Prednisolone suppresses collagen-encoding gene expression causing cartilage defects in zebrafish larvae

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Highlights

Abstract

Glucocorticoid-induced osteoporosis (GIOP) is a clinically important disease. Despite many studies, the intrinsic pathogenesis of GIOP is still not fully understood. Cartilage is the target tissue of the glucocorticoid prednisolone (PN). To explore the intrinsic mechanism of PN-induced cartilage damage, we performed cartilage staining and cell transfection experiments in zebrafish larvae treated with PN. The results showed that PN caused cartilage damage in zebrafish at 25 μM. Moreover, after treatment with PN, it was found that collagen-encoding gene expression was significantly reduced. Further research revealed that the glucocorticoid receptor (GR) mediates the transcriptional inhibition of collagen genes by PN. These results indicate that glucocorticoids cause cartilage damage by inhibiting the expression of collagen genes through their receptors. Our study provides new insights into GIOP.

Introduction

Glucocorticoids (GCs) and their analogues have been successfully used clinically for many years, mainly for the treatment of inflammation and some immune diseases (Barnes, 1998; Sannomiya et al., 1985; Schleimer, 2004). GCs act primarily by binding to the glucocorticoid receptor (GR) (Hollenberg et al., 1985). GR is a nuclear receptor that binds cortisol and other GCs (Adcock et al., 2004). GR is expressed in almost all vertebrate cells, directly up-regulating and down-regulating thousands of genes in different cell types, as well as regulating development, metabolism, stress response, inflammation, and other aspects of key tissue and tissue processes (Adcock et al., 2004). However, clinical studies have found that long-term or repeated doses of GCs analogues can cause many side effects, such as mood disorders (McEwen, 2005) and bone damage (Schacke et al., 2002; Weinstein, 2011). Clinical studies have found that long-term use of GCs drugs can reduce bone density, and induce glucocorticoid-induced osteoporosis (GIOP) (Dalle Carbonare et al., 2002; Dempster, 1989). These findings indicate that bone tissue is a target tissue of GCs.

Bone is a homeostasis tissue of the body, with osteoblasts compensating for lost bone mass, and osteoclasts resorbing excess bone tissue (Long, 2011). An imbalance of this homeostasis can lead to many bone diseases, of which osteoporosis, especially postmenopausal osteoporosis, is one of the most common, and can cause serious inconvenience to patients' lives (Pacifici, 1996). Studies have found that GCs drugs can inhibit osteoblast production and increase osteoblast apoptosis (Weinstein et al., 1998). Additionally, many osteoblast- and osteoclast-associated genes are altered after GC treatment (Leclerc et al., 2004).

Zebrafish is a model organism that has been successfully applied in many fields of biomedicine (Tavares and Santos Lopes, 2013). Zebrafish models have also been successfully applied in the fields of bone development and bone relative diseases. Similar to mammals, there are similar processes of intramembranous and intrachondral osteogenesis in zebrafish. Since embryos and larvae fish develop transparently, osteoblasts and osteoclasts can be monitored in much detail, using transgenic line. The key molecules that regulate bone development and remodeling in zebrafish are highly homologous to mammals. In 2006, GCs-induced osteoporosis model of zebrafish has been established (Barrett et al., 2006). PN treatment of zebrafish larvae fish showed significant bone mineralization reduction, and this bone loss could be rescued by the clinical drug alendronate (Barrett et al., 2006). Other GIOP-related zebrafish studies have also been reported. Zebrafish scales are also a good model to study GIOP (de Vrieze et al., 2014). Studies revealed that PN treatment alters osteoclast- and extracellular matrix-related gene expression (de Vrieze et al., 2014). Subsequent studies found that the osteoblasts and osteoclasts of zebrafish were significantly reduced after PN treatment, and that PN treatment affected the regeneration of zebrafish bone (Geurtzen et al., 2017). Two articles published this year found that extracellular matrix-related genes changed significantly after PN treatment, and found that almost all collagen-encoding gene expression is down-regulated (He et al., 2018; Huo et al., 2018), but the mechanism behind this is unknown.

Extracellular matrix is composed of macromolecules synthesized by the cell, secreted extracellularly, and distributed on the cell surface or between cells, and mainly includes polysaccharides and proteins, or proteoglycans (Badylak et al., 2015). Collagen is a key part of the extracellular matrix in bone tissue. Depending on the degree of mineralization, collagen is rigid in the bone (Viguet-Carrin et al., 2006). In the process of intramembranous ossification, mesenchymal cells directly differentiate into osteoblasts, and during endochondral ossification, mesenchymal cells first differentiate into chondrocytes, producing extracellular matrix proteins such as type II collagen and proteoglycans form a cartilage template, which is subsequently replaced by mineralized bone (Shen, 2005). Bone cells mainly produce type I collagen, and chondrocytes mainly produce type II collagen (Yamasaki et al., 2001). The main encoding genes are col1, col2, col7, col9, col10, col11, col12 and so on. A variety of collagen molecules constitute the main structural scaffold of the extracellular matrix, which make the cells adhesive, is very important for the correct assembly of the extracellular matrix, and plays an important role in bone signaling and bone remodeling (Viguet-Carrin et al., 2006).

Cartilage tissue is a target for GCs, supported by evidence both clinically and in model organisms (Nakazawa et al., 2002). However, the mechanism is unknown. In this study, we treated zebrafish larvae with PN and measured cartilage damage. Furthermore, to explore the maybe mechanisms of PN suppress the collagen encoding genes.

Section snippets

Fish maintenance and PN treatment

All procedures were approved by the Soochow University Animal Care and Use Committee and were in accordance with governmental regulations of China. Adult zebrafish of the AB strain were raised in a recirculating water system under a 14/10 h light/dark (L/D) cycle at 28 °C and fed three times per day with live baby brine shrimps (Snow mountainin, china) the morning and evening, and feed commercial feed at noon. To produce embryos, male and female zebrafish were paired in the evening, and

Concentration-dependent cartilage defects in zebrafish larvae in response to PN

Previous studies have shown that after PN treatment, zebrafish embryos show a significant reduction in bone mineralization. To further explore the effects of PN on cartilage development, we performed cartilage staining. Three concentrations of PN were used to treat 24 h post-fertilization (hpf) zebrafish embryos, and cartilage staining was performed at 4 dpf, 5 dpf, and 6 dpf. After staining, the ethmoid plate, Meckel's cartilage, ceratohyal cartilage, and ceratobranchial cartilage were

Discussion

Zebrafish, a good model for bone research, has the advantage of being easy to manipulate. Earlier studies reported that zebrafish can be used as a model for glucocorticoid-induced osteoporosis. PN treatment can lead to a significant reduction in the level of bone mineralization. Glucocorticoid-induced osteoporosis is a common bone disease in the clinic, involving many cellular and molecular mechanisms and its intrinsic pathogenesis is still not fully understood. In this study, PN treatment was

CRediT authorship contribution statement

Yu Jiang; Ning, Xin; Ninghan Feng; Guoxing Zhu design this project, Yu Jiang; Ning, Xin; Ninghan Feng; Jian Yang; Wen Wu; Mingyong Wang performed the experiment, Yu Jiang; Ning, Xin; prepare the manuscript, Ninghan Feng; Guoxing Zhu revise the manuscript, Ninghan Feng; Guoxing Zhu supervise whole project.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported in part by grants from the National Natural Science Foundation of China [grant number 81602864], Jiangsu Province Young medical personnel [grant number QNRC2016149], and Wuxi key clinical specialty [grant number ZDXK006].

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      These studies have shown that zebrafish exposed to dexamethasone (intraperitoneal injection of 30 μg per g fish body weight and 9811 μg/L) exhibited smaller regenerating scales with less circularity, as well as reductions in scale expression of mmp2, mmp9, rankl (osteoclast differentiation and activation) and ctsk (bone remodelling and resorption) (Chaichit et al., 2021; Saito et al., 2020). As well as effects on bone, prednisolone has been shown to damage the cartilage of zebrafish larvae (9011 μg/L) as well as reducing the expression of collagen-encoding genes through GR transcriptional inhibition (1802 μg/L) (col1a1a, col2a1a, col9a3, col10a1a, col11a1a, and col11a2) (Jiang et al., 2021). GCs also reduce bone regenerative capabilities in fish (Ando et al., 2017; Bohns et al., 2021).

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