Study. Carotenoids On The Regulation Of Osteoblast Metabolism

OBJECTHealing of fracture and bone metabolism are very important health problems.TCM consider that it is such an affinity between bone health and function of some viscera.At one time,free radical may affect on the biological function of these viscera.Furthermore,these recent researches indicated that free radical may take part in healing of fracture and bone metabolism.Thus,it suggests that the compounds of TCM,as antioxidants,heal bone diseases through inhibiting free radicals.Carotenoid is a natural kind of antioxidant compounds.They can inhibit different free radicals more effectively than that of Vitamin E.However its history of R&D has about 175 years,it has been unknown in the biological and medical functions of healing of fracture and bone metabolism.It was firstly reported about the relation of carotenoid and bone health in the 14^th international symposium on carotenoids (Edinburgh UK 2005).But it is hardly researched and reported,especially no-Vitamin A carotenoids,in the action on healing of fracture and bone metabolism.Effect of different molecular structure of carotenoids E/Z isomers(β,β-carotene, astaxanthin,lycopene,cantaxanthin,zeaxanthin and lutein) on the antioxidant function of the Wistar aged-rat induced by D-galactose,M3T3-E1 osteoblastic cell induced by H₂O₂ and DNA damaged by O₂^-,OH^-,¹O₂,which was studied in vivo and in vitro.Furthermore,it was studied the regulation of metabolism on M3T3-E1 Osteoblastic cell by different no-Vitamin A carotenoids(astaxanthin,lycopene, cantaxanthin) in vitro.The research will not only help us to understand deeply the mechanism of regulation on healing of fracture and bone metabolism by carotenoids, but also find a new way or method of TCM on the treatment of bone diseases.METHODS(1) Preparation,purification and identify different carotenoids E-monomers (β,β-carotene,astaxanthin,lycopene,cantaxanthin,zeaxanthin and lutein)(2) Transformation of different carotenoids Z/E isomers in vitro (3) Effect of different carotenoids E-monomers on the Wistar aged-rat induced by D-galactose.The experiments were studied including total antioxidant function (MDA,SOD,GSH-Px),distribution of different viscera(content,E/Z isomers proportion),serum metabolism kinetics,bone metabolism parameters(thighbone length,weight,BMD,bone calcium,bone phosphorus,bone magnesium,bone manganese,hydroxyproline,blood calcium,blood phosphorus,ALP activity,bone structural and biological character).(4) Effect of different carotenoids E-monomers on M3T3-E₁ Osteoblastic cell induced by H₂O₂.The experiments were studied including cell proliferation,SOD activity,ROS,LOP content,DNA damage and cell membrane biological physical parameters.(5) Effect of different carotenoids Z/E isomers on inhibit different free radical(O₂^-,OH^-,¹O₂) in vitro by chemical lighted method and biological micro-lighted method.(6) Effect of different no-Vitamin A carotenoids E-monomers on the proliferation and differentiation M3T3-E1 osteoblastic cell.The experiments were studied including cell proliferation and differentiation,total DNA content,total protein content,ALP activity and the expression of 10 key genes(ALP,Collagen I,RUNX2,TGFβ1,VEGF,BMP2,IGF-I,OPG,RANKL,RXR)byuse real-time PCR.RESULTS(1) The experiment results suggested that different carotenoids E-monomers (β,β-carotene,astaxanthin,lycopene,cantaxanthin,zeaxanthin and lutein) group, compared with D-galactose control group,can decrease the content of serum TC,TG,LDL-c,TC/HDL-c,LDL-c/HDL-c and visceral MDA(P＜0.01),and increase the activity of serum and visceral SOD,GSH-Px(P＜0.01).Furthermore,the different carotenoids can increase bone parameters(length,weight,BMD),content of bone mineral(calcium,phosphorus,magnesium,manganese) and hydroxyproline,activity of ALP in the rat thighbone.Thus they can affect on bone structural and biological character,so as to improve the metabolism of bone.(2) The experiment results suggested that different carotenoids E-monomers (β,β-carotene,astaxanthin,lycopene,cantaxanthin,zeaxanthin and lutein) group, compared with H₂O₂ control group,can decrease the content of ROS,LPO(P＜0.01) and increase the activity of SOD,DNA content and cell livability(P＜0.01). Furthermore,no-Vitamin A different carotenoids can stimulate the mRNA expression of ALP,Collagen I,RUNX2,TGFβ1,VEGF,BMP2,IGF-I,OPG,RANKLin M3T3-E1 Osteoblastic cell.Additional,the expression of RXR receptor mRNA by cultured carotenoids was differ from that of vitamin A.It suggested that there is an unknown special membrane receptor to recognize carotenoids,to enter into the cell metabolism and act or inhibit the expression of some gene,which can affect on proliferation and differentiation of osteoblastic cell.Also,it suggested that no-Vitamin A carotenoids(astaxanthin,lycopene,cantaxanthin) may not competitively inhibit the reaction of vitamin D with RXR receptor.Thus,the diet of no-Vitamin A carotenoids may not destroy the absorbability of vitamin D in osteoblastic cell.(3) As antioxidants,the different carotenoids E/Z isomers can well inhibit O₂^-,OH^-,¹O₂ in vitro.As to inhibit kinds of free radical:¹O₂＞OH^-＞O₂^-.As to inhibit ability of different carotenoids E-monomers:lycopene＞astaxanthin＞cantaxanthin＞lutein＞zeaxanthin＞β,β-carotene.As to inhibit ability of different E/Z carotenoids: all-E-carotenoids＞13-Z-i somer＞11-Z-i somer＞9-Z- isomer.CONCLUSION(1) Successfully prepared and purification different carotenoids E-monomers (β,β-carotene,astaxanthin,lycopene,cantaxanthin,zeaxanthin and lutein)(2) Successfully established a biological research system of antioxidant and inhibit different free radicals by carotenoids.(3) Successfully and firstly researched effect of no-Vitamin A carotenoids on the metabolism of M3T3-E1 Osteoblastic cell at level of gene expression and protein expression.