**4. Discussion**

The current study revealed several novel findings regarding the e ffect of Brazilian propolis on glycation stress in the skeletal muscle. Firstly, Brazilian propolis inhibited the formation of AGEs in vitro (Figure 1). Secondly, the 20-week ingestion of Brazilian propolis suppressed the accumulation of MGO-derived AGEs (Figure 2), promoted activity of glyoxalase 1 (Figure 3), and attenuated

mRNA expressions of proinflammatory cytokines IL-1β and IL-6 (Figure 4) in the EDL but not the soleus muscle.

Glycation stress is suppressed by several mechanisms such as inhibition of AGEs formation, MGO formation, and oxidative stress, detoxification of MGO, and blocked activation of AGEs receptors [30]. To date, many researchers have evaluated the inhibitory effect of natural compounds on the formation of AGEs, and many natural plants are confirmed to reduce glycation stress by inhibiting this formation [37,38]. In this study, we provided evidence for the inhibitory capacity of Brazilian propolis on formation of AGEs in vitro (Figure 1). To the best of our knowledge, this is the first study to demonstrate Brazilian propolis-induced anti-glycation activity. In accordance with this finding, European propolis, which differ from Brazilian propolis in terms of raw materials and components, have been revealed to inhibit glucose-derived and D-ribose-derived AGEs production [31–33]. These findings sugges<sup>t</sup> that various types of propolis have the capacity to inhibit AGEs formation in vitro.

We also provided a subsequent confirmation for the inhibitory effect of Brazilian propolis on formation of AGEs in vivo by showing that Brazilian propolis led to suppression of MGO-derived AGE accumulation in the skeletal muscle of MGO-loaded mice (Figure 2). This protective effect was seen in the fast-type EDL muscle but not the slow-type soleus muscle. Our previous study demonstrated that a 16-week glycation stress induced by a high-AGE diet in mice promoted the accumulation of AGEs in the EDL but not the soleus muscle [14]. Furthermore, another research has shown that the accumulation of AGEs in the diabetic rat skeletal muscle was greater in fast-type muscle [39]. These findings sugges<sup>t</sup> that fast-type muscles are susceptible to AGEs and that Brazilian propolis improves the inhibitory capacity against AGE formation in fast-type muscle. The potential mechanisms regarding the greater susceptibility of fast-type muscles to AGEs have been described. First, slow-type muscles have a higher protein turnover rate than fast-type muscles [40,41], thus indicating that AGEs are more easily broken down in slow-type muscle than fast-type muscle, and fast-type muscles have a tendency to accumulate AGEs. Second, fast-type muscles are more susceptible to changes in nutrients and hormones than slow-type muscles [42], thus indicating that fast-type muscles are more sensitive to AGEs and propolis than slow-type muscles. However, considering the finding that MGO tended to affect muscle mass with a large effect size in soleus muscle (Table 2), additional examinations using other muscles are needed to clear the fiber-type specific susceptibility to glycation stress.

Brazilian propolis increased muscle mass of soleus almost significantly (*p* = 0.054) with a moderate effect size (η<sup>2</sup> = 0.12), raising a possibility that Brazilian propolis has a hypertrophic effect in soleus muscle regardless of glycation stress. However, there was no significant difference in the calculated muscle CSA (Table S1), indicating that propolis-induced increase in soleus muscle mass was not caused by hypertrophy. In this regard, Brazilian propolis might stimulate glycogen accumulation, and thereby led to muscle mass gain, because it has been shown that Brazilian propolis stimulated glucose uptake in mouse skeletal muscle [43]. However, a previous study has shown that six-week intake of water extract of Korean propolis did not affect glycogen content in the gastrocnemius muscle of rat [44]. Another possibility is that Brazilian propolis increased connective tissue in muscle because it has been shown that propolis stimulated migration and proliferation of fibroblast cells [45]. At present, however, we have no clear explanation for the mechanism by which Brazilian propolis causes gain of soleus muscle mass without hypertrophy.

Detoxification of MGO is also important for reducing glycation stress. MGO is a highly reactive dicarbonyl compound and the major precursor in the formation of AGEs [46,47]. When MGO production exceeds the detoxification capacity, it can modify arginine residues to form MGO-derived AGEs [47]. The most important MGO detoxification system is the glyoxalase system and glyoxalase 1 functions as a rate-limiting enzyme in this system. Under normal physiological conditions, >99% of MGO is metabolized via the glyoxalase system [48]. In the present study, propolis enhanced glyoxalase 1 activity in the EDL muscle (Figure 3), indicating its capability to detoxify MGO, and thereby in inhibition of MGO-derived AGE production. Therefore, in addition to the inhibitory effect of AGE

formation, an enhancement of the glyoxalase system mediated by Brazilian propolis may contribute to the inhibitory effect of accumulation of MGO-derived AGEs in the skeletal muscle.

Inflammation is a crucial contributor toward pathology of diseases implicated in skeletal muscle dysfunction [25,49,50]. Binding of AGEs to AGE receptors including RAGE and TLR4 are potent inducers of inflammatory responses [22]. Inhibition of RAGE and TLR4 effectively reversed the AGE-induced inflammatory signaling [22,51]. In the present study, Brazilian propolis showed no effect of mRNA expression of RAGE, but prevented MGO-treated induction of IL-1β, IL-6, and TLR4 (Figure 4). Consistent with this observation, previous studies have shown that propolis inhibits production of IL-1β in human immune cells [52] and IL-6 in murine macrophages [53]. The current study is the first study that shows that Brazilian propolis has a protective effect on AGE-induced inflammatory responses in the skeletal muscle.

Among the various components of Brazilian propolis [27,28], kaempferide [54], ferulic acid [55], and caffeic acid derivatives [56] are established inhibitors of AGE formation. Furthermore, it has been shown that propolis-induced anti-inflammatory responses may occur due to the synergistic effect of its compounds, artepillin C [57], coumaric acid and cinnamic acid [53], and hesperidin, quercetin, and caffeic acid derivatives [52]. Flavonoid compounds also have a stimulating effect on the glyoxalase system and thereby contribute to neuroprotection [58]. Collectively, the protective activity of propolis against glycation stress in the skeletal muscle may be attributed to the combined biological activity of these phenolic compounds.

Food and fluid intakes were significantly affected by propolis and/or MGO treatment (Table 2). Food intake was reduced in the MGO + PRO group compared with that in the other groups, thus suggesting that MGO + PRO group received a lower contribution from propolis. However, the beneficial effects of propolis, including reduced AGEs accumulation and inflammatory responses, were confirmed in this group. Fluid intake was affected by treatment with MGO and/or propolis, but there was no difference between the MGO and MGO + PRO groups, thus indicating that the beneficial effects of propolis in the MGO + PRO group, including reduced MGO-derived AGEs content and inflammatory responses, were not caused by decreased MGO consumption. Therefore, we believe that the difference of food and fluid intakes does not influence the conclusions of this study.
