Study On The Effect Of Sea Cucumber Enzymatic Hydrolysates On Increasing Bone Mineral Density

Osteoporosis is a chronic bone disease that causes damage to bone microstructure and bone strength,increasing the risk of fractures and other complications.Improving bone mineral absorption and balancing bone metabolism is of great significance for the treatment of osteoporosis and improvement of bone mineral density.At present,relevant treatment methods often have certain limitations,such as limited efficacy and endocrine disorders.Therefore,there is an urgent need to develop healthy foods that come from natural sources and have the effect of increasing bone density.Sea cucumber has been considered as a rare medicine since ancient times.It contains a variety of active substances,such as protein and polysaccharide,and has antibacterial,antioxidant,anti-thrombotic and anti-tumor functions.Some polypeptides can promote the body’s absorption of calcium,while some active substances in sea cucumber,such as polysaccharide,have anti-inflammatory function,which is closely related to balance bone metabolism.Therefore,this topic uses Sea cucumber enzymatic hydrolysates（SCEH）as raw materials to explore whether they have the effect of increasing bone density and compare them with single peptide substances.After that,the specific mechanism of SCEH was explored to provide a new direction for promoting bone health and a theoretical basis for promoting the deep processing and utilization of sea cucumber resources.The main research contents of this paper are as follows:Firstly,SCEH of two different processes are obtained from the enterprise,and they are characterized,and the better ones are selected for subsequent animal experiments.To obtain more small peptides and collagen-characteristic amino acids to improve the bioavailability of sea cucumbers,sea cucumbers were enzymatically hydrolyzed.The crude protein content of SCEH was 64%,the crude polysaccharide content was 19%,and the heavy metal content was in accordance with the national standard.The protein was hydrolyzed into small peptide,and only 0.4%of the peptide with molecular weight above 2000 Da was obtained.The first animal experiments used low-calcium model rats to investigate whether SCEH could improve bone mineral density in low-calcium rats.The animals are grouped as follows:Control group（Control,n=10）,Model group（Model,n=10）,Ca CO₃ positive group（Ca CO₃,n=10）,pure sea cucumber group（SCEH,n=10）,sea cucumber calcium group（SCEH+Ca,n=10）,collagen polypeptide plus calcium group（FCP+Ca,n=10）.Animal growth showed that SCEH could promote the growth and development of low-calcium rats.Bone mineral density（BMD）results in rats showed that SCEH significantly improved BMD and bone strength reduction due to low calcium content.After determining the effect of SCEH on improving bone mineral density and relieving osteoporosis symptoms,the mechanism of action was explored.The data in the previous chapter showed that this mechanism may be related to calcium absorption promotion,so the second batch of animal experiments mainly used low-calcium rat model to explore the calcium absorption promotion ability of SCEH.The animal experiment groups are as follows:Blank Control group（Control,n=10）,Model group（Model,n=10）,Ca CO₃ positive group（Ca CO3,n=10）,pure sea cucumber group（SCEH,n=10）,sea cucumber low-dose calcium group（L,n=10）,sea cucumber medium-dose calcium group（M,n=10）,sea cucumber high-dose calcium group（H,n=10）.The data of bone mineral density and bone strength were consistent with that of the previous chapter,indicating that SCEH had the effect of improving bone mineral density.Three-dimensional reconstruction of trabecular bone showed that SCEH could improve bone microstructure.In terms of calcium metabolism,SCEH intervention significantly improved the apparent absorption and retention rate of calcium in rats,which directly proved its good ability to promote calcium absorption.SCEH can significantly up-regulate the expression levels of TRPV5 and TRPV6 genes in the intestine and kidney,to achieve the effect of improving calcium absorption in the body.The up-regulation of the two genes was positively correlated with the significantly increased level of 1,25（OH）₂D₃ of SCEH and significantly down-regulated level of PTH.In conclusion,SCEH can promote calcium absorption,and the effect of medium dose is the best.The ovariectomized rat model was used to study the balanced bone metabolism of SCEH.This model can simulate postmenopausal osteoporosis and is a classic model for studying bone metabolism.Animals were grouped as follows:Sham operation group（Sham,n=10）,ovariectomized model group（OVX,n=10）,estradiol positive group（E₂,n=10）,low dose group（L,n=10）,medium dose group（M,n=10）and high dose group（H,n=10）.The growth of rats showed that SCEH was equally safe for ovariectomized rats and had no significant effect on reproductive system and estrogen level.Bone mineral density,bone mass and bone strength data showed that SCEH could significantly improve bone mineral density in ovariectomized rats.HE and TRAP staining results showed that SCEH could repair bone microstructure,and reduce the number of osteoclasts in this model.SCEH can significantly down-regulate abnormally increased bone formation and bone resorption simultaneously,indicating that SCEH can improve bone mineral density and reduce bone loss by balancing bone metabolism.The effect of SCEH on inflammatory factors suggested that its improvement was closely related to RANK/RANKL/OPG and NF-κB pathway.The measurement results of OPG and RANKL gene and protein expression in rat bones confirmed the above speculation.SCEH can inhibit the proliferation and differentiation of osteoclasts by regulating the ratio of OPG and RANKL,leading to the reduction of bone resorption,thus achieving the effect of balancing bone metabolism and increasing bone mineral density,and the effect of medium dose is the best.In conclusion,efficacy evaluation and mechanism exploration of SCEH in animal experiments showed that SCEH has the effect of improving bone mineral density.The potential mechanism is as follows:firstly,it can regulate the levels of 1,25（OH）2D3 and PTH,thus up-regulating the expression of TRPV5 and TRPV6 in intestinal tract and kidney,and improving the absorption and storage rate of calcium in the body.Secondly,it can regulate the ratio of OPG and RANKL,inhibit the proliferation and differentiation of osteoclasts,balance bone metabolism,and ultimately achieve the purpose of improving bone mineral density and relieving osteoporosis.