Research On The Production Of Citric Acid In Bone And The Effect Of Citric Acid On Osteoblast Differentiation

Citric acid is a widely existed metabolite in the organism,mainly from the tricarboxylic acid cycle pathway.Studies have shown that high concentrations of citric acid are found in the prostate and bone,and the citric acid in the bones accounts for 70%of the total citric acid in the body,which is the largest citric acid library in the body.The citric acid is connected with apatite nanocrystals and collagen in bone,which has the effect of enhancing the strength and anti-fracture ability.Therefore,the presence of citric acid is important for bone,but the source of citric acid remains unknown.It is reported that the prostate has a unique pathway of producing and secreting citric acid.Large quantities of zinc+ is transported into cells from the blood plasma by zinc transporter ZIP1,located in prostate epithelial cells,and then enters the mitochondrial to suppress aconitase's activity of oxidation of citric acid,as wll as inhibits the generation of oxaloacetic acid.But the prostatic epithelium transports a large amount of aspartic acid to the mitochondria,and through the mitochondrial aspartate transaminase catalyzes the formation of oxaloacetic acid,which is used to synthesize citric acid.A large amount of citric acid is transported through the mitochondrial membrane by the citrite transport protein(CTP)into the cytoplasm.Na+/K+-ATPase using ATP,takes up K+ into the cytoplasm,elimimating Na+ at the same time.On both sides of the membrane form high K+ concentration gradient.K+concentration dependented on citric acid transporters(KCiT1).using K+concentration gradient,citrate co-transport to extracellular citric acid from the cytoplasm,prostate epithelial cells complete of the citric acid synthesis,transport process.Have been pointed out,and at the same time the preliminary test shows that osteoblasts could be a prime source of citric acid cells of bone,so we suspect that the osteoblast and prostate epithelial cells have a similar way to generate,secrete citrate.Objective:to study the origin of citric acid in bone,the pathway of citric acid formation and secretion,and the effect on osteogenesis.Research content:in prostate epithelial cells,ZIP1?CTP?Na+/K+-ATPase?citrate synthase play an important role in the production and secretion pathway of citric acid.in this experiment we use lentivirus to transfect MC3T3-E1 cells induced by osteogenic induction to knock down the expression of ZIP1?citrate synthase respectively.The changes of citric acid content and osteogenic differentiation and calcium nodules were observed by inhibiting the activity of CTP?Na+/K+-ATPase by inhibitors,1,3,5,trimesic acid and ubenosine.Results:the results showed that MC3T3-E1 transfected by lentivirus after induction,is compared with the control group,the citric acid content of experimental group is lower.Alkaline phosphatase staining and alizarin red staining are reduced,and osteogenesis and calcium nodules decrease.MC3T3-E1 added of Inhibitors after induction,compared with the control group,the citric acid content of inhibitor-added group is lower,alkaline phosphatase staining and alizarin red staining are reduced,and osteogenesis and calcium nodules are reduce.MC3T3-E1 added of Inhibitors after induction,compared with the control group,the expression of of runx2 of inhibitor-added group is lower,and inhibitor group is no obvious change.ZIP1,CTP,Na+/K+-ATPase,citrate synthase play an important role in the process of citrite in osteoblast,osteoblast could has a similar pathway with prostate epithelial cells in citric acid secretion.