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

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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.

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BMSC-derived exosomes carrying microRNA-122-5p promote proliferation of osteoblasts in osteonecrosis of the femoral head.

Wen He Liao, Yu Ning, Hai-Jia Xu … (2019)

Mesenchymal stem cells (MSCs) with multipotential differentiation capacity can differentiate into bone cells under specific conditions and can be used to treat osteonecrosis (ON) of the femoral head (ONFH) through cell transplantation. The current study aims to explore the role of bone marrow (BM) MSCs (BMSCs)-derived exosomes carrying microRNA-122-5p (miR-122-5p) in ONFH rabbit models.First, rabbit models with ONFH were established. ONFH-related miRNAs were screened using the Gene Expression Omnibus (GEO) database. A gain-of-function study was performed to investigate the effect of miR-122-5p on osteoblasts and BMSCs and effects of exosomes carrying miR-122-5p on ONFH. Co-culture experiments for osteoblasts and BMSCs were performed to examine the role of exosomal miR-122-5p in osteoblast proliferation and osteogenesis. The target relationship between miR-122-5p and Sprouty2 (SPRY2) was tested.MiR-122, significantly decreased in ONFH in the GSE89587 expression profile, was screened. MiR-122-5p negatively regulated SPRY2 and elevated the activity of receptor tyrosine kinase (RTK), thereby promoting the proliferation and differentiation of osteoblasts. In vivo experiments indicated that bone mineral density (BMD), trabecular bone volume (TBV), and mean trabecular plate thickness (MTPT) of femoral head were increased after over-expressing miR-122-5p in exosomes. Significant healing of necrotic femoral head was also observed.Exosomes carrying over-expressed miR-122-5p attenuated ONFH development by down-regulating SPRY2 via the RTK/Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Findings in the present study may provide miR-122-5p as a novel biomarker for ONFH treatment.

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miRNAs in bone tissue correlate to bone mineral density and circulating miRNAs are gender independent in osteoporotic patients

Sarah Kelch, Elizabeth Balmayor, Claudine Seeliger … (2017)

We previously demonstrated the aberrant expression of nine specific miRNAs in serum from osteoporotic patients. In the present study, we further evaluated the expression of these miRNAs in bone tissue, osteoblasts, and osteoclasts from 28 patients. We hypothesize that miRNA expression in serum from osteoporotic patients may be gender-independent. A further hypothesis is that the miRNA expression in bone could be correlated with BMD values. Moreover, intracellular expression of these osteoporosis-related miRNAs may indicate the role of these molecules during osteoporosis. Our results indeed show that miRNA expression in serum was gender-independent except for miR125b-5p. A correlation with BMD was confirmed for miR-21-5p, miR-24-3p, miR-93-5p, miR-100-5p and miR125b-5p with linear correlation coefficients r > 0.9. Intracellular studies revealed a simultaneous up-regulation of miR-21-5p, miR-93-5p, miR-100-5p and miR125b-5p in osteoblasts and in osteoclasts. miR-148a-3p up-regulation in cells was specific for osteoporotic osteoclasts. Altogether, miR-21-5p, miR-93-5p, miR-100-5p, and miR-125b-5p showed significant upregulation in serum, tissue and bone cells of osteoporotic patients. All except miR-125b-5p showed gender independent expression and good correlation to BMD values. Our results suggest that these miRNAs may be important for an earlier diagnosis of osteoporosis.

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Anti-inflammatory protein TSG-6 secreted by bone marrow mesenchymal stem cells attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia

Hao Yang, Lingmin Wu, Huimin Deng … (2020)

Background Neuroinflammation plays a vital role in the development and maintenance of neuropathic pain. Recent evidence has proved that bone marrow mesenchymal stem cells (BMSCs) can inhibit neuropathic pain and possess potent immunomodulatory and immunosuppressive properties via secreting a variety of bioactive molecules, such as TNF-α-stimulated gene 6 protein (TSG-6). However, it is unknown whether BMSCs exert their analgesic effect against neuropathic pain by secreting TSG-6. Therefore, the present study aimed to evaluate the analgesic effects of TSG-6 released from BMSCs on neuropathic pain induced by chronic constriction injury (CCI) in rats and explored the possible underlying mechanisms in vitro and in vivo. Methods BMSCs were isolated from rat bone marrow and characterized by flow cytometry and functional differentiation. One day after CCI surgery, about 5 × 106 BMSCs were intrathecally injected into spinal cerebrospinal fluid. Behavioral tests, including mechanical allodynia, thermal hyperalgesia, and motor function, were carried out at 1, 3, 5, 7, 14 days after CCI surgery. Spinal cords were processed for immunohistochemical analysis of the microglial marker Iba-1. The mRNA and protein levels of pro-inflammatory cytokines (IL-1β, TNFα, IL-6) were detected by real-time RT-PCR and ELISA. The activation of the TLR2/MyD88/NF-κB signaling pathway was evaluated by Western blot and immunofluorescence staining. The analgesic effect of exogenous recombinant TSG-6 on CCI-induced mechanical allodynia and heat hyperalgesia was observed by behavioral tests. In the in vitro experiments, primary cultured microglia were stimulated with the TLR2 agonist Pam3CSK4, and then co-cultured with BMSCs or recombinant TSG-6. The protein expression of TLR2, MyD88, p-p65 was evaluated by Western blot. The mRNA and protein levels of IL-1β, TNFα, IL-6 were detected by real-time RT-PCR and ELISA. BMSCs were transfected with the TSG-6-specific shRNA and then intrathecally injected into spinal cerebrospinal fluid in vivo or co-cultured with Pam3CSK4-treated primary microglia in vitro to investigate whether TSG-6 participated in the therapeutic effect of BMSCs on CCI-induced neuropathic pain and neuroinflammation. Results We found that CCI-induced mechanical allodynia and heat hyperalgesia were ameliorated by intrathecal injection of BMSCs. Moreover, intrathecal administration of BMSCs inhibited CCI-induced neuroinflammation in spinal cord tissues. The analgesic effect and anti-inflammatory property of BMSCs were attenuated when TSG-6 expression was silenced. We also found that BMSCs inhibited the activation of the TLR2/MyD88/NF-κB pathway in the ipsilateral spinal cord dorsal horn by secreting TSG-6. Meanwhile, we proved that intrathecal injection of exogenous recombinant TSG-6 effectively attenuated CCI-induced neuropathic pain. Furthermore, in vitro experiments showed that BMSCs and TSG-6 downregulated the TLR2/MyD88/NF-κB signaling and reduced the production of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in primary microglia treated with the specific TLR2 agonist Pam3CSK4. Conclusions The present study demonstrated a paracrine mechanism by which intrathecal injection of BMSCs targets the TLR2/MyD88/NF-κB pathway in spinal cord dorsal horn microglia to elicit neuroprotection and sustained neuropathic pain relief via TSG-6 secretion.

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Author and article information

Contributors

Xiao-yun Zhang

Hua-nan Li

Journal

Journal ID (nlm-ta): Saudi Pharm J

Journal ID (iso-abbrev): Saudi Pharm J

Title: Saudi Pharmaceutical Journal : SPJ

Publisher: Elsevier

ISSN (Print): 1319-0164

ISSN (Electronic): 2213-7475

Publication date PMC-release: 29 October 2021

Publication date (Print): December 2021

Publication date (Electronic): 29 October 2021

Volume: 29

Issue: 12

Pages: 1405-1415

Affiliations

[a ]Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China

[b ]Department of Orthopedics, Ruikang Hospital Affiliated with Guangxi University of Chinese Medicine, Nanning 530011, China

[c ]Department of Orthopedics, Hospital Affiliated with Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China

Author notes

[* ]Corresponding authors at: Jiangxi University of Traditional Chinese Medicine, No. 1688 Meiling Avenue, Nanchang 330004, China (X. Zhang). Department of Orthopedics, Hospital Affiliated with Jiangxi University of Traditional Chinese Medicine, No. 445 Meiling Avenue, Nanchang 330004, China (H. Li). zhangxiaoyun520@ 123456126.com 20086170@ 123456jxutcm.edu.cn

Article

Publisher Item ID: S1319-0164(21)00211-5

DOI: 10.1016/j.jsps.2021.10.009

PMC ID: 8720822

PubMed ID: 35002378

SO-VID: 70a7b476-de6a-4e4c-968c-3dcc3b5545ae

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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

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Most cited references 51

BMSC-derived exosomes carrying microRNA-122-5p promote proliferation of osteoblasts in osteonecrosis of the femoral head.

miRNAs in bone tissue correlate to bone mineral density and circulating miRNAs are gender independent in osteoporotic patients

Anti-inflammatory protein TSG-6 secreted by bone marrow mesenchymal stem cells attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia

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