Ferric ammonium citrate (FAC)-induced inhibition of osteoblast proliferation/differentiation and its reversal by soybean-derived peptides (SDP)

Ferric citrate has been used to treat hyperphosphatemia, a prevalent symptom in patients with chronic kidney disease while ferric ammonium citrate (FAC), a more dissolvable format, is widely used as food additive. However, excess iron is associated with osteoporosis. Dietary soybean products have been shown to prevent the progression of osteoporosis. In this study, a group of peptides, referred as P3, was identified from the enzymolysis of soybean protein isolates, and its biological functions were investigated. The results showed that MC3T3-E1 cell cycle progression from G0/G1 to S phase was accelerated by P3 treatment. MC3T3-E1 cell proliferation was enhanced by P3 via ERK1/2 activation. Importantly, P3 treatment abolished the antiproliferative effect of FAC on MC3T3-E1 cell. In addition, P3 treatment increased the expression of ALP, COL-1, OCN, consequently promoting the differentiation and mineralization of MC3T3-E1 cells via activation of p38 MAPK pathway. Consequently, P3 treatment was able to reverse the inhibitory effect of FAC on osteoblasts differentiation and mineralization. Our findings suggest P3, as a dietary supplement, has a potential therapeutic function to attenuate the adverse effects of FAC on bone metabolism and to prevent osteoporosis progression.


Ferric citrate has been approved, as a phosphate binder, for the improvement of hyperphosphatemia in patients with chronic kidney disease (CKD) (Yokoyama et al., 2014), or as an iron provider to treat an iron deficiency anemia in patients with CKD not requiring dialysis (Rebecca et al., 2020). Meanwhile, ferric ammonium citrate (FAC), a more dissolvable format, is widely used as a food additive. However, excess of iron being deposited in the body leads to hemochromatosis (Edison et al., 2008), which has been associated with osteoporosis (Kudo et al., 2008). Osteoporosis, a systemic bone metabolic disease, is results from the imbalance between bone resorption and formation, reduced bone mineral density and bone marrow enlargement, which increases the risk of bone brittleness and fracture (Friedman, 2006; Pang et al., 2015). Some drugs such as glucocorticoids, proton pump inhibitors, thiazolidinediones, antiepileptics, aromatase inhibitors, androgen deprivation therapy and heparin have detrimental effects on bone health. Glucocorticoids (GCs) promote the breakdown of protein, increase the excretion of calcium and phosphorus, inhibit the activity of osteoblasts, and cause osteoporosis. Osteoporosis induced by GCs is the most common type of secondary osteoporosis (Braun and Schett, 2012). Chronic use of proton-pump inhibitors is associated with lower bone mineral content and higher rates of osteopenia/osteoporosis (Mohammad et al., 2019). Osteoporosis is becoming a serious global public health problem, and the WHO estimates that greater than 200 million people worldwide suffer from osteoporosis (Lewiecki, 2018). Previous studies have shown that reasonable diet and dietary supplements can prevent the progression of osteoporosis (Xia et al., 2015; Yan et al., 2014; Zhao et al., 2017). Hence, the identification of bioactive compounds from daily foods that enhance bone formation is an effective, economic and safe approach for the prevention of osteoporosis.

Soybean is a common crop that is rich in proteins, oil and other nutrients. Soybean proteins, which account for 40% of soybean dry weight, are the most abundant plant proteins in nature. As a rich source of high-quality proteins and bioactive peptides, the ability of soybean proteins to prevent or relive osteoporosis has been recognized for a long time (Akao et al., 2015; Harrison et al., 1998; Kim et al., 2019). However, few studies have focused on the anti-osteoporotic activity of bioactive peptides from soybean.

Iron is a transition metal involving in many biological processes, including osteoblastic metabolism (Jaime et al., 2016). Excess iron from FAC has been shown to inhibit osteoblast metabolism (Yamasaki and Hagiwara, 2009). A recently study demonstrated that FAC dramatically inhibited the proliferation of osteoblast cells as a model agent to mimic a pathological environment for osteoporosis (Zhang et al., 2018). In the current study, P3, a group of bioactive peptides, were identified from soybean protein isolates by enzymolysis. Effects of P3 on MC3T3-E1 cell proliferation, differentiation and mineralization were determined. In addition, the protective effect of P3 on FAC-induced inhibition of MC3T3-E1 cells proliferation and differentiation were investigated. . Furthermore, the role of ERK1/2 activation and p38 MAPK signaling pathway in mediating P3-stimuated osteoblast proliferation and differentiation was also investigated.

Section snippets

Isolation of soybean-derived peptides with pro-osteogenic activity

Using the osteoblast proliferation assay as an indicator, peptides with pro-osteogenic activity from the soybean protein isolates were obtained by enzymolysis. Briefly, 5% (w/v) soybean protein isolates were hydrolyzed by papain (Beijing Solarbio Science & Technology Co., Ltd., China) (enzyme: substrate ratio at 3000 U/g) in phosphate buffer (pH 7.0) at 50 °C for 6 h. The enzymatic hydrolysates were fractionated using ultrafiltration membranes with different molecular weight cut-offs at 4 °C

Identification a group of soybean derived peptides that had the most potent ability to stimulate MC3T3-E1 cell proliferation

The initial experiments indicated that, after hydrolysis by papain, the fraction obtained with the 10-kDa cut-off ultrafiltration membrane exhibited the greatest ability to stimulate MC3T3-E1 cell proliferation. This fraction was further hydrolyzed with alkaline protease and separated by Sephadex G-15 gel filtration chromatography. Four fractions of soybean peptides, including Peak 1 (P1), Peak 2 (P2), Peak 3 (P3), and Peak 4 (P4), were obtained (Supplemental Fig. 1). As shown in Fig. 1A, P1,


Hyperphosphatemia is highly prevalent among patients with chronic kidney disease and is a major risk factor for cardiovascular disease and mortality. Oral phosphate binders, such as ferric citrate, for maintenance of normal serum phosphorus levels is a contemporary management of hyperphosphatemia. However, over excess of iron leads to hemochromatosis, which is associated with osteopenia and osteoporosis (Yamasaki and Hagiwara, 2009). Meanwhile the worldwide prevalence of osteoporosis is


In summary, a group of peptides named P3 were isolated from soybean protein isolates via two-step enzymatic hydrolysis. P3 stimulated MC3T3-E1 cell proliferation, differentiation and mineralization via activation of ERK1/2 and p38 MAP Kinase signaling pathway, respectively. In addition, P3 fraction peptides showed the protective role on FAC, ameliorating the adverse effect of FAC on bone metabolism. Therefore, this study provided an alternative dietary supplement for osteoporosis treatment and…

Source: Author links open overlay panelFang Wang a, Zebin Weng b, Haizhao Song a, Yifang Bao a, Huilin Sui a, Yong Fang a, Xiaozhi Tang a, Xinchun Shen a

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