In Vitro Proliferation and Anti-Apoptosis of the Papain-Generated Casein and Soy Protein Hydrolysates towards Osteoblastic Cells (hFOB1.19)

Abstract

Casein and soy protein were digested by papain to three degrees of hydrolysis (DH) 7.3%-13.3%, to obtain respective six casein and soy protein hydrolysates, aiming to clarify their in vitro proliferation and anti-apoptosis towards a human osteoblastic cell line (hFOB1.19 cells). Six casein and soy protein hydrolysates at five levels (0.01-0.2 mg/mL) mostly showed proliferation as positive 17β-estradiol did, because they conferred the osteoblasts with cell viability of 100%-114% and 104%-123%, respectively. The hydrolysates of higher DH values had stronger proliferation. Casein and soy protein hydrolysates of the highest DH values altered cell cycle progression, and enhanced cell proportion of S-phase from 50.5% to 56.5% and 60.5%. The two also antagonized etoposide- and NaF-induced osteoblast apoptosis. In apoptotic prevention, apoptotic cells were decreased from 31.6% to 22.6% and 15.6% (etoposide treatment), or from 19.5% to 17.7% and 12.4% (NaF treatment), respectively. In apoptotic reversal, soy protein hydrolysate decreased apoptotic cells from 13.3% to 11.7% (etoposide treatment), or from 14.5% to 11.0% (NaF treatment), but casein hydrolysate showed no reversal effect. It is concluded that the hydrolysates of two kinds had estradiol-like action on the osteoblasts, and soy protein hydrolysates had stronger proliferation and anti-apoptosis on the osteoblasts than casein hydrolysates.

Keywords: anti-apoptosis; casein hydrolysates; human fetal osteoblasts; proliferation; soy protein hydrolysates.

Figure 1

Proliferation of casein and soy protein hydrolysates (CH1–3 and SH1–3) on hFOB1.19 cells cultured for 24, 48 and 72 h, respectively. E denotes 17β-estradiol. CH1–3 denote casein hydrolysates with DH values of 7.3%, 10.8% and 13.3%, while SH1–3 denote soy protein hydrolysates with DH values of 7.5%, 10.9% and 13.2%, respectively. Different lowercase letters above the columns indicate that the mean values of different groups are significantly different (p < 0.05).

Figure 2

Effects of casein and soy protein and hydrolysates (CH3 and SH3) on cell cycle phase distribution of hFOB1.19 cells. (A) Representative histograms of DNA content in control group cells; (B,C) Representative histograms of DNA content in the cell incubated with CH3 and SH3 at 0.05 mg/mL for 48 h. Horizontal and vertical axes indicate the relative nuclear DNA content and number of cells, respectively; and (D) Percentage of cell populations in G0/G1-, S- and G2/M-phases.

Figure 3

Apoptotic prevention of casein and soy protein hydrolysates (CH3 and SH3) at 0.05 mg/mL on etoposide- and NaF-induced apoptosis of hFOB1.19 cells. Cell apoptosis was measured by Annexin FITC-V/PI double-stained flow cytometry. Q1, dead cells; Q2, later apoptotic cells; Q3, viable cells; Q4, early apoptotic cells. (A) The cells in the control group; (B,C) The cells treated by etoposide and NaF, respectively; (D,F) The cells first treated by CH3 and SH3, respectively, and then by etoposide; and (E,G) The cells first treated by CH3 and SH3, respectively, and then by NaF. The labeled data were obtained from one assaying only.

Figure 3

Apoptotic prevention of casein and soy protein hydrolysates (CH3 and SH3) at 0.05 mg/mL on etoposide- and NaF-induced apoptosis of hFOB1.19 cells. Cell apoptosis was measured by Annexin FITC-V/PI double-stained flow cytometry. Q1, dead cells; Q2, later apoptotic cells; Q3, viable cells; Q4, early apoptotic cells. (A) The cells in the control group; (B,C) The cells treated by etoposide and NaF, respectively; (D,F) The cells first treated by CH3 and SH3, respectively, and then by etoposide; and (E,G) The cells first treated by CH3 and SH3, respectively, and then by NaF. The labeled data were obtained from one assaying only.

Figure 4

Apoptotic reversal of casein and soy protein hydrolysates (CH3 and SH3) at 0.05 mg/mL on etoposide- and NaF-induced apoptosis of hFOB1.19 cells. Cell apoptosis was measured by Annexin FITC-V/PI double-stained flow cytometry. Q1, dead cells; Q2, later apoptotic cells; Q3, viable cells; Q4, early apoptotic cells. (A) The cells in control group; (B,C) The cells treated by etoposide and NaF, respectively; (D,F) The cells first treated by etoposide, and then by CH3 and SH3, respectively; and (E,G) The cells first treated by NaF, and then by CH3 and SH3, respectively. The labeled data were obtained from one assaying only.