*2.3. Inhibition of Mitochondrial Injury by Taurine in a Glucocorticoid-Administered Rabbit Osteonecrosis Model*

To determine any mitochondrial protective effect of taurine in a glucocorticoid-administered rabbit osteonecrosis model, using the percentage of positive cells (%PC) in TFAM and ATP5A, the inhibitory effect of taurine on intraosseous mitochondria injury was investigated. In the MP−/TAU− group, TFAM was 73.95 ± 1.20% and ATP5A was 57.22 ± 2.23%. In the MP+/TAU− group, TFAM was 41.15 ± 2.93%, and ATP5A 31.85 ± 3.61% in contrast to MP+/TAU+ group in which TFAM was 67.19 ± 1.57%, and ATP5A 59.71 ± 2.01%, with increased expression found in this order (Figure 3). In this way, the preservation by taurine of mitochondrial function could be confirmed also in vivo.

**Figure 3.** TFAM and ATP5A positive cell rate after taurine administration in a glucocorticoid-induced rabbit osteonecrosis model. (**a**) Immunohistochemical study of TFAM and ATP5A. Scale bar: 100 μm. (**b**) Graph indicates percentage of positive cells of TFAM and ATP5A in the indicated conditions. Columns and bars indicate means and S.E. As compared with MP−/TAU− group, in MP+/TAU− group the numbers of TFAM and ATP5A positive cells were significantly decreased (# *p* < 0.0001 vs. MP−/TAU−, ## *p* = 0.001 vs. MP−/TAU−). In MP+/TAU+ group, TFAM and ATP5A levels were maintained as compared with MP−/TAU− group. (\* *p* < 0.0001 vs. MP+/TAU−, \*\* *p* < 0.001 vs. MP+/TAU−).

#### **3. Discussion**

Recently, numerous studies have identified a role for oxidative stress in glucocorticoid-induced osteonecrosis [2,14,15]. Regarding mitochondria, which are the major site of oxidative stress generation, the intraosseous decrease of their TFAM levels induced by glucocorticoid administration has been implicated in osteonecrosis development, suggesting that mitochondrial stress plays a role in this process [5]. Accordingly, preservation of mitochondrial function may be an important factor helping to prevent osteonecrosis development [16].

The taurine used in this experiment has lately been recognized to be a useful therapeutic agent in various disorders, including cerebrovascular injury, cardiac disorders, and hepatic injury, by protecting cells from oxidative stress. Other beneficial effects lately emphasized include inhibition of cell injury and anti-ageing properties [9,12,17]. Taurine helps to keep Ca2<sup>+</sup> concentrations in cardiomyocyte mitochondria normal, even when they are increased by H2O2-induced oxidative stress, and a possible action on the apoptosis pathway of mitochondria exposed to oxidative stress has been proposed [17]. Moreover, in mitochondrial cytopathies such as MELAS, taurine has attracted attention as a therapeutic agent that provides symptomatic improvement [8]. These findings allow hope that taurine may also help to inhibit mitochondrial injury, which is now thought to be an important step in the course of development of glucocorticoid-induced osteonecrosis.

In this study, to evaluate the efficacy of taurine against mitochondrial injury present in bone and osteocytes, TFAM, a factor strongly involved in mitochondrial function [5,16,18,19] and ATP5A, a marker of mitochondrial function produced by mitochondria, were used.

First, in vitro studies using cultured osteocytes were conducted. In these osteocytes exposed to a sufficiently stressful H-D environment so as to induce cell necrosis, both TFAM and ATP5A expression levels were markedly decreased, proving that mitochondrial functional injury had been engendered. On the other hand, with the addition of taurine under the same conditions, the decrease in TFAM and ATP5A levels was attenuated. These results show that taurine is firmly protective of mitochondrial function in vulnerable osteocytes exposed to a stressful environment.

In various glucocorticoid-induced osteonecrosis models, the occurrence of oxidative and mitochondrial injuries within 48–72 h after glucocorticoid administration is considered to be a contributing factor [2,5,15]. The period from injury to osteonecrosis development has been estimated to be approximately 72–120 h [15,20,21]. Referring to these estimates, in the present glucocorticoid-induced rabbit osteonecrosis model, taurine was administered for 5 consecutive days, and its dose was set to be consistent with that routinely used clinically, 100 mg/kg/day. With glucocorticoid-alone administration (MP+/TAU− group), the intraosseous expression of TFAM and ATP5A was significantly decreased as compared with the MP−/TAU− group. On the other hand, in the MP+/TAU+ group, similar to the in vitro results, TFAM and ATP5A showed improvement up to the levels seen in MP−/TAU− group, as well as a significant decrease in the incidence of osteonecrosis. In this way, together with the mitochondrial injury associated with glucocorticoid use leading to osteonecrosis development, it was considered that taurine protected or preserved mitochondrial function, thereby in turn, possibly helping to prevent the development of osteonecrosis.

For the purposes of the present work, the taurine dose was set to be comparable to that routinely used in daily clinical practice, but because side effects are few, in conditions such as MELAS even larger doses are being administered. In this study, although the osteonecrosis incidence was 20%, to be able to achieve an incidence approaching 0%, factors such as the administered dose of taurine, and its timing and duration will need to be determined. Since a significant decrease in the incidence of glucocorticoid-induced osteonecrosis was attained, considering the ease of taurine administration and the paucity of associated side effects, its clinical application in the near future can be anticipated.

In conclusion, preservation of intraosseous mitochondrial function is vital for the prevention of glucocorticoid-induced osteonecrosis. From the in vivo and in vitro results of taurine obtained here, preservation of mitochondrial function was demonstrated, and was in turn, considered to be related to the prevention of osteonecrosis.

### **4. Materials and Methods**

#### *4.1. Cell Culture*

An established murine osteocytic cell line (MLO-Y4) (Kerafast, Boston, MA, USA) was maintained as a subconfluent monolayer culture in the alpha MEM medium (Gibco, Tokyo, Japan) supplemented with 10% fetal calf serum. When the culture reached 70% confluency being cultured at 37 ◦C under 20% O2 and 5% CO2, MLO-Y4 were treated with 1 μM dexamethasone (Dex, MSD, Tokyo, Japan) in 1% O2 (hypoxia) for 24 h (Dex+/hypoxia+ group). A quantity of 0.8 μM taurine was added to the medium of Dex+/hypoxia+ group exposed to taurine-free medium (Dex+/hypoxia+/taurine+ group). As a control group, cells were cultured under 20% O2 in the culture medium without either Dex or taurine (Dex-/hypoxia− group). Three independent experiments each were carried out.

#### *4.2. Immunocytochemical Study*

To determine the effect of taurine on cultured osteocytes in an H-D stress environment, an immunohistochemical study on TFAM which exerts a protective effect on mitochondria, and ATP5A which shows ATP production, which is the main function of mitochondria was conducted. Cells of each group were fixed in 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100 in phosphate buffered saline (PBS). Blocking was then achieved with 10% bovine serum albumin (Dako Cytomation, Santa Clara, CA, USA) in PBS, and the primary antibody, anti-ATP synthase (ATP5A) (Proteintech, Rosemont, IL), and anti-TFAM (LSbio, Seattle, MA, USA) antibody each at 10.0 μg/mL were made to react at room temperature for 2 h. The secondary antibody, anti-mouse Alexa 488 (Thermo Fisher Scientific, Tokyo, Japan), and anti-rabbit Alexa 594 (Thermo Fisher Scientific) each at 10.0μg/mL were made to react while being shielded from light, and nucleus staining was done with DAPI. Then after washing in PBS, they were mounted using a prolong diamond antifade mountant (Thermo Fisher Scientific, Tokyo, Japan). Images were taken with a Zeiss-LSM710 camera (Zeiss, Baden-Württemberg, Germany). The taurine non-administered and administered groups were then compared.
