RESUMO
OBJECTIVE@#This work aimed to study and identify the influence and target gene of microRNA-29a-3p (miR-29a-3p) in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high-fat environment in vitro and in vivo.@*METHODS@#1) In vitro: BMSCs were randomly allocated into two groups and were then induced to undergo osteogenic differentiation in a normal or high-fat environment. Next, a miR-29a-3p mimic/inhibitor was transfected into the two groups of cells. The mRNA expression levels of alkaline phosphatase (ALP), Runt related gene 2 (Runx2), and miR-29a-3p and the protein expression levels of ALP and Runx2 were detected before and after transfection through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Moreover, Frizzled (Fzd) 4 was predicted as the target gene of miR-29a-3p by using an online database (Target Scan, MiRNA.org). The interactive relationship between miR-29a-3p and Fzd4 was confirmed through dual-luciferase assays. 2) In vivo: Rats were randomly divided into two groups and fed with a standard or high-fat diet. Titanium implants were grown in rats. Then, the expression levels of miR-29a-3p, ALP, and Runx2 were detected in bone tissues surrounding implants. Moreover, hard tissue sections were subjected to methylene blue-acid magenta staining and observed under microscopy to study bone formation around implants. In addition, miR-29a-3p-overexpressing lentiviral vectors were transfected into rats, and the expression levels of ALP, Runx2, and miR-29a-3p in bone tissues surrounding implants were detected at 3 and 10 days after transfection.@*RESULTS@#The expression levels of ALP, Runx2, and miR-29a-3p and the osteogenic differentiation of BMSCs were suppressed in high-fat groups in vitro and in vivo.@*CONCLUSIONS@#MiR-29a-3p plays a positive role in the regulation of BMSCs in a high-fat environment. It can increase ALP and Runx2 expression levels in bone tissues surrounding implants in hyperlipidemia models. This result implies that miR-29a-3p can promote implant osseointergration in a rat model of hyperlipidemia.
Assuntos
Animais , Ratos , Diferenciação Celular , Implantes Dentários , Hiperlipidemias , MicroRNAs , Osseointegração , Osteoblastos , Osteogênese , Distribuição AleatóriaRESUMO
Objective To investigate the molecular mechanism of Loureirin A mediated anti-hepatic fibrosis by evaluting its effects on proliferation , secretion ofα-smooth muscle actin (α-SMA) and transforming growth factor-beta1 (TGF-β1), and expression of rat hepatic stellate cells in vitro . Methods Primary hepatic stellate cells were isolated and cultured from Sprague-Dawley rats. After activating and inducing primary hepatic stellate cells from qHSC to aHSC, the activated hepatic stellate cells model in vitro was established. Then we observed the morphological changes of static hepatic stellate cells and activated hepatic stellate cells with inverted phase contrast microscope. Cultured hepatic stellate cells were treated with different concentrations of loureirin A and the inhibitory rate of HSCs proliferation was measured by MTT assay. The expression of Frizzled-4 was measured by western blot analysis. The content ofα-SMA and TGF-β1 in the cultured HSCs'supernatant were measured by enzyme-linked immunosorbent assay (ELISA) . Results Loureirin A the proliferation of inhibited activated hepatic stellate cells in a time-dose-dependent manner compared with the control group,IC50=0.30 μg/μL. After loureirinA treatment of the HSCs, western blot analysis showed that Frizzled-4 expression level was obviously lower than control group. Loureirin A also inhibitedα-SMA and TGFβ1 (P<0.05) secretion in the cultured HSCs'supernatant in different degree by the assay of ELISA. Conclusions The molecular mechanism of Loureirin A and Wnt signaling pathway mediated anti-hepatic fibrosis and anti-angiogenesis may involve down-regulation the expression of Frizzled-4, inhibiting the synthesis and secretion ofα-SMA,TGF-β1and the proliferation of HSCs.