*Article* **Taurine Inhibits Glucocorticoid-Induced Bone Mitochondrial Injury, Preventing Osteonecrosis in Rabbits and Cultured Osteocytes**

**Hiroaki Hirata 1,**†**, Shusuke Ueda 1,**†**, Toru Ichiseki 1,\*, Miyako Shimasaki 2, Yoshimichi Ueda 2, Ayumi Kaneuji <sup>1</sup> and Norio Kawahara <sup>1</sup>**


Received: 30 July 2020; Accepted: 16 September 2020; Published: 20 September 2020

**Abstract:** Mitochondrial injury has recently been implicated in the pathogenesis of glucocorticoidinduced osteonecrosis. Using cultured osteocytes and a rabbit model, we investigated the possibility that taurine (TAU), which is known to play a role in the preservation of mitochondrial function, might also prevent the development of osteonecrosis. To reduplicate the intraosseous environment seen in glucocorticoid-induced osteonecrosis, dexamethasone (Dex) was added to MLO-Y4 cultured in 1% hypoxia (H-D stress environment). An in vitro study was conducted in which changes in mitochondrial transcription factor A (TFAM), a marker of mitochondrial function, and ATP5A produced by mitochondria, induced by the presence/absence of taurine addition were measured. To confirm the effect of taurine in vivo, 15 Japanese White rabbits were administered methylprednisolone (MP) 20 mg/kg as a single injection into the gluteus muscle (MP+/TAU− group), while for 5 consecutive days from the day of MP administration, taurine 100 mg/kg was administered to 15 animals (MP+/TAU+ group). As a control 15 untreated rabbits were also studied. The rabbits in each of the groups were sacrificed on the 14th day after glucocorticoid administration, and the bilateral femora were harvested. Histopathologically, the incidence of osteonecrosis was quantified immunohistochemically by quantifying TFAM and ATP5A expression. In the rabbits exposed to an H-D stress environment and in MP+/TAU− group, TFAM and ATP5A expression markedly decreased. With addition of taurine in the in vitro and in vivo studies, the expression of TFAM and ATP5A was somewhat decreased as compared with Dex−/hypoxia− or MP−/TAU− group, while improvement was noted as compared with Dex+/hypoxia+ or MP+/TAU− group. In rabbits, the incidence of osteonecrosis was 80% in MP+/TAU− group, in contrast to 20% in the taurine administered group (MP+/TAU+), representing a significant decrease. Since taurine was documented to exert a protective effect on mitochondrial function by inhibiting the mitochondrial dysfunction associated with glucocorticoid administration, we speculated that it might also indirectly help to prevent the development of osteonecrosis in this context. Since taurine is already being used clinically, we considered that its clinical application would also likely be smooth.

**Keywords:** osteonecrosis; taurine; mitochondrial function; glucocorticoid

#### **1. Introduction**

Glucocorticoids are excellent therapeutic agents that are used effectively in diverse conditions, notably autoimmune disorders and asthma. The price for this, however, includes many well-known serious side effects including glucocorticoid-induced femoral head osteonecrosis. Glucocorticoid-induced femoral head osteonecrosis occurs at both young and elder ages, and is considered an intractable condition in which destruction of the hip joint markedly impairs quality of life (QOL) by causing pain and impaired ambulation. Once femoral head osteonecrosis is established, surgical intervention such as artificial joint implantation cannot be avoided in most cases. This situation makes the devising of optimal prophylactic countermeasures and greater elucidation of the underlying pathogenetic mechanisms of glucocorticoid-related injury very important so as to make glucocorticoid use safer.

However, despite the extensive research being focused on glucocorticoid-induced femoral head osteonecrosis, its causes and pathophysiology are still far from clear. Various studies using rabbit, rat, and other animal models have been conducted, with models of rabbit osteonecrosis induced by glucocorticoid administration being the most common [1]. Hitherto, various causative factors such as oxidative stress, vascular endothelium injury, coagulopathy, and dyslipidemia have been implicated, and much work has been devoted to their prevention and management [2–4]. Recently, attention has been turned to mitochondria because they are the site of oxidative stress development, and the involvement of mitochondrial injury in glucocorticoid-induced osteonecrosis is being recognized [5].

In general, cells of the mitochondrial electron transport system in vivo account for ≥90% of oxygen consumption, of which 1–5% is converted into reactive oxygen species, representing the major intracellular source of reactive oxygen species generation [6]. The acceleration of reactive oxygen species production induced by mitochondrial and mitochondrial DNA injury has been shown to be involved in the development of diverse pathological conditions. Moreover, it has been recognized that mitochondria exist in a state characterized by constant exposure to oxidative stress, with this having a considerable impact on disease development and progression. For this reason, the possibility has been raised that mitochondria may be an ideal target for both therapeutic trials and attempts at elucidating the underlying pathogenetic mechanisms of various disorders [7].

Taurine (TAU, 2-aminoethanesulfonic acid) has been attracting increasing attention as an easy to administer therapeutic agent with few side effects that can help to prevent mitochondrial injury. Taurine is made up of free amino acids present in large quantities in vivo, and has been proven to be effective in the therapy of mitochondrial cytopathies such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS) syndrome [8]. Taurine's anti-inflammatory and anti-oxidative actions have been exciting interest [9,10], while its impact on mitochondrial function including regenerative and prophylactic actions is also being reported [11,12].

These properties of taurine suggest to us the realistic possibility that it may also be effective in preserving mitochondrial function and thereby help to inhibit the development of glucocorticoid-induced osteonecrosis in which mitochondrial dysfunction has been implicated. In a recent study conducted under in vitro conditions, osteocytes exposed to a stress environment (H-D stress environment), namely a hypoxic environment to which dexamethasone (Dex) had been added, an intraosseous environment with successful reduplication of glucocorticoid-induced osteonecrosis has been described [13]. Here, in an in vitro study, we first sought to determine whether taurine exerts any inhibitory effect on osteocyte mitochondrial functional injury in this kind of H-D stress environment. Furthermore, to document any in vivo inhibitory effect of taurine on the development of glucocorticoid-induced osteonecrosis, we investigated the incidence of osteonecrosis and intraosseous mitochondrial function using a glucocorticoid-induced rabbit osteonecrosis model.
