Icariin regulates miR-23a-3p-mediated osteogenic differentiation of BMSCs via BMP-2/Smad5/Runx2 and WNT/β-catenin pathways in osteonecrosis of the femoral head

Xiao-Yun Zhang; Hua-Nan Li; Feng Chen; Yue-Ping Chen; Yuan Chai; Jian-Zhao Liao; Bin Gan; Ding-Peng Chen; Song Li; Yong-Qian Liu

doi:10.1016/j.jsps.2021.10.009

Icariin regulates miR-23a-3p-mediated osteogenic differentiation of BMSCs via BMP-2/Smad5/Runx2 and WNT/β-catenin pathways in osteonecrosis of the femoral head

Saudi Pharm J. 2021 Dec;29(12):1405-1415. doi: 10.1016/j.jsps.2021.10.009. Epub 2021 Oct 29.

Authors

Xiao-Yun Zhang^{1

2}, Hua-Nan Li^{1

3}, Feng Chen², Yue-Ping Chen², Yuan Chai², Jian-Zhao Liao², Bin Gan¹, Ding-Peng Chen¹, Song Li¹, Yong-Qian Liu¹

Affiliations

¹ Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China.
² Department of Orthopedics, Ruikang Hospital Affiliated with Guangxi University of Chinese Medicine, Nanning 530011, China.
³ Department of Orthopedics, Hospital Affiliated with Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China.

Abstract

Icariin is commonly used for the clinical treatment of osteonecrosis of the femoral head (ONFH). miR-23a-3p plays a vital role in regulating the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). The present study aimed to investigate the roles of icariin and miR-23a-3p in the osteogenic differentiation of BMSCs and an ONFH model. BMSCs were isolated and cultured in vitro using icariin-containing serum at various concentrations, and BMSCs were also transfected with a miR-23a inhibitor. The alkaline phosphatase (ALP) activity and cell viability as well as BMP-2/Smad5/Runx2 and WNT/β-catenin pathway-related mRNA and protein expression were measured in BMSCs. Additionally, a dual-luciferase reporter assay and pathway inhibitors were used to verify the relationship of icariin treatment/miR-23a and the above pathways. An ONFH rat model was established in vivo, and a 28-day gavage treatment and lentivirus transfection of miR-23a-3p inhibitor were performed. Then, bone biochemical markers (ELISA kits) in serum, femoral head (HE staining and Digital Radiography, DR) and the above pathway-related proteins were detected. Our results revealed that icariin treatment/miR-23a knockdown promoted BMSC viability and osteogenic differentiation as well as increased the mRNA and protein expression of BMP-2, BMP-4, Runx2, p-Smad5, Wnt1 and β-catenin in BMSCs and ONFH model rats. In addition, icariin treatment/miR-23a knockdown increased bone biochemical markers (ACP-5, BAP, NTXI, CTXI and OC) and improved ONFH in ONFH model rats. In addition, a dual-luciferase reporter assay verified that Runx2 was a direct target of miR-23a-3p. These data indicated that icariin promotes BMSC viability and osteogenic differentiation as well as improves ONFH by decreasing miR-23a-3p levels and regulating the BMP-2/Smad5/Runx2 and WNT/β-catenin pathways.

Keywords: BAP, bone-specific alkaline phosphatase; BMP-2, bone morphogenetic protein-2; BMP-2/Smad5/Runx2 pathway; BMP-4, bone morphogenetic protein-4; BMSCs, bone marrow-derived mesenchymal stem cells; CTX-1, C-terminal telopeptides of type I collagen; DMEM, Dulbecco’s modified Eagle’s medium; DR, Digital Radiography; FBS, fetal bovine serum; HE, Hematoxylin‐eosin; Icariin; LPS, lipopolysaccharide; NTX-1, N-terminal telopeptides of type I collagen; OC, osteocalcin; ONFH, osteonecrosis of the femoral head; Osteonecrosis of the femoral head; RT-PCR, Real time PCR; SI, icariin-containing serum; TRACP-5b, tartrate-resistant acid phosphatase 5b; WNT/β-catenin pathway; miR-23a-3p.