Effect Of Tauroursodeoxycholic Acid(TUDCA) On Osteoblastic Differentiation And Mineralization In Nih3t3 Cells

Ectopic ossification is a pathological condition characterized by the formation of ectopic bone or the deposition of calcium phosphate complexes in aberrant locations. The molecular mechanism of ectopic ossification has always been concerned, but the exact pathogenic mechanism remains elusive. From the analysis of morphology, the formation of these ectopic bone is similar to normal embryonic bone formation and bone regeneration after fracture, these ectopic bone belong to endochondral ossification or intramembranous ossification in nature. The index change in ectopic ossification is in agreement with the formation of bone in normal physiological conditions, such as ALP activity, the secretion of collagen, the expression of osteogenic differentiation marker genes and the generation of mineralized nodule. And at present, in the surgical setting, the two most common preventative modalities are radiotherapy and indomethacin, a non-steroidal anti-inflammatory drug(NSAID), but both treatment modalities carry the risk of particular side effects, therefore, effective treatment is extremely urgent.Research shows that endoplasmic reticulum stress(ER Stress) can be activated in osteoblast differentiation and bone formation and stimulate signaling pathways: PERK/eIF2α/ATF4, IRE1/XBP1 s and ATF6 mediated signal pathway. Evidence are as followed, the Perk-/- and ATF4-/- mice show severe skeletal disorder because osteoblast differentiation has serious defects, important transcription factors can not effectively express, and the processing and secretion of collagen, osteocalcin and bone sialoprotein of bone matrix components is supressed, leading to the accumulation of premature protein in ER and cause severe skeletal dysplasia. In IRE1α-/- MEFs, the expression of bone related transcription factors decrease, osteogenic differentiation and the expression of extracellular bone matrix proteins(such as osteocalcin, bone sialoprotein, osteopontin) are inhibited. Although the ATF4-/- mice can not show severe skeletal disorder, in S1P-/- knockout mice, the expression of type II collagen in cartilage serious declines and abnormal development of cartilage and endochondral bone formation are shown. The main reason is that non-mature type II collagen is synthetized and accumulated in the endoplasmic reticulum which triggers ER Stress reaction, leading to the abnormal formation of endochondral bone.Tauroursodeoxycholic acid(TUDCA) is a bile acid present in human bile at a lower concentration and has a very good safety profile, the normal range of concentration in human plasma is 0.4-4 μM. It has been proved that TUDCA can enhance the ER folding ability to prevent protein aggregation and thus protect cells against ER stress. TUDCA has been widely used in treatment of disease, such as cholelithiasis, cholestatic liver disease, it works via preventing ER stress, as a classical ER stress inhibitor. In rescent study, it has been demonstrated that TUDCA could markedly prevent Aortic Valve(AV) calcification, and attenuate AV osteoblastic differentiation in both rabbit and mouse models of AV calcification via inhibition of ER stress and could suppress oxidized low density lipoprotein(oxLDL)-induced osteoblastic differentiation in cultured valvular interstitial cells(VICs).In view of the above, we propose the hypothesis that ER Stress may be involved in ectopic ossification and TUDCA inhibites ectopic ossification via preventing ER stress. Therefore, if true, TUDCA has the potential pharmacologic and therapeutic applications for treating ectopic ossification associated diseases.In this study, we induced NIH3T3 cells mineralization with 3 mM inorganic phosphate(Na3PO4, Pi) as a in vitro model for ectopic ossification and used different amount of TUDCA(0.1, 1, 5, 10 μM) to treat osteogenic inducted NIH3T3 cells. And next, we conducted the phenotype analysis to examine the effect of TUDCA on osteoblastic differentiation and mineralization in NIH3T3 and ER Stress response to investigate if ER Stress is associated with ectopic ossification and TUDCA can inhibit fibroblasts ectopic ossification via supressing the ER Stress response.Chapter 1: The effect of TUDCA on inorganic phosphate-induced osteoblastic differentiation and mineralization in NIH3T3 fibroblastsObjective: To examine the effect of TUDCA on inorganic phosphate-induced osteoblastic differentiation and mineralization in NIH3T3 fibroblasts.Methods: We used different amount of TUDCA(0.1, 1, 5, 10 μM) to treat osteogenic inducted NIH3T3 cells. In the indicated time, we detected the cell proliferation, ALP activity, the formation of mineralized nodules, calcium deposition and the expression of osteoblast-specific markers.Resultes: TUDCA can inhibite cell proliferation, ALP activity, the formation of mineralized nodules, calcium deposition and the expression of osteoblast-specific markers in a dose-dependent deference manner in osteogenic inducted NIH3T3 cells.Conclusions: TUDCA plays a role of inhibition on the osteoblastic differentiation and mineralization in inorganic phosphate-induced NIH3T3 fibroblasts.Chapter 2: TUDCA inhibites the osteoblastic differentiation and mineralization in inorganic phosphate-induced NIH3T3 fibroblasts by preventing ER stressObjective: The aim is to examine whether TUDCA inhibites the osteoblastic differentiation and mineralization in inorganic phosphate-induced NIH3T3 fibroblasts by preventing ER stress.Methods: The activitation of ER Stress stimulated by inorganic phosphate was examined and ER stress response mediated three main signal pathways were examined by Western-Blot in inorganic phosphate-induced NIH3T3 fibroblasts under the treatmend of TUDCA.Resultes: ER stress could be activitied by inorganic phosphate. TUDCA could inhibit the exprssion of important signal molecules(Phospho-e IF2α、ATF4、XBP1s、ATF6) in ERstress response mediated three main signal pathways, including Runx2.Conclusions: TUDCA inhibites the osteoblastic differentiation and mineralization in inorganic phosphate-induced NIH3T3 fibroblasts by preventing ER stress.