Aim. In routine histopathology, decalcification is an essential step for mineralized tissues. The purpose of this study is to evaluate the effects of different decalcification solutions on the morphological and antigenicity preservation in Sprague Dawley (SD) rat femurs. Materials and Methods. Four different decalcification solutions were employed to remove the mineral substances from rat femurs, including 10% neutral buffered EDTA, 3% nitric acid, 5% nitric acid, and 8% hydrochloric acid/formic acid. Shaking and low temperature were used to process the samples. The stainings of hematoxylin-eosin (HE) and immunohistochemical (IHC) were employed to evaluate the bone morphology and antigenicity. Key Findings. Different decalcification solutions may affect the quality of morphology and the staining of paraffin-embedded sections in pathological examinations. Among four decalcifying solutions, 3% nitric acid is the best decalcifying agent for HE staining. 10% neutral buffered EDTA and 5% nitric acid are the preferred decalcifying agents for IHC staining. Significance. The current study investigated the effects of different decalcifying agents on the preservation of the bone structure and antigenicity, which will help to develop suitable protocols for the analyses of the bony tissue.

Gut dysbiosis and oxidative stress may trigger senile osteoporosis. Fructus Ligustri Lucidi (FLL) has bone-preserving properties and affects the intestinal microecology. However, the mechanism of the anti-osteoporotic effect of FLL and its link to the gut microbiota remains to be elucidated. Here, we demonstrated that sustained exposure of ICR mice to D-galactose / sodium nitrite for 90 days causes aging-related osteoporosis and reduced cognitive performance. The aging phenotype is also characterized by increased oxidative stress in serum. This is likely triggered by abnormal changes in the gut microbiota population of Bifidobacterium and the ratio of Firmicutes/ Bacteroidetes that resulted in increased levels of flavin-containing monooxygenase-3 and trimethylamine-N-oxide (TMAO). Moreover, the increased oxidative stress further accelerated aging by increasing tumor necrosis factor-α levels in serum and reducing Sirtuin 6 (Sirt6) expression in long bones, which prompted nuclear factor kappa-B acetylation as well as over-expression and activation of cathepsin K. FLL-treated aging mice revealed a non-osteoporotic bone phenotype and an improvement on the cognitive function. The mechanism underlying these effects may be linked to the regulation of gut microbiota diversity, antioxidant activity, and the levels of TMAO and Sirt6. FLL may represent a potential source for identifying anti-senile osteoporotic drug candidates.

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Purpose: Increasing evidence supported that semaphorin 3A (Sema3A), insulin-like growth factor (IGF)-1 and β-catenin were involved in the development of osteoporosis and diabetes. This study is aimed to evaluate whether Sema3A/IGF-1/β-catenin is directly involved in the alterations of bone microarchitecture and bone strength of diabetic rats. Methods: Diabetic rats were induced by streptozotocin and high fat diet exposure. Bone microarchitecture and strength in the femurs were evaluated by micro-CT scanning, three-point bending examination and the stainings of HE, alizarin red S and safranin O/fast green, respectively. The alterations of lumbar spines microarchitecture were also determined by micro-CT scanning. Western blot and immunohistochemical analyses were used to examine the expression of Sema3A, β-catenin, IGF-1, peroxisome proliferator-activated receptor γ (PPARγ) and cathepsin K in rat tibias. Results: Diabetic rats exhibited decreased trabecular numbers and bone formation, but an increased trabecular separation in the femurs and lumbar spines. Moreover, the increased bone fragility and decreased bone stiffness were evident in the femurs of diabetic rats. Diabetic rats also exhibited a pronounced bone phenotype which manifested by decreased expression of Sema3A, IGF-1 and β-catenin, as well as increased expression of cathepsin K and PPARγ. Conclusions: This study suggests that diabetes could perturb bone loss through the Sema3A/IGF-1/β-catenin pathway. Sema3A deficiency in bone may contribute to upregulation of PPARγ and cathepsin K expression, which further disrupts bone remodeling in diabetic rats.

This review aimed to provide a general view of catalpol in protection against diabetes and diabetic complications, as well as its pharmacokinetics and safety concerns. The following databases were consulted with the retrieval of more than 100 publications through June 2019: PubMed, Chinese National Knowledge Infrastructure, WanFang Data, and web of science. Catalpol exerts an anti-diabetic effect in different animal models with an oral dosage ranging from 2.5 to 200 mg/kg in rats and 10 to 200 mg/kg in mice. Besides, catalpol may prevent the development of diabetic complications in kidney, heart, central nervous system, and bone. The underlying mechanism may be associated with an inhibition of inflammation, oxidative stress, and apoptosis through modulation of various cellular signaling, such as AMPK/PI3K/Akt, PPAR/ACC, JNK/NF-κB, and AGE/RAGE/NOX4 signaling pathways, as well as PKCγ and Cav-1 expression. The pharmacokinetic profile reveals that catalpol could pass the blood-brain barrier and has a potential to be orally administrated. Taken together, catalpol is a well-tolerated natural compound with promising pharmacological actions in protection against diabetes and diabetic complications via multi-targets, offering a novel scaffold for the development of anti-diabetic drug candidate. Further prospective and well-designed clinical trials will shed light on the potential of clinical usage of catalpol.

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Purpose: This study is designed to explore whether Fructus ligustri lucidi (FLL) exhibits antioxidant effect in ovariectomized (OVX) rats, and to identify the signaling pathway involved in this process.Methods: OVX rats were treated with FLL aqueous extract (3.5 g/kg) for 12 weeks. Serum, uteri, and tibias were harvested from the rats and the levels of total antioxidant capacity (TAC), nitric oxide (NO), malondialdehyde (MDA), 8-hydroxy-desoxyguanosine (8-OHdG), and superoxide dismutase (SOD) were determined. Changes in the levels of NF-κB-p65, phosphorylation of NF-κB-p65 (NF-κB-pp65), NF-κB inhibitor alpha (IκBα), phosphorylation of IκBα (p-IκBα), and NADPH oxidase 4 (Nox4) in uteri and tibias were determined by western blot, immunofluorescent and immunohistochemical analysis, respectively. In addition, the expression of cytochrome C (Cyto-C) and B-cell lymphoma-2 (Bcl-2) were determined in the tibias of rats. Histopathological changes in the bones were evaluated by hematoxylin-eosin staining. Bone mineral density (BMD) was determined in rat femurs by dual X-ray absorptiometry.Results: Treatment of OVX rats with FLL aqueous extract improved redox homeostasis by increasing the levels of TAC and NO as well as decreasing the levels of MDA and 8-OHdG in serum, tibias, and uteri. Further, FLL extract also downregulated the expression of Nox4, NF-κB-p65, NF-κB-pp65, and p-IκBα in the uteri and tibias. Furthermore, administration of FLL–OVX rats increased Bcl-2 expression and prevented cytoplasmic release of mitochondrial Cyto-C in the tibias. In addition, FLL treatment also improved bone microstructure and increased cortical bone thickness as well as increased BMD values in the femurs of OVX rats.Conclusions: FLL treatment may suppress oxidative stress response in OVX rats via regulating the Nox4/ROS/NF-κB signaling pathway. These results suggest the potential of using FLL as a natural antioxidant agent in preventing the development of osteoporosis.