Identification Of The Bone Red Material And The Bone Biologic Characteristic In Red-boned Guishan Goats

Guishan goats are a rare, wild native breed of Capra hircus in China, which are inthe "First Class National Protection Animals of China" category. Red-colored boneswere initially found in some Guishan goats since the1980's, and these goats weredesignated "red-boned" goats to distinguish them from common goats. Red-bonedGuishan goats are characterized as having whole red-colored bones. Further to this,red-boned Guishan goats are physically different from common Guishan goats. Forexample, the skeletal and overall body shapes of red-boned Guishan goats aredifferent, making them taller and stronger, although these differences can also have anadverse impact on their ability to climb mountains. However, it is unknown whatcauses the red color in the bone in the red-boned goats nor it is known whether the redcolor in bone is caused by the reason of endogenous or exogenous, and is not itunderstood whether the bone geometry architecture and metabolism in the red-bonedgoats are different from common goats. Therefore, in this study, red-colored materialof the bone were identifed and analyzed by High-performance liquidchromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) andNuclear magnetic resonance spectroscopy (NMR); Bone mineral elementscomposition and bone mineral density were measured and analyzed by An inductivelycoupled plasma optical emission spectrometer (ICP-OES), and an inductively coupledplasma mass spectrometer (ICP-MS) and Dual energy X-ray Absorptiometry (DEXA)in red-boned and common Guishan goats during the process of growth anddevelopment; The difference between red-boned and common Guishan goats on bonebiomechanical strength and micro-architecture parameters were measured by thematerials-testing machine and bone Micro-computed tomography scanning during theprocess of growth and development; The difference between red-boned and commonGuishan goats on bone metabolism-related genes were measured by the Real-timepolymerase chain reaction during the process of growth and development; The animal model after madder feeding was established and identified madder that impacts onrats on the regulation of bone mineral deposition and bone metabolism. The aboveresults provide systematic data on elucidating red-boned Guishan goats andcharacteristics of bone mineral deposition and bone metabolism.Identification of the red material from red-boned Guishan goats: we foundthat the carmine colored material of the bone in18-month old red-boned Guishangoats is pseudopurpurin, and that the red-colored material of the bone in36-month oldred-boned Guishan goats are pseudopurpurin and alizarin, but that pseudopurpurin ispredominant by NMR and HPLC-ESI-MS.Bone mineral density and mineral elements of red-boned Guishan goats:Analysis of femoral bone samples by dual-energy X-ray absorptiometry andinductively coupled plasma optical emission spectrometry revealed an increase inbone mineral density in the femoral diaphysis and distal femur of red-boned Guishangoats at6,12,18and36months of age. The data also indicated that the ratio of theBMD values of red-boned to common Guishan goats were higher at36months of agethan they were at18months of age. Furthermore, the levels of calcium, magnesium,iron, zinc, manganese and aluminium of femur were significantly higher in red-bonedGuishan goats than common Guishan goats at18and36months of age.Bone micro-structure and mechanical properties of red-boned Guishangoats: Analytic results of mechanical properties and micro-computed tomographyshowed that the red-boned goats displayed an increase in the trabecular volumefraction, the trabecular thickness and the trabecular number of the distal femur, andthe mean thickness, inner perimeter, outer perimeter, and area of the femur diaphysis.In the parallel, the trabecular separation and structure model index of the distal femurwere decreased, and the mechanical properties parameters of the femur diaphysiswere enhanced in the red-boned goats at18and36months of age.Bone metabolism-related genes in "red-boned" Guishan goats: The expressionlevel of OPG, RANKL, RANK, M-CSF, CaSR and c-Fms mRNA by real-timequantitative reverse-transcription polymerase chain reaction. These results showedthat bone metabolism-related genes mRNA expression were rised in red-boned Guishan goats. Osteoprotegerin, calcium-sensing receptor, alkaline phosphatase andosteocalcin mRNA expression was highest, and the ratio of the receptor activator ofthe nuclear factor kappa B ligand/osteoprotegerin was markedly lower in the femur ofthe red-boned goats compared with the common goats. We determined the completeCDS of Osteoprotegerin and calcium-sensing receptor in the femur of red-boned andcommon goats using5' and3'-RACE-PCR to amplify the complete sequence ofosteoprotegerin and calcium-sensing receptor expressed in bone tissues. The aminoacid sequences of osteoprotegerin, the receptor activator of the nuclear factor kappa Bligand, the receptor activator of the nuclear factor kappa B, macrophage colony-stimulating factor, calcium-sensing receptor and macrophage colony-stimulatingfactor receptor genes expressed in the femur of red-boned goats shared over98%homology with the respective genes in common goats. Therefore, the bone metabolicproperties of red-boned Guishan goats are better than common Guishan goats withoutduing to genetic factors.Effect of Rubia tinctorum L (madder) on femur of rats: To further confirm theeffect of pseudopurpurin on bone geometry architecture and metabolism Wistar ratswere fed diets in which0%,5%,10%and15%madder was added. Femurs of ratswere examined at0.5,1,3and6months after madder feeding. The long-term feedingof10%madder is safe for rats without disturbing bone growth and body weight. Thered matter from rats femur was one compound identified as similar to alizarin byHPLC analysis. The related to alizarin which has specific chemical affinities forcalcium in bone enhances bone strength and geometry architecture compared with0%groups. Similar changes were obtained in the femur of the treated rats.The above results demonstrate that pseudopurpurin and alizarin are maincomponents in Rubia cordifolia L, which is growing in the Guishan Mountains andare eaten by red-boned Guishan goats. Our results demonstrate that pseudopurpurin ismore effective than alizarin in staining bone tissue, it has a closer affinity for themineral salts of bone than other madder dyestuff, and the ability of pseudopurpurin tocompete with bone mineral salts is stronger than that of alizarin; therefore, a smalleramount of alizarin is deposited on the bone. The pseudopurpurin has a selectiveaffinity for the principal salts of bone, comes into contact with osteoid-tissue of developing bone, and subsequently a high level of mineral salts of the bone cause animprovement in the bone strength and an enhancement in the bone structure andmetabolic functions.