Puerarin promotes the viability and differentiation of MC3T3-E1 cells by enhancing LC3B-mediated autophagy through downregulation of miR-204

Abstract

Puerarin is a bioactive substance extracted from Pueraria lobata. It is known to promote the viability, differentiation and mineralization of osteoblasts. However, the molecular mechanisms involved in these activities are not well understood. The present study was conducted with the aim of elucidating the effect of puerarin on osteoblasts and to explore the underlying mechanism. CCK-8 analysis showed that puerarin (0.1, 1 and 10 µM) promoted the viability of osteoblastic MC3T3-E1 cells, with 1 µM of puerarin exhibiting the strongest effect. Moreover, 1 µM puerarin significantly increased the activity of alkaline phosphatase (ALP) and the formation of mineralized nodules in the MC3T3-E1 cells. Treatment with 1 µM puerarin for 72 h led to a significant upregulation in the expression level of microtubule-associated light chain 3 (LC3)B and Beclin1 proteins. This treatment was more effective in promoting LC3B expression than what was observed following treatment with rapamycin (overexpression for autophagy). The bilayer membrane structure of autophagosomes was observed by electron microscopy. Conversely, 3-methyladenine (3-MA, inhibitor of autophagy) reduced the cell viability as well as the activity of alkaline phosphatase (ALP) in MC3T3-E1 cells, although, there was no significant influence on mineralization. Prediction results of the biological information showed that LC3B could be a direct target of microRNA-204 (miR-204). In the present study, the expression level of miR-204 was decreased by puerarin. miR-204 mimics significantly decreased LC3B expression and inhibited auotophagosome formation, while the miR-204 inhibitor had the opposite effects. To conclude, the results of the present study suggest that puerarin promotes the viability and differentiation of MC3T3-E1 cells through autophagy, which is possibly associated with miR-204-regulated LC3B upregulation.

Keywords: LC3B; MC3TC-E1 cells; autophagy; differentiation; miR-204; puerarin; viability.

Figure 1.

Effects of puerarin on MC3T3-E1 cell viability, differentiation and mineralization. (A) Effects of 0.1, 1 and 10 µM puerarin on osteoblast cell viability at 24, 48 and 72 h detected by CCK-8 assay, compared with a control treated with serum-free medium. (B) Effect of 1 µM puerarin on ALP activity in osteoblasts at 72 h. (C) Alizarin red staining for detection of mineralized nodules under optical microscope (magnification, ×100). (D) Relative mineralized nodule area of each group was tested after treatment with 1 µM puerarin for 7, 14 and 21 days, respectively. *P<0.05 vs. the control. Pue, puerarin; ALP, alkaline phosphatase.

Figure 2.

Effects of puerarin on autophagy. (A) Expression levels of Beclin1 and LC3B proteins were measured after osteoblastic MC3T3-E1 cells were treated with 1 µM puerarin for 48 and 72 h by western blotting, compared with a control (treated with puerarin for 0 h). (B) Quantification of A. (C) The protein levels of Beclin1 and LC3B were tested after cells were treated with puerarin (Pue), 3-MA (75 µM), rapamycin (rap) (100 µM) or Pue+3-MA for 72 h. (D) Quantification of C. (E) Representative images of autophagosomes (black arrows) were captured by transmission electron microscopy (scale bar, 500 nm). (F) Number of autophagosomes in the same field of vision, compared with a control. *P<0.05 vs. the control. LC3, microtubule-associated light chain 3; Pue, puerarin; 3-MA, 3-methyladenine; rap, rapamycin.

Figure 3.

Effects of autophagy on osteoblastic MC3T3-E1 cell viability, differentiation and mineralization. (A) Cells were treated with puerarin (Pue) (1 µM), 3-MA (75 µM) or puerarin (Pue)+3-MA for 72 h. The cell viability was assessed by CCK-8 assay. (B) Alkaline phosphatase (ALP) activity was tested after cells were treatment with Pue (1 µM), 3-MA (75 µM) or Pue+3-MA for 72 h (C) Alizarin red staining was used to evaluate the mineralized nodules under an optical microscope (magnification, ×100). (D) Relative mineralized nodule area of each group following treatment with Peu (1 µM), 3-MA (75 µM) or Peu+3-MA for 14 days. *P<0.05 vs. the control. 3-MA, 3-methyladenine.

Figure 4.

miR-204 regulates LC3B and autophagosome formation. (A) Osteoblastic MC3T3-E1 cells were treated with 1 µM puerarin for 72 h, and the miR-204 expression level was examined by stem-loop reverse transcription-quantitative polymerase chain reaction. (B) Schematic of the miR-204 target sites within the mouse LC3B WT 3′-UTR. (C) The expression level of miR-204 mRNA following transfection with miR-204 mimics, miR-204 inhibitor or miR-204 negative control (NC). (D) The expression of LC3B in osteoblasts at 72 h following transfection with miR-204 mimics, miR-204 inhibitor or miR-204 NC. (E) Quantification of D. (F) Representative images of autophagosomes (black arrows) were captured by transmission electron microscopy (scale bar, 500 nm). (G) Number of autophagosomes in the same field of vision, compared with the control. *P<0.05 vs. the control. miR, microRNA; LC3, microtubule-associated light chain 3; Pue, puerarin; UTR, untranslated region.