Molecular Hydrogen Prevents Osteoclast Activation in a Glucocorticoid-Induced Osteoporosis Zebrafish Scale Model

Abstract

Antioxidants represent a powerful tool for many human diseases and, in particular, molecular hydrogen has unique characteristics that make it a very promising therapeutic agent against osteoporosis. Zebrafish scales offer an innovative model in which new therapeutic approaches against secondary osteoporosis are tested. Scale bone loss obtained by prednisolone (PN) treatment is characterized by increased osteoclast activity and decreased osteoblast activity highlighted with bone enzymatic assays. We used this read-out system to test the therapeutic effects of hydrogen-rich water (HRW), an innovative antioxidant approach. HRW prevented osteoclast activation and bone loss in PN-treated fish scales, as verified by both biochemical and histochemical tartrate-resistant alkaline phosphatase assays. On the other hand, HRW treatment did not prevent PN-dependent osteoblast suppression, as measured by alkaline phosphatase activity. Moreover, HRW treatment did not facilitate the reparation of resorption lacunae induced in scales by PN. Our study highlighted a specific effect of HRW on adult osteoclast activity but not in osteoblasts, introducing an intriguing new antioxidant preventive approach against osteoporosis.

Keywords: antioxidant; hydrogen; osteoporosis; zebrafish.

Figure 1

Molecular hydrogen counteracts oxidative stress through multiple pathways. -OH⁻: hydroxyl radical; -ONOO^-: peroxynitrite anion; SOD: superoxide dismutase; CAT: catalase, MPO: myeloperoxidase; HO-1: heme oxygenase; ARE: antioxidant responsive element; GPX: glutathione peroxidase.

Figure 2

(A) ORP measurements performed on FW and HRW right after production: dilution of HRW and withdrawal of FW from the ZEBTEC© system. FW ORP quickly drops from about 140 mV to a stable level of about 70 mV. (B) ORP difference between FW and HRW expressed as ΔORP (HRW ORP − FW ORP, mV), which reaches 0 after about 75 min.

Figure 3

(A) Histological TRAP activity assay highlighted by violet staining of enzymatic activity along the scale borders of PN and PN + HRW scales. Scale bar 0.22 mm. (B) Percentage of mineralized area of scales with PN scales area reduced by 11.8% (PN vs. CTR, p < 0.05 *; PN vs. HRW, p < 0.05 *; PN vs. PN+HRW, p < 0.05 *). (C) Percentage of TRAP positive scales equal to 76% in PN fish and 14% in PN + HRW fish (CTR vs. PN, p < 0.001 ***; CTR vs. PN+HRW, p < 0.01 **). (D) Biochemical TRAP assay shows increased TRAP levels only in PN fish scales (PN vs. CTR, p < 0.001 ***; PN vs. HRW, p < 0.001 ***; PN vs. PN+HRW, p < 0.001 ***).

Figure 4

(A) Histochemical ALP activity assay highlighted by blue staining of the localization of enzymatic activity in the scale. CTR and HRW scales show normal ALP activity, while PN and PN + HRW scales show decreased ALP activity, visualized as white unstained areas. Scale bar 0.11 mm. (B) Quantification of ALP activity performed with biochemical assay shows decreased ALP activity on PN and PN + HRW scales (PN vs. CTR, p < 0.05 *; PN vs. HRW, p < 0.05 *; PN + HRW vs. CTR, p < 0.01 **; PN + HRW vs. HRW, p < 0.05 *).

Figure 5

(A) Schematic representation of the experiment performed in PREVENTIVE MODE and REPAIR MODE with CTR, PN, HRW and PN + HRW samples (CTR: untreated, PN: treated with prednisolone, HRW: treated with hydrogen water, PN+HRW: treated with hydrogen water and prednisolone). (B) Prednisolone (pn) and hydrogen rich water (hrw) were administrated with different methods and timing in CTR, PN, HRW and PN + HRW samples in PREVENTIVE MODE or REPAIR MODE. (C) Exemplificative picture of the reparative process in resorption areas of HRW or untreated CTR fish after prednisolone treatment (REPAIR MODE). Green fluorescence indicates a new mineralized matrix deposed in the lacuna. Scale bar 0.02 mm. (D) % of scale mineralized area measured in 3rd and 4th weeks in HRW and untreated CTR fish after PN treatment (REPAIR MODE). The reparative process was not modulated by the HRW treatment.