| [Objective] Established the fluorosis and calcium supplementation rat models,2-DE quantitative proteomics techniques were used to compare the expression of proteins in rat’s bone with fluorosis and calcium supplementation to obtain differential proteins.Further,the function and the interaction of the proteins,and the pathway they may be involved were also analyzed with bioinformatics method.And by detecting the expression levels of genes and proteins related to PI3K/AKT signaling pathway,to provide a scientific basis for exploring the mechanism of calcium alleviating fluorosis.[Methods] In the test,40 healthy 120 g male rats were randomly divided into four groups,and named as normal control group(C,distilled water),fluorosis group(F,100 mg/L Na F-water),fluorosis group supplemented with 0.5% calcium(F+0.5% Ca,0.5% Ca,100 mg/L Na F-water)and fluorosis group supplemented with 1% calcium(F+1% Ca,1% Ca,100 mg/L Na F-water),respectively.After 120 days,the blood and bones of the rats was collected and the weights were weighed.1.Fluoride level were tested by fluoride iron selective electrode method;the serum calcium levels,alkaline phosphatase activity and tartrate resistant acid phosphatase activity were also detected;the bone histopathological changes were observed by HE stain.2.2-DE combined with MALDI-TOF MS mass spectrometry analysis was used to screen the differentially expressed proteins: Four groups of rat bone proteins were extracted and subjected to isoelectric focusing and SDS-PAGE electrophoresis.After image collection,protein spots with a difference of 2.5 times or more were digested with trypsin,and mass spectrometry and retrieval were performed to identify differential proteins.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 Tgm2 and Anxa5 were selected for the further validate.3.Detection of apoptotic DNA Ladder bands by agarose gel electrophoresis;in addition,the m RNA expression levels of Mylf、Pfkm、Tgm2、Anxa5、Col1a1、FAK、PI3K、PDK1、PTEN、AKT、BIM、Bax、Bcl-2 and Caspase-3 were researched by real-time RT-PCR.The protein expression of Tgm2、Anxa5、AKT、Foxo1 and Bcl-2 by Western-Blot.In addition,the protein expression of Foxo1 and Caspase-3 were studied by immunohistochemical staining.[Results] 1.Compared with the normal control group,the fluoride content in rats of fluorosis group and the calcium supplementation group significantly increased(p<0.01).Compared with the rats of fluorosis group,the fluoride content in the fluorosis group supplemented with 1% calcium was significantly decreased(p<0.05).Compared with the normal control group,alkaline phosphatase activity and the resistant tartrate acid phosphatase activity in rats of fluorosis group increased,the content of serum calcium in rats of fluorosis group significantly decreased.Compared with the rats of fluorosis group,alkaline phosphatase activity in the fluorosis group supplemented with 1% calcium decreased(p<0.05),the content of serum calcium significantly increased(p<0.05).Compared with the normal control group,we observed that the thickness distribution of bone trabecula was uneven.It appeared to be disordered and the shape was irregular.We also observed that the funicular structures of bone trabecula were disappeared.Compared with the fluorosis group,it showed that the bone trabecula distribution was uniform and had the funicular structures in the fluorosis group supplemented with calcium.2.Compared with the control group,17 significantly different expressed proteins were identificated in the fluorosis group and fluorosis group supplemented with 1% calcium,including type I collagen(Col1a1),actin(Actb),protein glutamine transferase 2(Tgm2).The biological processes,cell components and molecular functions involved with the significantly different expressed proteins are mainly related to cytoskeleton,energy metabolism,substance transport,ion channels,and apoptosis.These differential proteins are enriched into 38 bone metabolic pathways such as focal adhesion(FA),PI3K-Akt signaling pathway,and AMPK signaling pathway.15 differential proteins can construct gene network diagram,and their genes are direct or indirect connected.3.Compared with the normal control group,the bone cell DNA in rats of fluorosis group was broken,and the fluorosis group supplemented with 1% calcium was broken significantly lighter.Compared with the normal control group,the m RNA expression of Mylf、Pfkm、Tgm2、、Col1a1、FAK、PI3K、PTEN、AKT、Bax and Caspase-3 in rats of fluorosis group significantly increased(p<0.05,p< 0.01),the m RNA expression of Anxa5、PDK1 and Bcl-2 significantly decreased(p< 0.05,p<0.01).Compared with the fluorosis group,the m RNA expression of Pfkm、Tgm2、Col1a1、FAK、PI3K、PTEN、AKT、Bax and Caspase-3 in rats of the fluorosis group supplemented with calcium significantly decreased(p< 0.05,p < 0.01),and the m RNA expression of Anxa5、Bcl-2 significantly increased(p<0.05,p<0.01).4.Compared with the normal control group,the protein expression of Tgm2、AKT、Foxo1 and Caspase-3 in rats of fluorosis group significantly increased(p<0.05,p<0.01),the protein expression of Anxa5 and Bcl-2 in rats of fluorosis group significantly decreased(p<0.01).Compared with the fluorosis group,the protein expression of Tgm2、AKT and Foxo1 in rats of fluorosis group supplemented with calcium decreased significantly(p<0.05,p<0.01),the protein expression of Anxa5 and Bcl-2 in rats of fluorosis group supplemented with calcium significantly increased(p<0.05).[Conclusion] 1.In conclusion,the results of the present study demonstrated that the calcium could control bone metabolism related biochemical indicators in rats and alleviate the fluorosis.2.17 significantly different proteins,38 related pathways,15 interacting proteins were found that maybe involved with the molecular mechanism of calcium-relieving bone injury of chronic fluorosis.3.Calcium could regulate the expression of Tgm2、Anxa5 and Bcl-2 the PI3K-Akt signaling pathwayrelated genes and proteins to attenuate skeletal cell apoptosis in fluorosis rats.These findings could signify that calcium could attenuate the bone injury caused by fluorine,regulating the expression level of Tgm2 and Anxa5 and activating the PI3K-Akt signaling pathway. |
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