The Role Of Ca²⁺ Concentration,Oxidative Stress And ER Stress In Osteoblast Exposed To Fluoride

Endemic fluorosis can cause serious skeletal injury, including Osteosclerosis、Osteomalacia、Osteoporosis and soft tissue ossification, and can seriously affect theward residents’ quality of life. So far, however there are many problems in the studyof clinical prevention and treatment and the pathogenesis of basic research need to besolved. In the study of the pathogenesis of skeletal fluorosis, Our research group anddomestic and overseas related research prompt, there are close contacts between Ca2+level and oxidative stress、endoplasmic reticulum stress in osteoblas（tOB）, and keepa certain balance to maintain the body’s normal physiological function; otherwise, itwill lead to a series of pathological changes. Therefore, this research will be carriedout according to the following thinking:First prove that under the action of fluoride, OB calcium ion concentration willincrease significantly; and meanwhile detect oxidative stress state and endoplasmicreticulum stress state in osteoblasts culture supernatant, and the secretion of osteoblastbone growth factors. Synthetically analyzing above detective results, furthersystematically demonstrates that oxidative stress and endoplasmic reticulum stressparticipate in the hyperplasia and the activation of OB, and then leads to the changesof the affect of the bone metabolism local regulation network related factors, whichmakes it overexpression or ratio imbalance. In the end, it leads to the occurrence ofthe skeletal fluorosis bone lesions.Methods: Mice osteoblast cell lines（MC3T3-E1）were used. Experiment wasdivided into the control group、exposed to fluoride group1、exposed to fluoride group2and exposed to fluoride group3, and the concentration of fluoride respectively were0、1、5、10mg/L F-. The times of fluoride exposure were4h and48h。 The methods of fluorescence quantitative、real-time quantitative PCR、ELISAand biochemical kit were used to detect OB calcium ion concentration, glutathioneand the content of serum lipid peroxides phthalaldehyde （MDA), and detectendoplasmic reticulum stress system in fluoride exposure OB, which included theprotein contents of activating transcription factor4(ATF4)、binding immunoglobulinprotein(BIP)、 PKR-like ER kinase(PERK)、 X-box binding protein1（XBP1);meanwhile detect the expressions of osteocalcin(OCN) in OB、bone morphogeneticprotein (BMP)、basic fibroblast growth factor(bFGF)、receptor activator of nuclearfactor κB ligand (RANKL)/osteoprotegerin(OPG).Findings:1. When cells exposed to fluoride4h, OB Ca2+concentration increased in everyfluoride exposure comparing with the control group, and Ca2+concentration increasedobviously when fluoride exposure was10mg/L;2. The content of GSH in OB exposed to fluoride were increased.3. Comparing with the control group in the condition of fluoride exposure4h and48h, the expression of amount of protein in all the fluoride added groups of ATF4、BIP、PERK and XBP1significantly increased, and increased levels were related to the doseof fluoride exposure; that is to say, the higher fluoride exposure concentration is, themore obvious increase is; the test results of BIP and XBP1mRNA is similar to proteinexpression, but comparing with the control group, the expression of every the fluorideadded group of ATF4mRNA reduced. the changes of PERK is not obvious, except itsignificantly increased in fluoride exposure5mg/L group comparing with the controlgroup;4. RT-PCR results suggested that the expression of bFGF mRNA went up significantlyin fluoride exposure4h，5mg/L group and fluoride exposure48h,1、10mg/L group；the expression of BMP mRNA went up significantly in fluoride exposure48h,1、10mg/L group；OCN mRNA went up significantly in fluoride exposure4h,1mg/Lgroup and fluoride exposure48h,5mg/L group；RANKL/OPG. Results：1. Under this experimental condition, Ca2+concentration increased significantly whenOB was excited by fluoride, and that further proved the stimulating effect of thefluoride on osteoblast；2. Under this experimental condition, fluoride can stimulate oxidative stress systemand endoplasmic reticulum stress system in OB；3. Fluoride can stimulate the expression of bone growth factors significantly.Summing up the results， this study systematically proved that under thisexperimental conditions, fluorine can obviously increase OB calcium ionconcentration, and activate oxidative stress system and endoplasmic reticulum stresssystem. The final results was that OB function enhanced excessively, and bonemetabolism local regulation network related factors overexpressed, which became oneof the important component in leading to skeletal fluorosis bone lesions.