Proteomic Analysis On Screening Differentially Expressed Proteins Of Fluorosis Rats’ Bone By ITRAQ Combined With Mass Spectrometry And Bioinformatics Analysis

Objective: To explore the molecular mechanism of skeletal fluorosis,i TRAQ combined 2D LC-MS/MS technology was applicated to identify and quantitatively analyze the differentially expressed protein of fluorosis rat’s bone,further, the function and the interaction of the proteins, and the pathway they may be involved were also analyzed with bioinformatics method. Methods: 1. The establishment and validation of the chronic fluorosis model:(1) Fouty-eight healthy SD rats were randomly divided into 4 groups. Control group were fed with tap water,and low, mediumand high dose groups were fed with tap water supplemented with 50,150, 250 mg/L Na F treatment lasted for 6 months, respectively. Each rat was examed for the incidence of dental fluorosis, and the urine, bone, tooth and serum fluoride level were tested by fluoride iron selective electrode method.(2) Femoral inferior segment were embedded in paraffin and serially cut for staining with Hematoxylin eosin(HE) and observed under optical microscope.(3) Enzyme-linked immunosorbent(ELISA) was used to determine the contents of bone metabolic markers in rat serum. 2. i TRAQ combined with mass spectrometry analysis was used to screen the differentially expressed proteins:(1) Extracted protein from the bone of fluorosis rat were digested into peptides, then labelled with i TRAQ reagent. Peptide sepration was peformed rough HPLC reversed phase and RPLC.(2) Looking for related biological function, metabolic and signal transduction pathways, protein and protein interactive network of differential proteins were analyzed by GO functional classification, KEGG metobalic pathway classification analysis and STRING analysis.The key protein MMP8 and CRT were selected for the further validate. 3. Validation of MMP-8 and CRT expression in the cell of the femur tissue: Rat bone tissue paraffin sections were stained by immunohistochemical PV9001 method, the cell expressionparts of 2 proteins in the femur organizations were observed by microscope. The average optical density of protein expression levels were determined by IPP. Results:1.(1) The incidence of teeth fluorosis and the levels of urine fluoride, bone fluoride and blood fluoride in the treated rats were higher than those in the control group, the differences were statistically significant(P <0.05). With the increase of fluoride dose and the extension of experimental time, the fluoride in urine, bone and blood grew generally.(2) The femur histomorphology results: The bone cortex thickness of medium and high fluorine groups were higher than that of control group, the difference is statistically significant(P <0.05); In the low fluorine group, trabecular areas are less than the control group, the difference was statistically significant(P<0.05); the osteoblasts numbers on cortex of the 3 treated groups are lower than the control group, the difference was statistically significant(P <0.05).(3) BGP, BALP,PⅠNP test results: Compared with control group, low fluorine group shows downward trend, there is a significant difference in BALP(P <0.01); Both of BGP and BALP in medium and high fluoride groups are less than the control group, the difference was statistically significant(P <0.05); CTX-Ⅰ, TRACP-5b test results: The content of TRACP-5b in 3 treated groups were less than the control group, the difference was statistically significant(P <0.05); The content of CTX-Ⅰin medium and high fluoride groups are less than the control group, the difference was statistically significant(P <0.05). 2. Sixty three significantly different expressed proteins were identificated:(1) Compared with the control group, there are 13、35、33 different proteins in the low, medium and high fluoride groups respectively.(2)There are 37 kinds of biological processes, 39 kinds of cell components and 40 molecular functions involved with the significantly different expressed proteins,which take part in 15 pathways and construct gene network diagram, there is direct or indirect contaction during 51 of them. 3. The expressed level of MMP8 and CRT was detected by IHC:(1) The MMP-8 IOD values of bone cells in 3 treated groups was higher than the control group, the difference was statistically significant(P <0.05);The MMP-8 IOD values of osteoblast in low fluoride group was higher than the control group,the difference was statistically significant(P <0.05);(2) The CRT IODvalue of cartilage cells in medium fluoride group and of osteoblast in low fluoride group was lower than the control group, the difference was both statistically significant(P <0.05). Conclusions: 1. After adding fluoride into drinking water for 6months, The bone metabolism index reflect the activity of osteoblast and osteoclast in different fluoride dose groups showed different degree of down-regulated. Bone tissue histomorphometry shows that 150 mg/L, 250 mg/L Na F group rats bone mass increases; 50 mg/L Na F group of rats show indications of bone mass loss. It reveals that fluoride exert an impact on the activity of osteoblast and osteoclast which related to the fluoride dose. 2. Sixty three significantly different protein, 15 related pathways,51 interacting proteins were found that maybe involved with the molecular mechanism of bone injury of chronic fluorosis. 3. The MMP8 and CRT expression level was consistent with the results of MS identification. The expression of MMP-8and CRT changes may be closely related to the development of skeletal fluorosis.