Puerarin inhibits the osteoclastogenesis by inhibiting RANKL-dependent and -independent autophagic responses

Abstract

Background: Puerarin exerts therapeutic effect on osteoporosis due to its inhibitory effect on the formation of osteoclasts. Puerarin is also widely established as an autophagy inhibitor. The study aimed to investigate the significance of autophagy in Puerarin-treated osteoclast formation.

Methods: Osteoclast precursors (OCPs) derived from bone marrow-derived macrophages (BMMs) were treated with Puerarin along with RANKL or without RANKL, and then the autophagic parameters of OCPs (including autophagic proteins, LC3 transformation, autophagosome or LC3-puncta) were observed through Western Blotting, Transmission Electron Microscopy and Immunofluorescence assays. Next, after using overexpression vectors of autophagic genes (Atg7, Atg5 and BECN1) to alter autophagy activity, OCP proliferation was measured by Ethynyl deoxyuridine (EdU) assays and Cell Counting Kit-8 (CCK-8) kit, and osteoclast differentiation was assessed by Tartrate-resistant acid phosphatase (TRAP) staining.

Results: The results showed that Puerarin could directly inhibit the autophagy and proliferation of OCPs. Importantly, overexpression of autophagic genes Atg5, Atg7 and BECN1 reversed Puerarin-inhibited OCP autophagy and proliferation. What's more, RANKL could promote the autography of OCPs, which was recovered by Puerarin treatment. Interestingly, different from single-Puerarin treatment, we found that in the presence of RANKL, only BECN1 overexpression significantly reversed Puerarin-inhibited osteoclast differentiation and OCP autophagy.

Conclusion: In conclusion, Puerarin could inhibit the OCP autophagy in the presence or absence of RANKL, which blocked the OCP proliferation and osteoclast differentiation respectively. Moreover, BECN1 plays an essential role in Puerarin-inhibited osteoclastogenesis. Our study provides potential clue to further complete the intrinsic mechanism of Puerarin in treating osteoporosis.

Keywords: Autophagy; BECN1; Osteoclast; Osteoporosis; Puerarin.

Fig. 1

Puerarin directly inhibits OCP autophagy. a The protein levels of Atg7, Beclin1 and Atg5 in the OCPs treated with different levels of Puerarin for 6 h without RANKL were measured by Western Blotting. Compared between Group 0 μM and Group 10 μM, Group 10 μM and Group 25 μM, Group 25 μM and Group 50 μM, respectively, *P < 0.05. b The ratio of LC3II/I in the OCPs treated with Puerarin (50 μM) in the presence or absence of E64d + PEPS A for 8 h without RANKL. c mRNA expression of Atg5, BECN1 or Atg7 in the treated OCPs after infection with lentivirus encoding Atg5, BECN1 or Atg7 (LV- Atg5, LV-BECN1 or LV-Atg5) and the corresponding control vector (LV-Cont). d Viruses-transduced OCPs were treated with Puerarin (50 μM) for 6 h without RANKL, and LC3II/I was detected by Western Blotting. e The autophagosomes (yellow arrows) in the viruses-transduced OCPs treated with Puerarin (50 μM) for 24 h without RANKL were observed using TEM. Scale bar, 1 μm. f Statistical diagrams showed the quantitative results of autophagosomes from E (45 cells from three independent assays). Data are presented as mean ± SEM from three independent experiments. Compared between Group LV-Cont and Group LV-Cont+PR, Group LV-Cont+PR and Groups LV-Atg5/BECN1/Atg7 + PR, respectively, *P < 0.05. PR, Puerarin; Cont, control groups; E, E64D; P, PEPS A

Fig. 2

Overexpression of autophagic gene reverses Puerarin-reduced OCP proliferation. a The proliferation of viruses-transduced OCPs treated with Puerarin (50 μM) for 24 h was detected using EdU kit. Scale bar, 250 μm. b Statistical diagram displayed the percentages of EdU-positive cells in A (50 cells per field, n = 10). c After treatment as described for (a), the OCP proliferation was measured using CCK-8 kit. The group without Puerarin were regarded as the control group (100%). Data are presented as mean ± SEM from three independent experiments. Compared between Group LV-Cont and Group LV-Cont+PR, Group LV-Cont+PR and Groups LV-Atg5/BECN1/Atg7+ PR, respectively, *P < 0.05. PR, Puerarin

Fig. 3

Puerarin reverses RANKL-enhanced OCP autophagy. a Following treatment with or without Puerarin (50 μM) for 6 h in the presence of RANKL, the protein level of Atg7, Beclin1 or Atg5, and the ratio of LC3II/I were detected by Western Blotting. b After treatment as described for (a), LC3-puncta in each group was imaged using the immunofluorescence staining and observed using the fluorescence microscope. Scale bar, 20 μm. c Statistical diagrams exhibited the percentages of LC3-puncta positive cells (more than five dots, 50 cells/field, n = 5). Data are presented as mean ± SEM from three independent experiments. Compared between control group and RANKL group, RANKL group and RANKL+PR group, respectively, *P < 0.05. ns, no significance; PR, Puerarin; Cont, control groups

Fig. 4

Under RANKL intervention, BECN1 overexpression reverses Puerarin-inhibited OCP autophagy and osteoclast differentiation. a-b LC3II/I in viruses-transduced OCPs treated with Puerarin (50 μM) along with RANKL for 6 h was detected by Western Blotting. Compared between Group LV-Cont and Group LV-Cont+PR, Group LV-Cont+PR and Groups LV-Atg5/BECN1/Atg7+ PR in the presence of RANKL, respectively, *P < 0.05. c Typical TRAP staining of mature osteoclasts derived from viruses-transduced OCPs under the joint intervention of M-CSF, RANKL and Puerarin for 5 days. Scale bar, 100 μm. d The quantitative results regarding TRAP⁺ multinucleated cells in C. e-g mRNA expression of MMP-9, TRAP or CTSK in OCPs treated with indicated treatments. Data are presented as mean ± SEM from three independent experiments. Compared between Group LV-Cont and Group LV-Cont+PR, Group LV-Cont+PR and Groups LV-Atg5/BECN1/Atg7+ PR in the presence of RANKL plus M-CSF, respectively, *P < 0.05. ns, no significance; PR, Puerarin

Fig. 5

The current working model regarding Puerarin-regulated autophagy during the osteoclastogenesis