In Vitro Proliferation And Anti-apoptotic Effects Of Three Protein Hydrolysates Towards Osteoblasts

Osteoporosis is a chronic disease caused by multiple factors, such as age, estrogen, malnutrition, immune and genetic factors. Osteoporosis is characterized by low bone mass and micro-architectural deterioration of bone tissue. Traditional osteoporosis drugs such as strontium preparation and osteoprotegerin have certain curative effects, but the doses these drugs used in clinical treatment, their reasonable combination and possible side-effects are still plagued us. It is worthy of researching and developing functional food ingredients with health function on the bone but without any side-effect on the body. Both food proteins and amino acids are essential materials for the organic synthesis in the bone. Long-term insufficiency in protein intake can cause decreased bone matrix synthesis, leading to osteoporosis. On the contrary, long-term excessive protein intake is also detrimental to bone health, as it causes greater calcium loss and bone absorption, together with an inhibition on matrix mineralization. The role of food proteins in bone health thereof needs detailed and systematic investigation.In the present study, chum salmon skin gelatin, soybean protein and casein were digested in vitro by papain to generate gelatin, soybean protein and casein hydrolysates with three degrees of hydrolysis(DH), respectively. The impacts of these nine hydrolysates on proliferation, cell cycle and apoptosis of an osteoblastic cell line(h FOB1.19) were investigated in vitro. CCK-8 assaying results indicated that all hydrolysates had the capacities to promote cell proliferation, as they elevated cell viability to 103%–134%, 104%–123% or 100%–114%, respectively. More important, the hydrolysates with higher DH values seemed to possess stronger osteoblast proliferation. Flow cytometric analysis results revealed that the three kinds of protein hydrolysates could accelerate cell cycle progression, by keeping the cells in S-phase from previous proportion of 50.5% to much higher proportions of 67.4%, 60.5%, and 56.5%, respectively. This result further confirmed that the three kinds of protein hydrolysates could promote osteoblast proliferation. Moreover, gelatin, soybean protein and casein hydrolysates could antagonize etoposide- or Na F- induced apoptosis, showing apoptotic prevention and reversal. In the assaying of apoptotic prevention, apoptotic cellswere decreased from 31.6% to 13.6%, 15.6% and 22.6%(by etoposide treatment), or from 19.5% to 11.3%, 12.4% and 17.7%(by Na F treatment), respectively. In the assaying of apoptotic reversal, gelatin and soybean protein hydrolysates decreased apoptotic cells from 13.3% to 11.8% and 11.7%(by etoposide treatment), or from 14.5% to 12.4% and 11.0%(by Na F treatment), respectively. However, casin hydrolysate did not show any reversal effect on etoposide- or Na F- induced apoptosis. In total, gelatin hydrolysate showed the greatest proliferative promotion and anti-apoptotic effect than soybean protein and casein hydrolysates.DNA microarray technology and RT-PCR assaying were used to detect the changes of gene expression in the osteoblasts. The analysis results revealed that after the treatment by gelatin hydrolysates, related genes involve cell proliferation, cell cycle and apoptosis genes were changed in the osteoblasts. Among these genes, MPHOSPH1 and PAK1LP1 were up-regulated, but RPS6KA2, JNK1, JNK3 and JNKK were down-regulated. The expression of JNK’s family proteins were analyzed by Western-blot,. The obtained result demonstrated that gelatin hydrolysate inhibited the apoptosis in the osteoblasts via regulating JNK signaling pathway.In surmmary, three kinds of protein hydrolysates(especially gelatin hydrolysate) prepared in the present study have in vitro proliferation and anti-apoptotic effects towards the studied osteoblasts. In addition, gelatin hydrolysate can antagonize induced osteoblast apoptosis via regulating JNK signaling pathway, thereof show potential to prevent osteoporosis. The present results provide a new scientific evidence for food proteins and bioactive peptides with potential prevention on osteoporosis.