Xiaolong Gu

Cytoprotective effect of chlorogenic acid against hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells through PI3K/Akt-mediated Nrf2/HO-1 signaling pathway

Osteoporosis is a disorder of bone and its development is closely associated with oxidative stress and reactive oxygen species (ROS). Chlorogenic acid (CGA) has potential antioxidant effects and its pharmacological action in osteoblasts is not clearly understood. The present study aimed to clarify the protective effects and mechanisms of CGA on hydrogen peroxide (H2O2)-induced oxidative stress in osteoblast cells. MC3T3-E1 cells were treated with H2O2 to induce oxidative stress model in vitro. Cells were treated with CGA prior to H2O2 exposure, the intracellular ROS production, malondialdehyde content, nitric oxide release and glutathione level were measured. We also investigated the protein levels of heme oxygenase-1 (HO-1), the nuclear translocation of transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation levels of Akt in CGA-treated cells. The results showed that pretreatment of CGA could reverse the inhibition of cell viability and suppress the induced apoptosis and caspase-3 activity. Additionally, it significantly reduced H2O2-induced oxidative damage in a dose-dependent manner. Furthermore, it induced the protein expression of HO-1 together with its upstream mediator Nrf2, and activated the phosphorylation of Akt in MC3T3-E1 cells. LY294002, a PI3K/Akt inhibitor, significantly suppressed the CGA-induced Nrf2 nuclear translocation and HO-1 expression. Reduction of cell death mediated by CGA in presence of H2O2 was significantly inhibited by Zinc protoporphyrin IX (a HO-1 inhibitor) and LY294002. These data demonstrated that CGA protected MC3T3-E1 cells against oxidative damage via PI3K/Akt-mediated activation of Nrf2/HO-1 pathway, which may be an effective drug in treatment of osteoporosis.

SIRT1-mediated FoxOs pathways protect against apoptosis by promoting autophagy in osteoblast-like MC3T3-E1 cells exposed to sodium fluoride

Fluorine may result in damage to teeth, bones and other body tissues, and is a serious public health problem. SIRT1 deacetylates FOXOs, which brings about apoptosis and autophagy promotion or suppression. Fluorine may induce cell apoptosis, however, the role of autophagy in apoptosis induced by fluorine is still poorly understood, and the interaction between SIRT1 and FOXOs should be further illustrated. Therefore, this study investigated the mechanisms underlying the NaF- induced apoptosis and autophagy in osteoblast-like MC3T3-E1 cells in vitro through activating or inhibiting SIRT1. Via RT-PCR, western blot, flow cytometry assays, fluorescence and laser confocal microscopy, it was found that NaF induced both cell apoptosis and autophagy. Results also showed that NaF up-regulated SIRT1 expression in a dose-dependent manner. The autophagy of MC3T3-E1 was also up- regulated indirectly whilst apoptosis was significantly attenuated when incubated with the SIRT1 activator SRT1720. When SIRT1 inhibitor Ex-527 was used, the latter effects were reversed. Furthermore, SIRT1 increased deacetylation of FoxO1 and promoted the up-regulation of its target substrate Rab7, as well as increase of Bnip3 which was substrate of FoxO3, and we hypothesize that these pathways may cause an increase in autophagic flux and a reduction in apoptosis. In conclusion, SIRT1-induced autophagy enhancement protects against fluoride-induced apoptosis through autophagy induction in MC3T3-E1 cells, which may be associated with a SIRT1-FoxO1-Rab7 axis and a SIRT1-FoxO3-Binp3 axis. The role of SIRT1 in selecting between cell survival and death provides a potential therapeutic strategy in fluorosis.

The role of selenium in insulin-like growth factor I receptor (IGF-IR) expression and regulation of apoptosis in mouse osteoblasts

Selenium (Se) is an essential component for animals and human beings. The chemoprotective role of Se, via the regulation of the cell cycle, stimulation of apoptosis and activation of some cytokines among others, is well known; however, the comprehensive effects of Se on the expression of IGF-IR and its regulation of apoptosis have not been investigated. Thus the aim of this study was to report on the effects that different concentrations of Se extert on body weight, blood serum IGF-IR levels and histopathology in mice; and on IGF-IR expression, proliferation and apoptosis in mouse osteoblasts. In vivo experiments showed a significant decrease in body weight, serum levels of IGF-IR and prominent toxicant effects on the liver, kidney, heart and spleen following the administration of defined concentrations of Se for 30 d. However, moderate levels (0.1 mg/kg) of Se gradually improved weight and serum IGF-IR. In vitro osteoblast experiments revealed that at concentrations of 5 × 10(-6) and 10(-5) mol/L Se, MTT activity decreased in comparison with control cells. Cell cycle, TEM and caspase-3 activity supported these observations including an increase in the sub-G1 phase and notable apoptosis in osteoblasts, along with a decrease in the expression of mRNA and protein levels of IGF-IR. Moreover, the MTT activity, mRNA and protein levels of IGF-IR in osteoblasts were decreased and caspase-3 activity was increased in siRNA groups as compared with non-siRNA groups. These data suggest that Se significantly affects IGF-IR expression, and that it contributes to the proliferation and regulation of apoptosis in osteoblasts.

Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway

Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01) and casepase-3 (p < 0.05) levels, significantly (p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin. The present study is the first comprehensive insight into patho-morphological ultrastructural features of apoptosis/necrosis induced by N. cyriacigeorgica, which concluded that the clinical N. cyriacigeorgica induced apoptotic changes in the bMECs through mitochondrial-caspase dependent apoptotic pathway.

P21 Waf1/Cip1 depletion promotes dexamethasone-induced apoptosis in osteoblastic MC3T3-E1 cells by inhibiting the Nrf2/HO-1 pathway

Dexamethasone (Dex), a glucocorticoid with strong anti-inflammatory and immunosuppressive activities, has been shown to exhibit marked cytotoxicity and apoptosis in osteoblasts, but the underlying mechanisms have not yet been comprehensively investigated. P21Waf1/Cip1 (p21) plays a critical role in the regulation of cell cycle progression and apoptosis. The present study aims to investigate the role of p21 in Dex-induced apoptosis in osteoblastic MC3T3-E1 cells, and to explore its mechanisms. Results demonstrated that Dex-induced apoptosis decreased the phosphorylation of Akt in a concentration-dependent manner. Moreover, LY294002, an inhibitor of the PI3K/Akt pathway enhanced the Dex-induced apoptosis of osteoblasts. On the contrary, insulin-like growth factor-1 (IGF-1), an activator of PI3K/Akt, attenuated the apoptosis of Dex in MC3T3-E1 cells. The protein level of p21 was downregulated by shortening its half-life, which was associated with inhibition of the PI3K/Akt pathway by Dex. Furthermore, depletion of p21 by siRNA enhanced Dex-induced caspase-3 activation and ROS generation, and promoted apoptosis of MC3T3-E1 cells. In addition, suppression of p21 led to a reduction of Dex-induced upregulation of nuclear Nrf2 and heme oxygenase-1 (HO-1) protein levels. These findings demonstrate that p21 depletion promotes Dex-induced apoptosis of MC3T3-E1 cells by inhibiting the antioxidant Nrf2/HO-1 pathway, which highlights the anti-apoptotic effect of p21 in MC3T3-E1 cells.