Effect Of CASR/MAPK Signaling Pathway On The Pathogenesis Of Fluorosis

Background:Fluorosis is an endemic disease,which seriously affects the health of the body.The etiology is clear but the molecular mechanism of the disease is still unclear.we found that the function of osteoblast(OB)was actived and the turnover of bone was accelerated.That is all important features of fluorosis and the pathological basis of orthopedic diseases(including bone sclerosis,osteomalacia,osteoporosis,and periosteum soft tissue calcification),while changes in OB calcium homeostasis is the important of osteoblast's priming links.The calcium-sensitive receptor,Ca SR play a key role in the regulation of Ca2+ homeostasis in the body.As well as MAPK signaling pathways are significant in osteogenic differentiation including the signaling pathways in P38,JNK and ERK.Previous studies have found that fluoride can significantly stimulate the expression of Ca SR and OB specific transcription factor,RUNX2.And it is considered that fluoride stimulation of OB function enhancement is one of the vital mechanisms for the pathogenesis of skeletal fluorosis,but there are still unclear between the specific pathway and the correlation.Therefore,this paper will start with Ca SR/MAPK signaling pathway,whether fluorine can enhance the expression RUNX2 OB by stimulating the signaling pathway promoted the enhancement of OB osteogenic function and to try to find the vital role of Ca SR/MAPK signaling pathway in the pathogenesis of fluorosis.Finally,this reaearch provide a significant research background and theoretical basis for the study of the pathogenesis of fluorosis.Methods:In this experiment,we used the method in vitro experiment to study MC3T3-E1 Subclone 14 mouse preosteoblast cell line.There are 4 groups including control group,low fluoride group,medium fluoride group,high fluoride group.Fluorine concentration are 0 mg/L,2 mg/L,5 mg/L,10 mg/L.Duration: 48 h.There are three methods of this research.Firstly,the Gomori calcium-cobalt method was used to detect OB alkaline phosphatase activity.Secondly,the cell related factors(such as Ca SR,PKC,KRAS,JNK,P38 MAPK,ERK,RUNX2)were detected by real-time quantitative PCR and Western-Blot.At last,the cell related factors were taken as the core to analyze the correlation.Results:1.Changes of CASR signaling pathways in fluoride OB(1)CaSR m RNA levels were significantly up-regulated in the high-dye fluoride group,but the levels of protein were significantly increased in all dose groups.(2)PLC m RNA expression was obviously enhanced in all dose groups.(3)The protein expression of Gq/11/14 was increased in the middle and high fluoride groups.Gna11 m RNA has an upward trend in the middle and high fluoride groups.But Gna14 m RNA was increased in the low and high fluoride groups.(4)The m RNA expression of PKC(Prkca,Prkcb and Prkcg)showed an upward trend except for the low fluoride dye group.(5)The KRAS m RNA showed high expression in low and middle fluoride group,by contrast the expression of KRAS protein decreased in fluoride group.2.Variation of MAPK signaling pathways in fluoride OB(1)In fluorinated OB,P38 MAPK m RNA and protein expression showed a significant increase trend,significant difference in statistics.(2)In fluorinated OB,the expression of ERK protein was significantly increased in the middle and high fluoride groups,but m RNA expression was down-regulated in the three fluoride groups.(3)Fluoride-dyeing stimulated JNK m RNA and protein increased to a statistically significant degree.3.The changes of RUNX2 in fluoride OB(1)The RUNX2 m RNA levels significantly increased in middle and high fluorinated groups.(2)The protein levels of RUNX2 were increased in middle and high fluoride.(3)The m RNA level of RUNX2 and Ca SR showed positive correlation in low dose group.4.The m RNA levels of Ca SR and CT were positively correlated in the low-dose group,but negatively correlated in the high-dose group.5.Bone growth factors of fluoride groups were overexpressed to varying degrees in OB.6.RUNX2 was positively correlated with Ca SR m RNA in the low-dose group.Conclusion:1.Fluoride significantly affects CaSR signaling pathways in OB(1)Fluoride can significantly stimulate the expression of Ca SR,PKC protein and m RNA in OB.(2)Fluoride observably affected the expression of Gq/11/14 m RNA and protein in OB.Exceptly for the decrease of Gq m RNA expression,others was mainly up-regulated.(3)Fluoride dramatically stimulated the expression of PLC m RNA levels in OB.(4)Fluoride can make a increase for KRAS at m RNA levels and decreased protein levels in OB.2.Fluoride significantly affects MAPK signaling pathways in OB(1)Fluoride can stimulate the protein levels of JNK and P38MAPK;(2)Fluoride stimulates the production of ERK protein at high concentrations.3.CASR/MAPK signaling pathway factors have correlations with RUNX2 at m RNA and protein levels.There are the experimental results obtained in this paper,which basically confirm our experimental expectation.The enhancement of osteoblast function in fluorosis is the initial stage of the development of fluorosis,while there are the association and influence between CASR/MAPK signaling pathway factors and RUNX2.And also they play an important role in the enhancement of osteogenic function.Although there are still some unclear points,the results of this experiment suggest that in CASR/MAPK signaling pathway we should be paid attention to and studied more deeply in the work of exploring the pathogenesis of fluorosis.