**4. Discussion**

This study reports new data on the role of BJ phytochemicals and polyphenolic antioxidants in the activation of osteogenic factors and in the induction of the mineralization process in the presence or not of oxidative stress induced by GSH depletion. In particular, the polyphenolic content of the juice was characterized, and it was shown that BJ performs an important antioxidant action and protects from damage induced by oxidative stress, as well as upregulates factors such as ALP and RUNX-2, related to di fferentiation and the mineralization process, in normal intracellular redox state conditions. The involvement of SIRT1 in these events was also demonstrated.

In this study, the e ffect of BJ containing certain amounts of TSP was evaluated in GSH-depleted SaOS-2 cells, an in vitro condition that mimics what happens in vivo in the bone environment in the presence of oxidative stress due to microdamage and/or estrogen deficiency, [4,5,8,22,51]. Indeed, GSH is involved in osteoblast and osteoclast di fferentiation and, together with other thiol antioxidants, it may play a crucial role in estrogen deficiency-associated bone loss [1,22,51,52]. In fact, some data show that bone loss due to a lack of estrogen is related to the lowered thiol antioxidants in osteoclasts, and this activates osteoclastogenic signals which induce ROS-enhanced expression of cytokines promoting osteoclastic bone resorption [51].

We used BJ given that BBs are commercialized in di fferent ways, mainly as fresh or frozen products, also in addition to juices or dry extracts. However, the drying process and treatment with solvents (i.e., for the production of dry extracts) might partially destroy anthocyanins and their antioxidant effects [53], and the anthocyanins seem to be more stable over time in a juice with acidic pH than in a dry extract [53]. The results obtained from the qualitative and quantitative analysis of TSP in BJ show that the main polyphenolic component was represented by anthocyanins belonging to the flavonoid family, which were present mainly as glucosides, galactosides, and arabinosides; these data are similar to those found in the literature [42,54]. Moreover, TSP content in BJ obtained by HPLC-PDA was very similar to the value obtained with the Folin–Ciocalteu method, indicating that quantification via this spectrometric method is feasible and realistic.

Some studies demonstrated that many BB polyphenols are bioavailable; in fact, after various processes of ingestion, they were found in the plasma [36,54,55]. It was also demonstrated that anthocyanins or cyanidins, after oral administration, can be absorbed in intact form as glycosides and/or aglycones [34,35,55]. Moreover, even if BB polyphenols undergo complex metabolic modifications, their derivatives have the same functional characteristics [34,36]. The flavonoids and anthocyanins present in BB have strong antioxidant capacity [24,31,32,54], and these, along with the other polyphenol compounds and their derivatives, favor the formation of bone mass [18–20,30,54].

The data of this study demonstrate that TSPs, together with other phytochemicals contained in BJ, are able to prevent BSO-induced oxidative stress, and the results partly correlate with what was previously obtained with thiol antioxidants, such as GSH and *N*-acetyl cysteine, in SaOS-2 cells under similar conditions of oxidative stress [13]. The BJ antioxidant e ffect in SaOS-2 cells was obtained with the same concentrations of TSP used in osteocytes in which oxidative stress was induced by starvation [33], and ROS reduction was achieved by BJ treatment very quickly both in osteocytes and in SaOS-2 cells [13,33], similarly to thiol antioxidants [6]. Therefore, thanks to their antioxidant action, BJ phytocompounds may e ffectively contribute to preventing and/or eliminating oxidative stress damage present in bone pathologies, particularly in osteoporosis, as reported in the literature for various polyphenols or their derivatives [18,24,25,30]. Our results support these data; in fact, the

initial stimuli that induce osteogenic activity in SaOS-2 cells are sensitive to changes in the oxidative state. Indeed, both ALP and RUNX-2 are important markers related to the activation of the first phase of the osteoblast di fferentiation process and to the subsequent induction of calcium and matrix deposition by di fferentiated osteoblasts [13,37,43,44]. In fact, we observed a strong initial increase of ALP activity that subsequently remained high and then decreased, indicating the achievement of a high degree of di fferentiation, as also previously observed [13]. Both ALP activity and RUNX-2 expression and activation significantly decreased in the presence of BSO-induced oxidative stress, as well as the mineralization process. These events are e fficiently prevented by BJ antioxidant action.

The results of this study also show that BJ treatment in the presence of normal ROS levels has a remarkable non-redox-regulated osteogenic action that occurs through a significant upregulation of ALP and RUNX-2 activity. It is worthy to note that BJ maintains ALP activity levels higher than those in untreated cells for a long time, and this seems to be related to the activation of the mineralization process that is evident only in the late phase of this event. Finally, the similar and high levels of calcium, obtained in cells treated with BSO + BJ or with BJ alone after 24 days, show that the elimination of the oxidative state in the initial phase of the di fferentiation allows BJ to perform the long-term activation of this process. Indeed, modulation of ALP and RUNX-2 activity in BJ-treated SaOS-2 cells, in the presence or not of BSO-induced oxidative stress, is similar to that previously observed in these cells treated with thiol antioxidants, even if the e ffect of the latter on the mineralization process became evident more quickly [13]. These di fferences may be due to the di fferent chemical characteristics and action mechanisms of the antioxidants. However, in both studies, thiol and non-thiol antioxidants regulated the osteogenic activity of osteoblasts through mechanisms unrelated to their antioxidant activity. In fact, the activating e ffect of BJ or of thiol antioxidants on the mineralization process occurred in cells in which there was no alteration in the redox state. It is worthy to note that the BJ e ffects on osteogenic factors were found using TSP concentrations similar to those used to demonstrate the osteogenic activity of the polyphenolic component of dried plums [56]. In fact, these antioxidants restore the TNF α-induced suppression of ALP activity and upregulate RUNX-2 expression, influencing mineralized matrix formation under normal and inflammatory conditions [56]. Moreover, our data agree with the increased expression of RUNX-2 and ALP in pre-osteoblast cells treated with serum from BB-fed rats and with the subsequent increase in osteoblast activity and bone formation [57]. These data are also related to the ability of BB-rich diets administered in young female rats to prevent bone loss in ovariectomized adult female rats [19,20,24,30], where the e ffects of dietary BB appear similar to those of estrogens [57]. In fact, the polyphenols seem to interact with the estrogen receptors and induce their e ffects through redox-independent factors and signaling pathways related to the regulation of bone cell activity [36,57].

Moreover, many data sugges<sup>t</sup> that anthocyanins, in addition to their antioxidant activity, can also have other beneficial health e ffects [20,24–26]. This may agree with the possible involvement of SIRT1 in ALP and RUNX-2 activation, as well as the increase in calcium deposition, due to BJ treatment in the presence or not of a normal redox state. Indeed, SIRT1 levels did not change in the presence of BSO-induced oxidative stress, indicating that SIRT1 expression does not seem to be a redox-regulated mechanism. BJ was e ffectively able to upregulate SIRT1 expression in BSO-treated and untreated cells, and BJ antioxidant action was partly related to SIRT1 activity. Previously, we similarly demonstrated a significant relationship between BJ phytocompounds and molecular events related to apoptosis and expression of osteoclastogenic factors induced by oxidative stress and SIRT1 activation [33]. Indeed, dietary BBs increase SIRT1 levels in mammals [49,50], and SIRT1 overexpression is also related to the inhibition of osteoclastogenic factors [58,59]. Moreover, SIRT1 activity promotes osteogenic di fferentiation of mesenchymal stem cells and activation of RUNX-2 [46–48]. Previously, we also demonstrated that SIRT1 activity contributes, in part, to the BJ protective e ffect in MSCs against cytotoxicity due to oxidative damage [33]. Therefore, the possible role of SIRT1 can explain, at the molecular level, the positive action of TSP and/or other phytocompounds contained in BJ on the

osteogenic activity and the mineralization process of osteoblasts, although other experiments will have to be performed to validate SIRT1's involvement in these events.

The activation of osteogenic factors and mineralization due to BJ treatment is also in agreemen<sup>t</sup> with the increase in bone mass found in young subjects fed with diets rich in blueberries or fruits rich in antioxidant phytochemicals [17,20,24,60,61]. It was also shown that BBs stimulate the growth of bone in growing rats, and this appears to be due to polyphenols and their metabolic derivatives [18,62]. Indeed, phenolic acid derivatives present in the diet promote the di fferentiation of osteoblasts and bone growth in young mice [63]. Some studies demonstrated that daily consumption of these compounds may be important in increasing the bone mass peak [20,60,64], and this is an independent predictor of increased bone mass in early pubertal children [61].
