Differential bone remodeling mechanism in hindlimb unloaded and hibernating Daurian ground squirrels: a comparison between artificial and natural disuse within the same species.

Loss of bone mass can occur in mammals after prolonged disuse but the situation for hibernators that are in a state of torpor for many months of the year is not yet fully understood. The present study assesses the bone remodeling mechanisms present in Daurian ground squirrels (Spermophilus dauricus) during hibernation as compared with a model of hindlimb disuse. Differences in microstructure, mechanical properties, bone remodeling-related proteins (Runx2, OCN, ALP, RANKL, CTK and MMP-9) and key proteins of Wnt/ß-catenin signaling pathway (GSK-3ß and phospho-ß-catenin) were evaluated in ground squirrels under 3 conditions: summer active (SA) vs. hibernation (HIB) vs. hindlimb unloaded (HLU). The results indicated that the body weight in HLU ground squirrels was lower than the SA group, and the middle tibia diameter in the HLU group was lower than that in SA and HIB groups. The thickness of cortical and trabecular bone in femurs from HLU ground squirrels was lower than in SA and HIB groups. Most parameters of the tibia in the HLU group were lower than those in SA and HIB groups, which indicated cortical bone loss in ground squirrels. Moreover, our data showed that the changes in microscopic parameters in the femur were more obvious than those in the tibia in HLU and HIB ground squirrels. The levels of Runx2 and ALP were lower in HLU ground squirrels than SA and HIB groups. The protein levels of OCN were unchanged in the three groups, but the protein levels of ALP were lower in the HLU group than in SA and HIB groups. RANKL, CTK and MMP-9 protein levels were significantly decreased in tibia of HLU ground squirrels as compared with SA and HIB groups. In addition, the protein expression levels of RANKL, CTK and MMP-9 showed no statistical difference between SA and HIB ground squirrels. Thus, the mechanisms involved in the balance between bone formation and resorption in hibernating and hindlimb unloading ground squirrels may be different. The present study showed that in femur, the Wnt signaling pathway was inhibited, the protein level of GSK-3ß was increased, and the protein expression of phospho-ß-catenin was decreased in the HIB group as compared with the SA group, which indicates that the Wnt signaling pathway has a great influence on the femur of the HIB group. In conclusion, the natural anti-osteoporosis properties of Daurian ground squirrels are seasonal. The squirrels do not experience bone loss when they are inactive for a long time during hibernation, but the mechanisms of anti-osteoporosis did not work in HLU summer active squirrels.

Differential bone metabolism and protein expression in mice fed a high-fat diet versus Daurian ground squirrels following natural pre-hibernation fattening.

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Kunming mice were fed a high-fat diet to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Micro-computed tomography (micro-CT) and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone metabolism (Runt-related transcription factor 2 (RunX2), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-|κB ligand (RANKL), cathepsin K, matrix metallopeptidase 9 (MMP9), patched protein homolog 1 (Ptch1), phosphorylated ß|-|catenin (P|-|ß|-|catenin), and glycogen synthase kinase-3ß (GSK-3ß)). Compared with controls, there was no obvious bone loss in the OB mice, and the stiffness of the femur was increased significantly. Compared with summer active squirrels, bone formation was enhanced but the mechanical properties did not change in the PRE group squirrels. In OB mice, western blots showed significantly increased expression levels of all proteins except RunX2, OPG, and Ptch1. PRE ground squirrels showed significantly increased expression of most proteins except OCN and Ptch1, which decreased significantly, and P|-|ß|-|catenin and OPG, which did not change. In conclusion, for non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, increasing both bone loss and bone formation. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones, and also enhanced the expression levels of proteins related to bone formation, bone resorption, and Wnt signaling.

Biodegradable polymer-curcumin conjugate micelles enhance the loading and delivery of low-potency curcumin.

PURPOSE: To utilize a novel type of polymer-drug conjugate micelle to enhance the delivery of low-potency curcumin. METHODS: Multiple curcumin molecules were conjugated to poly(lactic acid) (PLA) via tris(hydroxymethyl)aminomethane (Tris) linker producing the hydrophobic drug-binding block; methoxy-poly(ethylene glycol) (mPEG) was employed as the hydrophilic block. Micelles were characterized by size, loading capacity, stability, and critical micelle concentration (CMC). Human hepatocellular carcinoma (HepG2) cells were employed to assess cytotoxicity and intracellular targeting ability of micelles. RESULTS: mPEG-PLA-Tris-Cur micelles were within nanorange (<100 nm). CMC of such micelles (2.3 ± 0.4 µg/mL) was 10 times lower than mPEG-PLA micelles (27.4 ± 0.8 µg/mL). Curcumin loading in mPEG-PLA-Tris-Cur micelles reached 18.5 ± 1.3% (w/w), compared to traditional mPEG-PLA micelles at 3.6 ± 0.4% (w/w). IC(50) of mPEG-PLA-Tris-Cur micelles (~22 µg/mL at curcumin-equivalent dose) was similar to unmodified curcumin. Placebo and drug-encapsulated conjugate micelles could be efficiently internalized to cytoplasmic compartment of HepG2 cells. CONCLUSIONS: Micelle-forming polymer-drug conjugates containing multiple drug molecules were an efficient means to increase loading and intracellular delivery of low-potency curcumin.