3.1. GlcN delays bone microstructure destruction and biomechanical properties loss of senile osteoporotic mice
In order to explore the effect of GlcN on D-Gal-induced osteoporosis in vivo, we firstly established the senile osteoporosis model in mice by chronic administration of D-Gal (125mg/kg/day) for 12 weeks, and then additionally treated with GlcN (9mg/kg/day) for 12 weeks (Figure 1A). As shown in Figure 1B, there were no significantly differences between control group and D-Gal group on mice weight after 12 weeks of feeding (P > 0.05). However, mice in D-Gal group showed decreased food intake, decreased activity, darkened hair color and hair loss over time. The results of three-dimensional µCT images of femur metaphysis in mice showed that the trabeculae were sparse, irregularly arranged, and widened trabecular spaces in D-Gal group, compared to control group. In addition, the results of µCT data analysis showed that the bone density (BMD, 0.40±0.03 mg/cc), bone volume (BV/TV, 20.94±2.98%) and trabecular number (Tb.N, 1.62±0.09 mm) were significantly decreased, whereas the structural model index (SMI, 1.70±0.26) and trabecular space (Tb.Sp, 0.54±0.10 mm) were significantly increased in D-Gal group, compared to control group (BMD, 0.56±0.03 mg/cc; BV/TV, 45.48±1.53%; TB.N, 3.27±0.13 mm; SMI, 0.85±0.13; Tb.Sp, 0.21±0.01 mm) (***P < 0.001). Meanwhile, the results of µCT data analysis showed that the BMD (0.47±0.03 mg/cc), BV/TV (29.45±3.52%) and Tb.N (2.11±0.24 mm) were significantly increased, whereas the SMI (1.30±0.15) and Tb.Sp (0.40±0.06 mm) were significantly decreased in GlcN group, compared to D-Gal group (#P < 0.05, ##P < 0.01, ###P < 0.001). Interestingly, there were no significantly differences among the three groups on trabecular thickness (Tb.Th, Control, 0.14±0.01 mm; D-Gal, 0.13±0.02 mm; GlcN, 0.14±0.01 mm) (P > 0.05) (Figure 1C). Moreover, the results of three-point bending test showed that the break load (23.25±2.74 N), maximum load (23.93±2.29 N) and stiffness (146.48±19.78 N.mm−1) were significantly decreased in D-Gal group, compared to control group (break load, 35.32±3.11 N; maximum load, 34.58±3.21 N; stiffness, 232.74±34.61 N.mm−1) (***P < 0.001). While the maximum load (27.92±1.78 N), break load (28.7±0.89 N) and stiffness (191.33±13.87 N.mm−1) were significantly increased in GlcN group, compared to D-Gal group (#P < 0.05, ##P < 0.01). Interestingly, there were no significantly differences among the three groups on elastic load (control, 22.30+3.92 N; D-Gal, 18.22±2.37 N; GlcN, 21.83±2.57 N) (P > 0.05) (Figure 1D). Therefore, we successfully established the senile osteoporosis model in mice by chronic administration of D-Gal. Meanwhile, we also found that GlcN significantly inhibits D-Gal-induced osteoporosis in mice, as shown by GlcN not only significantly improved the microstructure of the distal femur trabecular bone, but also significantly improved the biomechanical properties of the bone.
3.2. GlcN delays senescence of senile osteoporotic mice
In order to further explore the role of GlcN in delaying the progression of D-Gal-induced osteoporosis, we conducted some follow-up studies. The results of HE staining showed that the bone trabeculae in D-Gal group were thin and sparse, and filled with a large number of adipocytes, and presented the phenotype of osteoporosis. We further measured the area of adipocytes per square millimeter of femoral bone trabecular to evaluate the degree of adipogenesis. The results showed that the percentage of adipocytes volume in the femoral bone marrow cavity (65.50±3.27%) was significantly increased in the D-Gal group, compared to control group (7.17±1.84%) (***P < 0.001). Meanwhile, we found that although the percentage of adipocytes volume in the femoral bone marrow cavity (49.67±3.39%) was also increased in GlcN group, but the increased volume was significantly smaller than that in D-Gal group (###P < 0.001) (Figure 2A). Next, we detected the senescence-associated indicators in serum. The results showed that the levels of IL-1β (34.58±3.26 pg/ml), IL-6 (7.68±0.73 pg/ml) and MDA (3.01±0.68 µmol/L) (pro-senescence) were significantly increased in D-Gal group, compared to control group (IL-1β, 18.68±1.39 pg/ml; IL-6, 4.15±0.31 pg/ml; MDA, 1.67±0.28 µmol/L) (***P < 0.001). Meanwhile, the levels of IL-1β (24.08±2.49 pg/ml), IL-6 (4.83±0.40 pg/ml) and MDA (2.02±0.13 µmol/L) (pro-senescence) were significantly decreased in GlcN group, compared to D-Gal group (##P < 0.01, ###P < 0.001). Interestingly, there were no significantly differences among the three groups on the level of SOD (anti-senescence) (control, 50.48±2.38 U/ml; D-Gal, .42.58±4.37 U/ml; GlcN, .45.65±1.86 U/ml) (P > 0.05) (Figure 2B). In addition, we detected the expression of senescence-associated proteins in mice bone tissue by WB and IHC. The results of WB showed that the expression of Ki-67 (pro-proliferation/anti-senescence) was significantly decreased, whereas the expression of p16 (pro-senescence) was significantly increased in D-Gal group, compared to control group (*P < 0.05, ***P < 0.001). Meanwhile, the expression of Ki-67 was significantly increased, whereas the expression of p16 was significantly decreased in GlcN group, compared to D-Gal group (#P < 0.05, ###P < 0.001) (Figure 2C). Similar results were found in IHC. The results showed that the expression of Ki-67 was significantly decreased in D-Gal group, compared to control group (**P < 0.01). Although the expression of Ki-67 was also decreased in GlcN group, but the decreased expression of Ki-67 was significantly smaller than that in D-Gal group (#P < 0.05) (Figure 2D). Studies showed that apoptosis is closely related to the occurrence and development of senescence-associated diseases, including osteoporosis [24]. Therefore, we detected the expression of apoptosis-associated mRNAs and proteins in mice bone tissue by qRT-PCR and WB, respectively. The results of qRT-PCR showed that the mRNAs of Bax and Cleaved Caspase-3 (pro-apoptosis) were significantly upregulated, whereas the mRNA of Bcl-2 (anti-apoptosis) was significantly downregulated in D-Gal group, compared to control group (***P < 0.001). Meanwhile, the mRNAs of Bax and Cleaved Caspase-3 were significantly downregulated, whereas the mRNA of Bcl-2 was significantly upregulated in GlcN group, compared to D-Gal group (#P < 0.05, ##P < 0.01) (Figure 2E). Similar results were found in WB. The results of WB showed that the proteins of Bax and Cleaved Caspase-3 were significantly upregulated, whereas the protein of Bcl-2 was significantly downregulated in D-Gal group, compared to control group (***P < 0.001). Meanwhile, the proteins of Bax and Cleaved Caspase-3 were significantly downregulated, whereas the protein of Bcl-2 was significantly upregulated in GlcN group, compared to D-Gal group (###P < 0.001) (Figure 2F). Taken together, our results demonstrated that GlcN down-regulate the level of aging and apoptosis level in bone tissue of D-Gal-induced osteoporotic mice.
3.3. GlcN delays the progress of osteoporosis in senile osteoporotic mice by promoting autophagy
Our previous study demonstrated that GlcN promoted proliferation and inhibited apoptosis of human osteoblasts through upregulating autophagy in vitro [14]. Therefore, our subsequent studies would further explore whether autophagy was involved in anti-osteoporosis of GlcN in vivo. We measured IF, qRT-PCR and WB to evaluate the expression of autophagy-related genes and proteins in bone tissue. Studies reported that LC3 is mainly located on the surface of pre-autophagosomes and autophagosomes and is a general biomarker of autophagy [25]. Therefore, we detected the expression of LC3 in proximal tibia by IF. The results of IF showed that the punctate aggregations (autolysosomes) of LC3 was significantly decreased in D-Gal group, compared to control group (**P < 0.001). Meanwhile, the punctate aggregations of LC3 was significantly increased in GlcN group, compared to D-Gal group (##P < 0.001). Interestingly, we found the punctate aggregations of the LC3 were located on the surface of the bone trabecula (Figure 3A). As shown in Figure 3B, the results of qRT-PCR showed that the mRNA expression of Beclin-1 was significantly downregulated, whereas the mRNA expression of p62 was significantly upregulated in D-Gal group, compared to control group (**P < 0.001, ***P < 0.001). Meanwhile, the mRNA expression of Beclin-1 was significantly upregulated, whereas the mRNA expression of p62 was significantly downregulated in GlcN group, compared to D-Gal group (###P < 0.001). Similar results were found in WB. As shown in Figure 3C, the results of WB showed that the proteins expression of LC3 II and Beclin-1 were significantly downregulated, whereas the protein expression of p62 was significantly upregulated in D-Gal group, compared to control group (**P < 0.001, ***P < 0.001). Meanwhile, the proteins expression of LC3 II and Beclin-1 were significantly upregulated, whereas the protein expression p62 was significantly downregulated in GlcN group, compared to D-Gal group (###P < 0.001). Autophagy is a self-protection strategy and plays an important role in the body growth and development by maintaining the body homeostasis, however the level of autophagy is associated with aging. From the above studies, we found that autophagy levels significantly reduced in D-Gal treatment group, however we also found that GlcN up-regulated the level of autophagy to delay the progress of osteoporosis in D-Gal-induced osteoporosis mice.
3.4. Inhibition of autophagy reverses the anti-osteoporosis effect of GlcN
To further clarify the role of autophagy in anti-osteoporosis of GlcN in vivo, the osteoporotic mice were additionally treated with 3-MA (4mg/kg/day) for 12 weeks (Figure 3A). There were no significantly differences among the four groups on mice weight after 12 weeks of feeding (P > 0.05) (Figure 3B). Moreover,we evaluated the level of adipogenic differentiation by HE staining. The results showed that the percentage of adipocytes volume in the femoral bone marrow cavity (69.17±3.67%)was significantly increased in the GlcN+3-MA group, compared to GlcN group (48.67±3.20%) (###P < 0.001). Meanwhile, there were no significantly differences of the percentage of adipocytes volume in the femoral bone marrow cavity between PBS group (63.92±2.78%) and 3-MA group (64.00±1.55%) (P > 0.05) (Figure 3C). Next, we detected the senescence-associated indicators in serum. The results showed that the levels of IL-1β (43.20±6.84 pg/ml), IL-6 (9.60±1.52 pg/ml) and MDA (3.16±0.22 µmol/L) were significantly increased, whereas the levels of SOD (39.98±1.87 U/ml) was significantly decreased in GlcN+3-MA group, compared to GlcN group (IL-1β, 23.63±1.55 pg/ml; IL-6, 5.25±0.35 pg/ml; MDA, 2.22±0.16 µmol/L; SOD, 45.65±1.86 U/ml) (#P < 0.05, ##P < 0.01, ###P < 0.001). Meanwhile, there were no significantly differences between PBS group and 3-MA group on the levels of IL-1β (PBS, 19.21±0.23 pg/ml; 3-MA, 37.43±2.27 pg/ml), IL-6 (PBS, 4.35±0.45 pg/ml; 3-MA, 8.32±0.50 pg/ml), MDA (PBS, 2.98±0.75 µmol/L; 3-MA, 3.03±0.38 µmol/L) and SOD (PBS, 42.73±3.97 U/ml; 3-MA, 41.08±5.19 U/ml) (P > 0.05) (Figure 3D). In addition, we detected the expression of senescence-associated proteins in mice bone tissue by WB and IHC. The results of WB showed that the expression of Ki-67 was significantly decreased, whereas the expression of p16 was significantly increased in GlcN+3-MA group, compared to GlcN group (##P < 0.01, ###P < 0.001). Meanwhile, there were no significantly differences of the expression of Ki-67 and p16 between PBS group and 3-MA group (P > 0.05) (Figure 3E). Similar results were found in IHC. The results showed that the expression of Ki-67 was significantly decreased in GlcN+3-MA group, compared to GlcN group (###P < 0.001). Meanwhile, there were no significantly differences between PBS group and 3-MA group on Ki-67 expression (P > 0.05) (Figure 3E). Last, we detected the expression of apoptosis-associated proteins in mice bone tissue by WB. The results showed that the expression of Bax and Cleaved Caspase-3 were significantly increased, whereas the expression of Bcl-2 was significantly decreased in GlcN+3-MA group, compared to GlcN group (##P < 0.01, ###P < 0.001). Meanwhile, there were no significantly differences between PBS group and 3-MA group on apoptosis related proteins (P > 0.05) (Figure 3F). In conclusion, our results demonstrated that inhibition of autophagy reverses the anti-osteoporosis effect of GlcN in D-Gal-induced osteoporotic mice.
3.5. GlcN delays osteoporosis by promoting osteoblast autophagy
Along with aging, bone marrow mesenchymal stem cells in bone marrow cavity decreased osteogenic differentiation whereas increased adipogenic differentiation. Our previous study found that GlcN reduced adipogenic differentiation of bone marrow mesenchymal stem cells in senile osteoporotic mice. Meanwhile, we also found that autophagosomes gathered around trabecular bone after GlcN activated autophagy. While osteoblasts were attached around bone trabeculae [26, 27]. In addition, our previous in vitro studies reported that GlcN increased the level of osteoblast autophagy. Therefore, we conducted the follow-up studies to explore whether GlcN exerts an anti-osteoporosis effect by activating osteoblast autophagy. Firstly, we confirmed the inhibition of autophagy did reverse the anti-osteoporosis effect of GlcN by IF, qRT-PCR and WB. IF results showed that the autophagy activation of GlcN was significantly reversed by 3-MA (GlcN group VS. GlcN+3-MA group, ##P < 0.01) (Figure 5A). Furthermore, the results of qRT-PCR showed that the mRNA expression of Beclin-1 was significantly downregulated, whereas the mRNA expression of p62 was significantly upregulated in GlcN+3-MA group, compared to GlcN group (#P < 0.05) (Figure 5B). Similar results were found in WB. As shown in Figure 5C, the results of WB showed that the proteins expression of LC3 II and Beclin-1 were significantly downregulated, whereas the protein expression of p62 was significantly upregulated in GlcN+3-MA group, compared to GlcN group (##P < 0.01, ###P < 0.001). In addition, the results showed that the protein expression of LC3 II was significantly downregulated in 3-MA group, compared to PBS group (*P < 0.05). Next, we measured IF, qRT-PCR, WB, ALP staining and Goldner staining to evaluate whether GlcN maintains osteogenic activity and promotes differentiation, proliferation and mineralization of osteoblast by activating autophagy, thus exerting anti-osteoporosis effect. The results of IF showed that the expression of COL1A1 (indicator of osteoblast, pro-osteogenesis) was significantly increased in GlcN group, compared to PBS group (***P < 0.001). While the expression of COL1A1 was significantly decreased in GlcN+3-MA group, compared to GlcN group (##P < 0.01). Meanwhile, there were no significantly differences between PBS group and 3-MA group on the expression of COL1A1 (P > 0.05) (Figure 6A). The results of qRT-PCR showed that the mRNAs expression of RUNX2, BMP-2 and OCN (indicators of osteogenesis differentiation) were significantly increased in GlcN group, compared to PBS group (*P < 0.05, ***P < 0.001). While the mRNAs expression of RUNX2, BMP-2 and OCN were significantly decreased in GlcN+3-MA group, compared to GlcN group (##P < 0.01, ###P < 0.001). Meanwhile, there were no significantly differences between PBS group and 3-MA group on the mRNAs expression of RUNX2, BMP-2 and OCN (P > 0.05) (Figure 6B). Similar results were found in WB. The results of WB showed that the proteins expression of RUNX2, BMP-2 and OCN were significantly increased in GlcN group, compared to PBS group (**P < 0.01, ***P < 0.001). while the proteins expression of RUNX2, BMP-2 and OCN were significantly decreased in GlcN+3-MA group, compared to GlcN group (##P < 0.01, ###P < 0.001). Meanwhile, there were no significantly differences between PBS group and 3-MA group on the proteins expression of RUNX2, BMP-2 and OCN (P > 0.05) (Figure 6C). As shown in ALP staining (Figure 6D), the number of osteoblast was significantly increased in GlcN group, compared to PBS group (***P < 0.001). While the number of osteoblast was significantly decreased in GlcN+3-MA group, compared to GlcN group (##P < 0.01). Meanwhile, there were no significantly differences between PBS group and 3-MA group on the number of osteoblast (P > 0.05). Furthermore, osteoblast mineralization was assessed by Goldner staining. As is shown in Figure 6E, the osteoblast mineralization was significantly increased in GlcN group, compared to PBS group (**P < 0.01). While the osteoblast mineralization was significantly decreased in GlcN+3-MA group, compared to GlcN group (##P < 0.01). Meanwhile, there were no significantly differences between PBS group and 3-MA group on osteoblast mineralization (P > 0.05). In summary, the above results demonstrated that GlcN maintains osteogenic activity and promotes osteoblast proliferation and mineralization by activating autophagy, so as to delay the progression of osteoporosis in D-Gal-induced osteoporotic mice.