP-Ca reached 2.64 × 10 mg/kg. The test results of Ultraviolet-Visible absorption spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) indicated that carboxyl oxygen and amino nitrogen atoms of CPPs might be chelated with calcium during the chelation. Compared with the control group, the proliferation of MC3T3-E1 cells treated with 250 μg/mL of CPP-Ca increased by 21.65%, 26.43%, and 28.43% at 24, 48, and 72 h, respectively, and the alkaline phosphatase (ALP) activity and mineralized calcium nodules of MC3T3-E1 cells were notably increased by 55% and 72%. RNA-seq results showed that 321 differentially expressed genes (DEGs) were found in MC3T3-E1 cells treated with CPP-Ca, including 121 upregulated and 200 downregulated genes. Gene ontology (GO) revealed that the DEGs mainly played important roles in the regulation of cellular components. The enrichment of the Kyoto Encyclopedia of Genes and Genomes Database (KEGG) pathway indicated that the AMPK, PI3K-Akt, MAPK, and Wnt signaling pathways were involved in the differentiation of MC3T3-E1 cells. The results of a quantitative real-time PCR (qRT-PCR) showed that compared with the blank control group, the mRNA expressions of Apolipoprotein D (APOD), Osteoglycin (OGN), and Insulin-like growth factor (IGF1) were significantly increased by 2.6, 2.0 and 3.0 times, respectively, while the mRNA levels of NOTUM, WIF1, and LRP4 notably decreased to 2.3, 2.1, and 4.2 times, respectively, which were consistent both in GO functional and KEGG enrichment pathway analysis. This study provided a theoretical basis for CPP-Ca as a nutritional additive in the treatment and prevention of osteoporosis.
酪蛋白磷酸肽（CPPs）具有良好的钙结合能力和促进肠道钙吸收的作用，但关于CPPs成骨活性的研究较少。本研究在前期研究基础上优化了酪蛋白磷酸肽钙螯合物（CPP-Ca）的制备工艺，并对其肽-钙螯合活性进行了表征。随后，研究了CPP-Ca对MC3T3-E1细胞增殖、分化和矿化的影响，并通过RNA测序（RNA-seq）进一步阐明了CPP-Ca对MC3T3-E1细胞的分化机制。结果表明，CPPs的钙螯合率为23.37%，CPP-Ca的钙含量达到2.64×10 mg/kg。紫外可见吸收光谱（UV）和傅里叶变换红外光谱（FTIR）测试结果表明，CPPs的羧基氧原子和氨基氮原子在螯合过程中可能与钙发生螯合。与对照组相比，250μg/mL CPP-Ca处理的MC3T3-E1细胞在24、48和72 h时的增殖率分别提高了21.65%、26.43%和28.43%，MC3T3-E1细胞的碱性磷酸酶（ALP）活性和矿化钙结节显著增加了55%和72%。RNA-seq结果显示，在CPP-Ca处理的MC3T3-E1细胞中发现了321个差异表达基因（DEGs），其中上调基因121个，下调基因200个。基因本体（GO）分析表明DEGs主要在细胞成分调节中发挥重要作用。京都基因与基因组百科全书数据库（KEGG）通路富集表明，AMPK信号通路、PI3K-Akt信号通路、MAPK信号通路和Wnt信号通路参与了MC3T3-E1细胞的分化。定量实时PCR（qRT-PCR）结果显示，与空白对照组相比，载脂蛋白D（APOD）、骨甘蛋白（OGN）和胰岛素样生长因子（IGF1）的mRNA表达分别显著增加了2.6、2.0和3.0倍，而Notum蛋白、Wnt抑制因子1（WIF1）和低密度脂蛋白受体相关蛋白4（LRP4）的mRNA水平分别显著降低至2.3、2.1和4.2倍，这与GO功能分析和KEGG富集通路分析结果一致。本研究为CPP-Ca作为营养添加剂用于骨质疏松症的治疗和预防提供了理论依据。
