Puerarin inhibits the osteoclastogenesis by inhibiting RANKL-dependent and \u2013independent autophagic responses

Guoyou Zhang,Yuanzheng Ma,Guoke Tang,Yu Wang

doi:10.1186/s12906-019-2691-5

Abstract

BackgroundPuerarin 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.MethodsOsteoclast 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.ResultsThe 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.ConclusionIn 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.

Highlights

Puerarin exerts therapeutic effect on osteoporosis due to its inhibitory effect on the formation of osteoclasts
This study showed a role of Puerarin in inhibiting the Osteoclast precursors (OCPs) autophagy in the absence or presence of Receptor activator for nuclear factor-kB (RANKL), which contributed to the reduction in OCP proliferation or OCP differentiation, respectively
The results showed that directly intervened by Puerarin, the expression of autophagic proteins Atg5, Atg7 and Beclin1 in OCPs decreased in a concentration-dependent manner (Fig. 1a)

Summary

Introduction

Puerarin exerts therapeutic effect on osteoporosis due to its inhibitory effect on the formation of osteoclasts. Previous studies disclosed that the protective autophagy exerts an indispensable effect on the osteoclast formation as well as bone absorption activity of osteoclast [8,9,10]. Some reports have shown that Puerarin upregulates autophagy [11,12,13], but some studies showed that it inhibits autophagy [14,15,16]. He et al [14] suggested that Puerarin had neuroprotective effect against cerebral ischemia, which was related to the decrease of autophagic activity in neurons after its intervention. Whether the effect of Puerarin on inhibiting the osteoclastogenesis is by mediating the change of autophagic activity is worth further exploring

Methods

Results

Discussion

Conclusion