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Results include junk files and folders and description why they are junk. You will be able to remove the detected junk files by hand or automatically. After using disk cleaner you will have more disk space at your disposal so you can install more programs and games, have more music and video, and you will be amazed that your computer will work faster. Windows All. Pakay Crack With License Key Windsty DiskCleaner reviews April 30, , Daniele wrote: cheers, thanks. Leave a reply Your email will not be published. Popular Posts FileBot 4. Crack4Windows Copyright c This is an open access article distributed under the terms of Creative Commons Attribution License. Due to the outstanding performance with respect to immune response and anti-inflammatory effect, glucocorticoids GCs are routinely prescribed to treat autoimmune and noninfectious inflammatory diseases. However, the long-term and excessive administration of GCs can lead to many serious complications, such as osteoporosis. GC-induced osteoporosis GIOP , which is considered to be the most common form of secondary osteoporosis 1 , 2 , increases the risk of fracture in patients treated with GCs. Clinical studies have indicated that GCs dysregulated bone metabolism and led to a rapid reduction in trabecular bone mass within the first year of treatment 3 , 4. However, this reduction in trabecular bone mass cannot entirely explain the observed increase in fracture risk because the bone mineral density BMD value of patients treated with GCs is higher than those of women with postmenopausal osteoporosis 4. Therefore, it is necessary to more comprehensively elucidate the pathology of GC-induced bone loss. A better understanding of this process will help clinicians effectively prevent and treat GIOP. Osteoblasts, which are derived from mesenchymal progenitor cells, are mainly responsible for bone formation and development 5. Prior studies have suggested that the administration of GCs induced the apoptosis of osteoblasts and osteocytes, which was regarded as the main cause of GIOP 6. Nevertheless, it is well known that GCs are indispensable for normal bone metabolism and development under physiological condition 7. A recent study demonstrated that different dose of GCs led to different osteocyte fates 8. In addition, Shi et al 9 concluded that GCs had dose-related effect on osteoclast formation and function. Based on these studies, GC dose is a crucial factor affecting the function and viability of cells. Unfortunately, few studies have addressed the relationship between osteoblast viability and GC dose, particularly for low-GC dose. Autophagy is a highly conserved intracellular cleaning process in eukaryotic cells that is responsible for removing damaged organelles and counteracting harmful stimuli 10 , Generally, physiological autophagy exists in all cells to maintain normal cellular metabolism and viability Once cells encounter stress stimuli, such as hypoxia 13 , oxidative stress 14 , or ER stress 15 , autophagy is induced to prevent cells from undergoing apoptosis by eliminating stress inducers. Autophagy reportedly plays a protective role in maintaining osteoblast viability by allowing cells to survive various stresses 16 , Microtubule-associated protein light chain 3 LC3 and beclin 1 are important biological signal to identify autophagy In the process of autophagy, cytoplasmic proteins, organelles and so on, are engulfed by autophagosomes. LC3II is simultaneously formed by conjugating LC3I with phosphatidylethanolamine, which is fixed on the membrane of autophagosome. After the formation of autolysosome consisting of lysosome and autophagosome, the cytoplasm in autophagosome is degraded. Meanwhile, LC3II is also degraded in the autolysosome. Therefore, the activity of autophagy could be characterized by detecting activity of LC3II 18 , Beclin 1 is also an essential protein for the formation of autophagosome. It can guide other autophagy-related proteins to move into vacuoles, and thus regulates the formation of autophagosome It is well-known that GCs can interrupt mitochondrial function and thereby increase reactive oxygen species ROS in many cells 21 , Generally, ROS is produced by mitochondria during cellular metabolism. However, ROS level is significantly increased when cells are exposed to noxious stimuli, such as cytotoxic drugs. At normal level, ROS plays a crucial role in the energy cycling of cells; however, excessive ROS induces cell apoptosis, even death, which are closely associated with many diseases 23 , Certain studies have indicated that reduced bone formation and decreased BMD were induced by increased oxidative stress in aged humans and mice 25 , Moreover, a recent study demonstrated that the GC-induced upregulation of ROS could lead to osteoporosis by inducing osteoblast apoptosis 2. However, studies have claimed that ROS, as a source of oxidative stress, also contributed to the initiation of autophagy to prevent osteoblasts from undergoing apoptosis We hypothesize that the biphasic effect of ROS on osteoblast viability may be attributable to the difference in ROS level, which is associated with GC dose. However, this hypothesis must be verified by additional research. The primary aim of this study is to investigate the effect of GC dose on the viability of osteoblasts, assess whether autophagy is involved in this process, and examine the relationship between ROS and autophagy. The human fetal osteoblastic cell line hFOB 1. After the cells reached a steady-state of exponential growth in the media, they were exposed to variable concentrations of DEX for specific time. The hFOB 1. Cell viability was determined by 3-[4,5-dimethylthiazoly]-2,5-diphenyltetrazolium bromide MTT; Keygen, Nanjing, China assay following the manufacturer's instructions. After treated with indicated treatment for 24 h, cells were collected and stained with the Annexin V-FITC Apoptosis Detection kit Keygen according to manufacturer's instructions, and the apoptosis was analyzed by the flow cytometer equipped with Modfit LT 3. Average fluorescence intensity was calculated based on the data from the six fields of each treatment. Three independent experiments were conducted. The treated hFOB 1. Next, the membrane was incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase ,; ZSGB-BIO, Beijing, China for 1 h at room temperature. The band density was quantified using the ImageJ image processing program. Cells were photographed using the inverted fluorescence microscope. LC3-positive cells, defined as cells with visible LC3 foci, were quantified by manually in three randomly-selected fields; nuclei were enumerated by counting DAPI-stained nuclei in the same field. The number of LC3-positive cells in each microscopic field was divided by the number of nuclei in the same field, and was regarded as the rate of LC3-positive cells. Subsequently, the cells were conventionally dehydrated, embedded, sectioned, and stained. The number of intracellular autophagosomes in every 10 fields was counted. All the experiments were conducted in triple. In contrast, the viability of hFOB1. Cell viability was estimated using an MTT assay. C Quantification analysis of apoptotic cells. Viable cells, early apoptotic cells, late apoptotic cells and necrotic cells appear in the bottom left quadrant Q3 , bottom right quadrant Q4 , top right quadrant Q2 and top left quadrant Q1 , respectively. Based on these results, we suggest that DEX impacts osteoblast viability in a dose-dependent manner in vitro. To investigate whether increased viability of hFOB1. Therefore, we hypothesize that the dose-dependent effect of DEX on osteoblast viability is closely related to autophagy. To verify this hypothesis, we treated osteoblasts with the specific autophagy agonist Rap. It was observed that the decrease in the viability of hFOB1. All of these findings demonstrated that autophagy protected hFOB1. Effect of autophagy modulators on the viability of DEX-treated cells. B Quantification analysis of apoptotic cells in 3-MA studies. E Quantification analysis of apoptotic cells in Rap studies. DEX only group. According to guideline for monitoring autophagy, autophagy level can be accurately evaluated by detecting the autophagic flux, which can be assessed based on protein level of LC3II and beclin 1. The level of LC3II and beclin 1 increased gradually starting from the 3 h time-point and peaked at the 6 h time-point Fig. Higher and lower concentration of DEX could also induce autophagy but at lower level, indicating that DEX modulated autophagy in a dose-dependent manner. The assessment of autolysosome presence and number via transmission electron microscopy TEM is recognized as the gold standard for monitoring autophagy intensity. The quantity of autolysosomes was significantly elevated in hFOB1. Furthermore, large numbers of mitochondria that were undergoing phagocytosis were observed. GCs have been reported to increase intracellular oxidative stress by damaging mitochondria In response to this oxidative stress, autophagy is upregulated to help to clear damaged mitochondria with autophagosomes The number of autolysosomes was significantly lower in the group treated with 3-MA 5 mM than that in the corresponding group treated with DEX alone Fig. This result confirmed that autophagy protected osteoblasts against oxidative stress by clearing damaged organelles In addition, cells with punctate aggregates autolysosomes was defined as LC3-positive cells and they were observed via fluorescence microscopy Fig. This result was consistent with the finding obtained via TEM. A Transmission electron microscopy showing autophagosomes in hFOB 1. Arrows indicate autophagosomes. B Quantitation of autophagosomes. Arrows indicate LC3-positive cells. D Quantitation of LC3-positive cells. DEX only treatment. After hFOB1. The result showed that intracellular ROS level increased Fig. B Quantification analysis of ROS. D Quantification analysis of ROS. In particular, hFOB1. Rap significantly decreased intracellular ROS level. These results indicated that autophagy could protect osteoblasts by decreasing ROS level. Western blot analysis showed that catalase remarkably inhibited the autophagy induced by low-dose DEX Fig. Furthermore, the result of immunofluorescence staining revealed that the rate of LC3-positive cells was lower in the group treated with catalase and DEX than that treated with DEX alone Fig. B Quantitative analysis of beclin 1 and LC3 protein expression. Taken together, the aforementioned results suggested that a low dose of DEX induced autophagy by upregulating intracellular ROS level. Significantly lower viability was observed in hFOB1. A Cell viability was estimated using the MTT assay. Many studies have suggested that GCs contributed to the development and progression of GIOP by repressing viability and promoting apoptosis among osteoblasts 2 , 6. However, there is controversy regarding the effect of GCs on osteoblasts in vivo and in vitro. Endogenous GCs, which are secreted by the adrenal cortex, are essential for bone development 31 , Moreover, investigations have found that in vitro , DEX can accelerate the osteogenic activity of osteoblasts 33 and the proliferation of bone marrow stromal cells 34 , which are precursors and source of osteoblasts. This result is similar to the finding obtained by Jia et al 8 and Shi et al 9 , who claimed that the effect of GCs on cell viability was determined by GCs dose. In fact, the treatment dose of GCs used for autoimmune and inflammatory diseases is higher than the physiological level of these compounds, and treatment duration is often months or even years. This observation may partially resolve the controversy regarding the opposite effect of GCs on osteoblast viability. As we know, basal autophagy occurs at low level in all cells and is considered as a quality control mechanism of protein and organelle, maintaining normal cellular homeostasis When encountering some stresses, autophagy is usually upregulated If autophagy is excessively upregulated, autophagosomes will devour the cellular proteins and organelles, and cause cells apoptosis. Hence, autophagy is also regarded as type II form of programmed cell death However, under some moderate stimulus conditions, autophagy can be moderately induced to promote cell survival and avert apoptosis by eliminating stress inducers Autophagy ensures the delivery of metabolic substrates to cells in order to fulfill their energy demand during stress, thus supports cell growth and survival 38 , This function is critical for the survival of some terminally differentiated cells, such as osteoblasts and osteoclasts Many recent studies have demonstrated the protective effect of autophagy with respect to facilitating osteoblast viability under various stress 16 , 17 , Moreover, the involvement of GC-induced autophagy in modulating the viability of various cells has been well demonstrated 42 — However, the role of autophagy in affecting the viability of osteoblasts exposed to low dose of GCs has rarely been reported. Based on prior studies and our results, we hypothesize that the increased viability of osteoblasts exposed to low-dose DEX may be associated with autophagy. In conclusion, all of the aforementioned results indicated that a low dose of GCs increased osteoblast viability by inducing autophagy at early stage. DEX, as an inducer of oxidative stress, can lead to mitochondrial dysfunction and upregulate oxidative stress in cells 21 , To respond to this oxidative stress, autophagy was upregulated to protect osteoblasts from apoptosis due to DEX exposure; this phenomenon could be observed by detecting autophagy-related proteins and autophagosomes in osteoblasts incubated with low-dose DEX. If the DEX dose and the corresponding oxidative stress are low and the treatment time is short, this attempt to protect osteoblasts will be successful. However, as DEX dose and treatment time increase, the antioxidant capacity of autophagy will be overwhelmed, and the ability of protecting against stress will be reduced, potentially leading to osteoblast apoptosis. Our study demonstrated that osteoblast viability was strictly determined by GCs dose. A low dose of GCs activated autophagy to protect cells from apoptosis, whereas a high dose of GCs induced apoptosis. This finding was consistent with the results of an in vivo investigation by Jia et al 8 , who reported that autophagic gene expression and osteocyte autophagy increased when mice were treated with a low dose of GCs but the expression of apoptotic genes increased when mice were treated with higher dose of GCs. The generation of ROS is known to be a normal physiological activity of cellular metabolism However, researches on the pathological mechanism of GCs have demonstrated that excessive and long-term treatment with GCs caused the excessive accumulation of intracellular ROS by dysregulating mitochondria function, leading to the upregulation of oxidative stress The excessive accumulation of ROS might disturb cellular homeostasis, resulting in cell injury and death 47 , Both in vitro and in vivo studies have demonstrated that excessive ROS and oxidative stress reduce bone formation by inhibiting the differentiation and viability of osteoblasts 45 , Therefore, we concluded that the reduced viability of osteoblasts treated with high-dose DEX was associated with excessive intracellular ROS. However, studies have showed that physiological ROS acted as intracellular signalling molecule, and moderate ROS was thought to modulate cell viability by upregulating autophagy 17 , 46 , Based on our investigation and works by other researchers, we hypothesize that low-dose DEX increased osteoblast activity by inducing autophagy via intracellular ROS. The results showed that the increased cell activity and autophagy induced by a low dose of DEX was significantly inhibited by catalase. In addition, increased autophagy can decrease or eliminate intracellular ROS In our study, the upregulation of autophagy by Rap caused the reduction of intracellular ROS in osteoblasts treated with low-dose DEX, whereas pretreatment with 3-MA produced the opposite result. In summary, our data implied that the low-dose GCs induced autophagy via intracellular ROS and autophagy protected osteoblasts from apoptosis by reducing ROS level. In conclusion, the present study demonstrated that GCs modulated osteoblast viability in a dose-dependent manner. High-dose of GCs induced osteoblast apoptosis, whereas a lower dose of GCs enhanced osteoblast viability by inducing autophagy via intracellular ROS. Our findings suggested that the modulation of osteoblast autophagy could be a novel approach for the prevention and treatment of GIOP. Next, further studies should be performed to explore the underlying mechanism of low-dose GC-induced autophagy via ROS. Expert Opin Drug Saf. Adv Exp Med Biol. J Bone Miner Res. Arthritis Rheum. Arch Biochem Biophys. Moriishi T and Komori T: Glucocorticoid and bone. The inhibition of osteoblast differentiation and induction of osteocyte apoptosis through the regulation of Bcl-2 by glucocorticoids. Clin Calcium. In Japanese. Komori T: Glucocorticoid signaling and bone biology. Horm Metab Res. Sci Rep. EMBO J. Cancer Cell. Biochem Pharmacol. Cell Death Differ. Yang L, Meng H and Yang M: Autophagy protects osteoblasts from advanced glycation end products-induced apoptosis through intracellular reactive oxygen species. J Mol Endocrinol. Methods Mol Biol. Int J Biochem Cell Biol. J Biol Chem. Clin Exp Pharmacol Physiol. Osteoarthritis Cartilage. Prog Neurobiol. Trends Mol Med. Free Radic Biol Med. Wohlgemuth SE, Calvani R and Marzetti E: The interplay between autophagy and mitochondrial dysfunction in oxidative stress-induced cardiac aging and pathology. J Mol Cell Cardiol. Ishida Y, Tertinegg I and Heersche JN: Progesterone and dexamethasone stimulate proliferation and differentiation of osteoprogenitors and progenitors for adipocytes and macrophages in cell populations derived from adult rat vertebrae. Atmani H, Chappard D and Basle MF: Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: Effects of dexamethasone and calcitriol. J Cell Biochem. Can J Cardiol. Cell Signal. Cell Prolif. Adv Cancer Res. Trends Endocrinol Metab. Toxicol Lett. Mol Med Rep. Eur Rev Med Pharmacol Sci. Antioxid Redox Signal. J Cell Physiol. Manolagas SC: From estrogen-centric to aging and oxidative stress: A revised perspective of the pathogenesis of osteoporosis. Endocr Rev. Cell Mol Life Sci. Trends Cell Biol. March Volume 17 Issue 3. Sign up for eToc alerts. You can change your cookie settings at any time by following the instructions in our Cookie Policy. To find out more, you may read our Privacy Policy. I agree. Home Submit Manuscript My Account. Advanced Search. Register Login. Molecular Medicine Reports. This article is mentioned in:. Introduction Due to the outstanding performance with respect to immune response and anti-inflammatory effect, glucocorticoids GCs are routinely prescribed to treat autoimmune and noninfectious inflammatory diseases. Cell culture and treatment The human fetal osteoblastic cell line hFOB 1. Cell viability assay The hFOB 1. Cell apoptosis analysis After treated with indicated treatment for 24 h, cells were collected and stained with the Annexin V-FITC Apoptosis Detection kit Keygen according to manufacturer's instructions, and the apoptosis was analyzed by the flow cytometer equipped with Modfit LT 3. Protein extraction and western blot analysis The treated hFOB 1. Statistical analysis All the experiments were conducted in triple. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Related Articles. This site uses cookies. Spandidos Publications style. Mol Med Rep , Zhang, S. Low-dose dexamethasone affects osteoblast viability by inducing autophagy via intracellular ROS. Molecular Medicine Reports, 17, Molecular Medicine Reports Molecular Medicine Reports 17, no.
 

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