Caffeine-induced neurotoxicity mediated by Nrf2 pathway in PC12 cells and zebrafish larvae.

Caffeine is one of the most widely used psychostimulants in the world and possesses central excitative, anti-depressive, and neuroprotective properties. However, excessive ingestion or abuse of caffeine can lead to intoxication. Many toxic effects are attributed to oxidative damage, and nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical intracellular regulator of the oxidative stress response. Here, we investigated the neurotoxicity of caffeine in rat pheochromocytoma PC12 cells and zebrafish larvae. It was found that caffeine inhibited the viability of PC12 cells in a dose- and time-dependent manner. Furthermore, it induced PC12 cell apoptosis and elevated reactive oxygen species (ROS) production. Quantitative polymerase chain reaction (qPCR) and western blotting revealed that caffeine also inhibited the expression levels of Nrf2 mRNA and protein and its target genes (e.g., NADPH quinone oxidoreductase 1 [NQO1]). Furthermore, Nrf2 silencing attenuated the toxic effects of caffeine. In addition, zebrafish larvae were treated with different doses of caffeine. Behavioral experiments showed that a low dose of caffeine (0.05 to 0.3 mM) increased the average distance of movement and promoted excitation. Survivorship curves showed that caffeine (0.2 to 1.5 mM) caused lethality. Finally, qPCR revealed that a higher dose of caffeine inhibited mRNA levels in the Nrf2 pathway. Based on these results, this study identified for the first time that overuse of caffeine can induce neurotoxicity by inhibiting the Nrf2 pathway. These results will provide a new perspective for studies on caffeine toxicity.

Glucocorticoid-induced dose-related and site-specific bone remodelling, microstructure, and mechanical changes in cancellous and cortical bones.

The study investigated the effects of long-term glucocorticoid (GC) administration on bone remodelling, microstructure, and biomechanical strength in cortical and cancellous (trabecular) bones. Thirty-one female Sprague-Dawley rats were randomly divided into three dexamethasone (Dex) dosage groups, 1.0, 2.5, and 5.0 mg/kg twice a week for 8 weeks, and one control group treated with saline. At the end of the experiment, the tibia of one side and the fourth lumbar vertebrae were processed into sections for a histomorphometric analysis, while the femur of the same side and the fifth vertebrae were isolated for a biomechanical test. A dose-dependent decline in bone formation was observed in both trabecular and cortical (periosteal and endosteal) bones. In contrast, bone resorption was inhibited only in cancellous bone in the two higher dose groups and not dose-related. The ratio of Node/Termini increased, while marrow star volume (MSV) decreased in all Dex groups in metaphyseal trabecular bones, both of which were dose-dependent. Subendosteal cortex porosity increased in parallel with non-uniform trabecular distribution, but cortical thickness remained unchanged. Interestingly, there were no significant changes in microstructure or mechanical strength in lumbar trabecular bone. The cortical elastic load was dose-independently reduced in all three Dex groups when compared with the control group. In summary, bone remodelling was dose-dependently inhibited in cancellous bones but enhanced in intracortical bones. The non-uniform distribution of trabecular bone and increased porosity in the inner edge of cortical bone were both in parallel with GC dosage, and the porosity increase was more likely to occur, leading to reduced cortical mechanical strength.

Effect of prednisone treatment for 30 and 90 days on bone metabolism in collagen-induced arthritis (CIA) rats.

Glucocorticoids (GCs) are often prescribed to treat rheumatoid arthritis (RA) in the long term, but there is still controversy in the administration of GCs, mainly because of the adverse reactions such as osteoporosis. Numerous studies have demonstrated that osteoporosis could be induced by GCs in normal rats. However, few experiments have focused on whether osteoporosis could be induced or aggravated by GCs in collagen induced arthritis (CIA) rats. We have investigated bone changes in CIA rats treated with prednisone at 4.5 mg/kg/day for 30 and 90 days by bone histomorphometry, bone mineral density (BMD), micro-CT, biomechanical test, and enzyme-linked immunosorbant assay. We found that high bone turnover osteoporosis was shown in CIA rats. Prednisone treatment for 30 and 90 days improved articular structure and decelerated the degeneration of the femur in CIA rats, but did not improve BMD and bone biomechanics. We conclude that osteoporosis was not aggravated in CIA rats treated with prednisone for 30 and 90 days. On the contrary, prednisone treatment for 30 and 90 days could prevent bone loss of the femur in CIA rats. There was a negative effect on bone metabolism in CIA rats treated with prednisone for 90 days.

Effects of salvianolate on bone metabolism in glucocorticoid-treated lupus-prone B6.MRL-Fas (lpr) /J mice.

AIM: To investigate the bone-protective effects of salvianolate (Sal), a total polyphenol from Radix Salviae miltiorrhizae, on bone tissue in the spontaneous lupus-prone mouse model, B6.MRL-Fas (lpr) /J, undergoing glucocorticoid (GC) treatment. METHODS: Fifteen-week-old female B6.MRL-Fas (lpr) /J mice were administered either a daily dose of saline (lupus group), prednisone 6 mg/kg (GC group), Sal 60 mg/kg (Sal group); or GC plus Sal (GC + Sal group) for a duration of 12 weeks. Age-matched female C57BL/6J wild-type (WT) mice were used for control. Micro-computed tomography assessments, bone histomorphometry analysis, bone biomechanical test, immunohistochemistry and immunoblotting analysis for bone markers, and renal histology analysis were performed to support our research endeavor. RESULTS: Lupus mice developed a marked bone loss and deterioration of mechanical properties of bone due to an increase in bone resorption rather than suppression of bone formation. GC treatment strongly inhibited bone formation in lupus mice. Sal treatment significantly attenuated osteogenic inhibition, and also suppressed hyperactive bone resorption, which recovered the bone mass and mechanical properties of bone in both the untreated and GC-treated lupus mice. CONCLUSION: The data support further preclinical investigation of Sal as a potential therapeutic strategy for the treatment of systemic lupus erythematosus-related bone loss.

Secondary osteoporosis in collagen-induced arthritis rats.

Numerous studies have demonstrated that rheumatoid arthritis (RA) is often associated with bone loss; however, few experiments have focused on cancellous and cortical bone changes in rats during the process of arthritis. We have investigated bone changes in rats with collagen-induced arthritis (CIA) and have explored the characteristics of how RA induces osteoporosis by means of bone histomorphometry, bone biomechanics studies, bone mineral density studies, micro computer tomography, enzyme-linked immunosorbant assay, immunohistochemistry, and Western blot analysis. Bone mineral density of the femur and lumbar vertebrae and biomechanical properties of the femur were decreased in CIA rats. Trabecular bone volume of the tibia and lumbar vertebrae was decreased whereas bone resorption was increased in CIA rats. Bone formation of the tibial shaft in periosteal surfaces was decreased in CIA rats. Furthermore, the trabecular bone loss in CIA rats was severer at 16 weeks than at 8 weeks, as was cortical bone loss. The serum level of tumor necrosis factor α in CIA rats was increased, and the expression of dickkopf 1 and that of receptor activator of nuclear factor κB (RANKL) ligand (RANKL) in the ankle joints were also increased, but the expression of osteoprotegerin (OPG) was decreased. We conclude that CIA rats developed systemic osteoporosis, and that osteoporosis became more serious with CIA development. The mechanism may be related to the increase of bone resorption in cancellous bone cause by upregulation of the expression of DKK-1 and regulation of the RANKL/RANK/OPG signaling pathway, and the decrease of bone formation in cortical bone caused by an increase in the expression of DKK-1.

[Effects of DanShenGuBao on biomechanical properties and bone mineral density of femur induced by retinoic acid in rats].

This investigation was directed to the effects of DanShenGuBao on biomechanical properties and bone mineral density (BMD) of femur induced by retinoic acid (RA) in rats. Forty 4-month-old virgin female Sprague-Dawley rats were randomly divided into 5 groups(8 rats each) control group, RA group and different doses of DanShenGuBao groups. Rats in control group were given vehicle, rats in other four groups were given RA at 70 mg x kg(-1) x d(-1) in the morning and given different drugs in the afternoon at the same time. Rats in RA group were given vehicle, rats in other groups were given different doses of DanShenGuBao which contained 10 mg x kg(-1) x d(-1), 5 mg x kg(-1) x d(-1), 2.5 mg x kg(-1) x d(-1) tanshinol, respectively. All of rats were treated at 5 ml x kg(-1) by oral gavaged for 28 days. In preparation for the determination of dynamic changes in bone tissues, all rats were given subcutaneous injections of 30 mg x kg(-1) tetracycline on the 14th, 13th day and 5 mg x kg(-1) calcein on the 4th, 3rd day before death. At the experimental endpoint, the rats were sacrificed by cardiac puncture under sodium pentobarbital anesthesia. Physical parameters, BMD and biomechanical properties of femur were assessed. Compared with those in control group, the physical parameters (cross-sectional diameter, wet and dry weight), BMD and biomechanical properties (max-load, elasticity-load, break-strain, rigid coefficient and bending-energe) were significantly decreased in RA group. Compared with that in RA group, the BMD of femur was increased significantly in medium and high dose of DanShenGuBao group, but there was no significant change in physical parameters and biomechanical properties of femur. RA could decrease the physical parameters, BMD and biomechanical properties of femur. DanShenGuBao could increase BMD, but it was found with no obvious effect on physical parameters and biomechanical properties.