| ObjectiveThe healing processes of tooth extraction wounds are affected by systemic conditions. As an established etiological factor of cardiovascular diseases, hyperlipidemia is also reported to be actively involved in the regulation of bone remodeling. However, it is unclear whether or how the hyperlipidemic microenvironment affects the healing processes of tooth extraction wounds. Moreover, although considerable efforts have been dedicated to the investigations of hyperlipidemia-regulated bone metabolism, the underlying molecular mechanisms are still largely unknown. Secreted frizzled-related protein 1 (SFRP1) is a competitive suppressor of Frizzled, the specific receptor of canonical Wnt signaling pathway. It has been reported that SFRP1 plays an important role in regulating bone metabolism through manipulating canonical Wnt signal pathway. Unfortunately, evidence is still unavailable indicating whether the regulatory effects of hyperlipidemia on bone metabolism are, at least partly, mediated through its effects on SFRP1. In this study, hyperlipidemic mouse models will be established using the wild type C57BL/6 mice and the apolipoprotein E knockout mice (ApoE-/-mice). Tooth extraction wounds will be established and left untreated in these mice and the healing processes of these wounds will be evaluated. In addition, the 3'untranslated region (3'UTR) of SFRP1 gene will be amplified and subcloned into a luciferase expression vector. The newly constructed plasmid will be used in future studies aimed at investigating the effects of hyperlipidemic microenvironment on the regulatory roles of SFRP1 in canonical Wnt signaling and osteogenic differentiation.MethodsPart I:Six-week-old male C57BL/6 mice and ApoE-/-mice were randomly assigned to four groups:C57BL/6 mice fed with standard chow diet (named as WT+S group), C57BL/6 mice fed with high-fat diet (named as WT+HFD group), ApoE-/-mice fed with standard chow diet (named as ApoE+S group), and ApoE-/-mice fed with high fat diet (named as ApoE+HFD group). Four weeks after fed with the corresponding diet, blood samples were collected from the angular vein and serum lipid leves were determined. Five weeks after feeding, bilateral second and third mandibular molars were extracted, and the animals were sacrificed at lweek and 2 weeks, respectively, after the surgery. Blood samples were collected using the above-mentioned method for serum lipid level determination. The mandibles were isolated, processed, and subjected for histomorphometic analysis to evaluate new bone formation and the position of the alveolar crest during the healing processes.Part II:The mRNA and protein levels of SFRP1 geneinMC3T3-E1 cells were measured using real time quantitative polymerase chain reaction and Western Blot at 0 day,7days and 10days after osteogenic differentiation was induced in these cells using 50 ?g/ml of ascorbic acid. GAPDH and ?-actin served as controls, respectively. The 3'UTR of mouse SFRP1 gene was amplified from total RNA isolated from MC3T3-E1 cells using PCR. The SFRP1 3'UTR fragment was then subcloned into the vector, pMIRREPORT, resulting in a new plasmid named as pMIR-REPORT-SFRP1UTR. pMIR-REPORT-SFRP1UTR and pMIR-REPORT were transfected into MC3T3-El and NIH3T3 cells, and the luciferase levels were determined.ResultsPart I:Four weeks after fed with the corresponding diet, we found that serum levels of total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-c) in the WT+HFD group were 1.54-fold (p< 0.05),2.08-fold (p<0.05) and 2.32-fold (p<0.05), respectively, higher than those in the WT+S group. When compared with those in the ApoE+S group, the serum levels of TC, TG and LDL-c in the ApoE+HFD group were increased by 2.06 folds (p<0.05),3.51 folds (p<0.05) and 2.52 folds (p< 0.05), respectively. One week and 2 weeks after tooth extraction, the serum lipid level determination showed similar results. In addition,1 week after tooth extraction, new bone formation in the WT+HFD group was significantly inhibited when compared with that in the WT+S group (p<0.05). Although the newly formed bone area in the ApoE+HFD group was slightly lower than that in the ApoE+S group, no statistically significant difference was detected (p> 0.05). Two weeks after tooth extraction, new bone formation in the WT+HFD group was significantly inhibited when compared with that in the WT+S group (p<0.05). Similarly, the new bone formation in the ApoE+HFD group was also significantly retarded when compared with that in the ApoE+S group (p<0.05). Furthermore, we found that the loss of the alveolar crest height in the HFD-fed groups is more prominent when compared with the corresponding standard diet-fed groups at both time points after tooth extraction (p<0.05).Part ?:SFRP1 mRNA levels in MC3T3-E1 cells treated with ascorbic acid for 0 day,7 days and 10 days were 1.003±0.119,1.973±0.344 (p>0.05, vs.0 d group) and 4.976±0.990 (p<0.05, vs.0 d group), respectively. After treated with ascorbic acid for 0 day,7 days and 10 days, the protein levels of SFRP1 in MC3T3-E1 cells were 0.937±0.132,1.257±0.155 (p>0.05, vs. 0d group) and 1.295±0.150 (p>0.05, vs. 0d group), respectively. Luciferase levels in MC3T3-E1 cells transfected with pMIR-REPORT-SFRP1UTR and pMIR-REPORT were 74±10 and 487±34, respectively (p<0.05). Luciferase levels in NIH3T3 cells transfected with pMIR-REPORT SFRP1UTR and pMIRREPORT were 2240±175 and 2707±117, respectively (p<0.05).ConclusionsPart ?:Hyperlipidemic mouse models were successfully established by feeding the animals with a high fat diet. New bone formation was inhibited and bone healing was delayed in tooth extraction wounds created in the hyperlipidemic mice. Furthermore, hyperlipidemia promotes the loss of the alveolar crest height after tooth extraction.Part ?:The 3'UTR of mouse SFRP1 gene was successfully amplified and subcloned into pMIR-REPORT. The 3'UTR of SFRP1 gene is involved in the cell-specific post-transcriptional regulation of SFRP1 gene. |
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