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

Zhang et al. BMC Complementary and Alternative Medicine https://doi.org/10.1186/s12906-019-2691-5 (2019) 19:269 RESEARCH ARTICLE Open Access Puerarin inhibits the osteoclastogenesis by inhibiting RANKL-dependent and – independent autophagic responses Guoyou Zhang1,2,3†, Yu Wang1,4†, Guoke Tang5 and Yuanzheng Ma1,2* 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: Puerarin, Osteoclast, Autophagy, BECN1, Osteoporosis Background Pueraria lobata is a leguminous plant in China, which is widely used in the treatment of cardiovascular diseases, diabetes, osteonecrosis and neurodegeneration [1]. As an extract from Pueraria lobate, Puerarin is a phytoestrogen with significant bone-protective effect. Its therapeutic * Correspondence: † Guoyou Zhang and Yu Wang contributed equally to this work. 1 Department of Orthopedics, Southern Medical University, Guangzhou 510515, Guangdong, China 2 Department of Orthopaedics, The 8th medical center of chinese PLA general hospital, No. 17 Heishanhu Road, Haidian District, Beijing 100091, China Full list of author information is available at the end of the article effect has been broadly reported in the treatment of osteoporosis. Cho et al. [2] found that the decrease in the femoral bone density of ovariectomized mice was inhibited after feeding Puerarin-containing diet for 4 weeks. Another study found that Puerarin could alleviate streptozotocininduced osteoporosis in rats via HDAC1/HDAC3 signaling [3]. In addition, Puerarin could also prevent bone loss in castrated male rats [4]. The inhibitory effect of Puerarin on osteoclasts matters a lot in its treatment of bone loss. Pueraria extracts are known to inhibit RANKL-stimulated osteoclastogenesis in the dose-dependent manner and to reduce bone resorption activity of osteoclasts [5]. Puerarin also reduced the formation of mature osteoclasts in RANKL-induced © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Complementary and Alternative Medicine (2019) 19:269 RAW264.7 cells [6]. In addition, Puerarin can also prevent lipopolysaccharide-stimulated osteoclastogenesis and bone loss [7]. However, the mechanism regarding the effect of Puerarin on inhibiting the osteoclastogenesis remains unclear. Previous studies disclosed that the protective autophagy exerts an indispensable effect on the osteoclast formation as well as bone absorption activity of osteoclast [8–10]. In addition, a considerable number of studies suggested that Puerarin could regulate the autophagic activity. Some reports have shown that Puerarin upregulates autophagy [11–13], but some studies showed that it inhibits autophagy [14–16]. 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. It was also reported that Puerarin could prevent rat brain from ischemia/reperfusion injury through repressing the autophagic response [15]. In addition, Puerarin pretreatment reduced the hypoxia/reoxygenation injury via inhibiting the Akt-autophagy signaling in the myocardium [16]. Therefore, whether the effect of Puerarin on inhibiting the osteoclastogenesis is by mediating the change of autophagic activity is worth further exploring. Osteoclastogenesis consists of the proliferation and differentiation of OCPs. RANKL is a key inducing factor in osteoclast differentiation. Autophagy not only plays an important role in OCP proliferation [17], but also regulates OCP differentiation under RANKL intervention [8, 9]. Thus, we can clarify the overall significance of autophagy in Puerarintreated osteoclastogenesis by observing the effects of Puerarin on the autophagic activity of OCPs in the presence and absence of RANKL, respectively. During the autophagy response, a cytosolic form of LC3 (LC3I) forms membranebound LC3 (LC3II) by conjugating to phosphatidyl inositol. Thus, LC3 transformation and LC3 puncta are pivotal parameters for observing autophagic activity [18], including the osteoclastogenesis [8, 10, 19, 20]. On the one hand, as important autophagy parameters, LC3 conversion rate and LC3 puncta number were upregulated by RANKL in OCP [8, 19]; On the other hand, LC3 plays a significant role in the osteoclastogenesis. In this study, the detection of autophagic activity was focused on LC3-related indicators [10]. This study showed a role of Puerarin in inhibiting the OCP autophagy in the absence or presence of RANKL, which contributed to the reduction in OCP proliferation or OCP different (...truncated)