ObjectiveTo study the effects of 17β-estradiol (E2) on the regulation of the proliferation of condylar chondrocytes and provide a preliminary discussion on the role of phosphorylate-mammalian target of rapamycin (p-mTOR) in this regulatory process.
MethodsCondylar chondrocytes were isolated from 6-week-old female rats for primary culture. Drug treatment with different concentrations of E2 and/or rapamycin (RAPA) was carried out on second-generation cells. Cell Counting Kit 8 was used to measure the cell viability of condylar chondrocytes after culture for 24, 48, or 72 h, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the relative gene expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), collagen type Ⅱ (COLⅡ), autophagy-related gene 6 (Beclin-1), and autophagy-related gene 5 (ATG-5). Western blot was employed to determine the relative protein expression of ERα, ERβ, Beclin-1, lipid-modified light chain 3B (LC3-Ⅱ), and p-mTOR.
ResultsE2 could significantly promote the proliferation of chondrocytes culturedin vitro, and maximum promotion was achieved at a concentration of 10-8mol·L-1. RAPA could significantly inhibit cell proliferation. E2 at aconcentration of 10-8mol·L-1could greatly improve the gene expression levels of ERα and COLⅡ (P< 0.01) with the protein levels of ERα and p-mTOR (P< 0.05), and decrease the gene expression levels of Beclin-1 and ATG-5 (P< 0.05) with the protein levels of Beclin-1 and LC3-Ⅱ (P< 0.05). RAPA could also enhance the relative protein expression of Beclin-1 and LC3-Ⅱ (P< 0.01), and reduce the expression of p-mTOR (P< 0.01). Treatment with the ERα antagonist significantly reduced the expression of p-mTOR in cells (P< 0.01).
ConclusionAt a concentration of 10-8mol·L-1, E2 could effectively activate the phosphorylation of mTOR through the ERα-p-mTOR pathway, inhibit cell autophagy, and promote the proliferation of condylar chondrocytes.
