The regulation of the cholesterol metabolism in tumoral cells is complex. It is known that cancer cells have the capacity to increase intracholesterol via a feedback regulation mechanism of the HMGCR enzyme, which controls de novo cholesterol synthesis36,37. Importantly, the expression of this gene was downregulated in association with the degree of malignancies of the tumors. These changes may be related with similar free cholesterol levels found in the more aggressive tumors compared with those of benign tumors. It is noteworthy that the analyses in normal and ATC cell lines revealed a gene expression pattern like that found in the thyroid tumors from the patients. In contrast, one of the metabolites of cholesterol with greater relevance in both inflammatory and tumor processes is 27-HC, the most abundant oxysterol in the systemic circulation. In 2013, Nelson et al. demonstrated that 27-HC acted as an estrogen receptor agonist in breast cancer, inducing tumor growth and metastasis. These effects were reproduced in mice by overexpressing the CYP27A1 gene, which regulates 27-HC synthesis18. In contrast, decreased expression of CYP7B1 triggers accumulation of 27-HC, and CYP7B1 was downregulated in breast cancer compared with normal breast tissue19,20. In line with these findings, CYP7B1 was strongly downregulated in the aggressive tumor tissues (PTC high risk and PDTC/ATC) in relation to the benign tumors, in close association with the higher concentration of 27-HC. These data indicate that the accumulation of 27-HC in the thyroid cells may be promoting the development and progression of TC. In fact, downregulation of CYP7B1 was also detected in the anaplastic cell line (CAL-62) and its overexpression reduced cell growth and migration. Furthermore, downregulation of CYP27A1 completely blocked LDL-mediated cell proliferation. However, 27-HC by itself only promoted cell migration in non tumoral cells. We also treated the cells with DHEA, which may be hydroxilated by CYP7B138 and it did not produce any effect in the cells with anaplastic phenotype. Nevertheless, in non-cancer cell (Nthy-ori 3.1 cells) we found a doses dependent decline in cell proliferation, as occurred with 27-HC. These results rather indicate a dual effect of 27-HC in cells with aggressive behaviors in contrast with immortalized cells with non tumoral phenotype. It should be noted that 27-HC increased ROS levels and reduced the antioxidant defense system levels in non pathological astrocytes, thereby affecting cell viability39. In addition, 27-HC may downregulate the expression of the nuclear factor E2-related factor 2 signaling pathways40. Moreover, in immortalized retinal pigment epithelial cell line (RPE cells), 27-HC also caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death41.
Our results indicate that resveratrol potently reduced LPS-induced PGE2 synthesis and the formation of 8-iso-PGF2α, a measure of free radical production. Interestingly, resveratrol dose-dependently reduced the expression (mRNA and protein) of mPGES-1, which is a key enzyme responsible for the synthesis of PGE2 by activated microglia, whereas resveratrol did not affect the expression of COX-2. Resveratrol is therefore the first known inhibitor which specifically prevents mPGES-1 expression without affecting COX-2 levels. Another important observation of the present study is that other COX-1 selective inhibitors (SC-560 and Valeroyl Salicylate) potently reduced PGE2 and 8-iso-PGF2α production by LPS-activated microglia.
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8-isoprostanes are formed in response to free radical attack on arachidonic acid on membrane phospholipids and are considered as a reliable and highly sensitive measure of free radical formation [41]. Microglial cells were pre-treated for 30 min with different concentrations of resveratrol, Trolox C, α-tocopherol, SC-560, or VAS (see Results and Figures). After 30 min pre-stimulation, cells were added 10 ng/ml LPS for 24 h. Control experiments consisted of cells treated only with the solvent of each compound without LPS. Supernatants were harvested and the levels of 8-iso-PGF2α (IUPAC nomenclature: 15-F2t-IsoP) were measured by an enzyme immunoassay according to the manufacturer's instructions (Cayman Chemicals, Ann Arbor, MI, USA). The standards were used in the range of 3.9 to 500 pg/ml (detection limit of 5 pg/ml).
Since resveratrol has been proven to have potent antioxidant properties, we aimed to determine the efficacy of this compound in reducing free radical production by activated microglia. LPS produced a very significant increase in the formation of 8-iso-PGF2α, a very sensitive marker of cellular free radical generation, as assessed by enzyme immunoassay (Fig. 5). More importantly, resveratrol potently reduced LPS-mediated formation of ROS, an effect that was seen at very low doses (starting at 1 μM). This reduction in 8-iso-PGF2α by resveratrol showed a concentration-dependent response between 1 and 10 μM (Fig. 5A), an effect also seen in our first experiment evaluating PGE2 levels (Fig. 1A). We then tested the ability of two well-known antioxidants, Trolox C and α-tocopherol, to reduce free radical formation in LPS-activated microglia. These antioxidants significantly reduced 8-iso-PGF2α as well, but not as potent as resveratrol (Fig. 5B).
The ability of resveratrol to inhibit the peroxidase activity of COX-1 is a well-known pharmacological effect of this compound [43, 44]. It was then very important to investigate the ability of other COX-1 inhibitors to reduce PGE2 and 8-iso-PGF2α production in microglia activated with LPS. Interestingly, two structurally different highly selective COX-1 inhibitors (SC-560 and VAS) potently reduced PGE2 production (Fig. 6A and 6C) and free radical formation (Fig. 6B and 6D) in activated microglia in a dose-dependent manner.
In the present study, we have found that resveratrol is a potent inhibitor of PGE2 and free radical formation by activated microglial cells. These findings add significant information on the molecular mechanisms involved in the neuroprotective effect of this compound. The ability of resveratrol to reduce PGE2 production comes from the modulation of multiple events in the COX/PGE2 pathway: 1) resveratrol is a potent inactivator of the peroxidase reaction of COX-1 [43], and thus is considered a relatively selective inhibitor of this isozyme; 2) resveratrol significantly diminished LPS-induced expression of mPGES-1 (Figs. 3 and 4), the most important terminal synthase responsible for PGE2 synthesis in activated microglia [42]; and 3) production of 8-iso-PGF2α, a reliable indicator of free radical generation, is dramatically reduced by low concentrations of resveratrol (Fig. 5).
The relative contribution of each of these mechanisms to the overall reduction in PGE2 by resveratrol is difficult to address based on present data. However, some issues deserve further discussion. The ability of resveratrol to inhibit the peroxidase activity of COX-1 is a well-known pharmacological effect of this compound [43, 44]. COX-1 is constitutively expressed in microglia under resting conditions, and its expression is not induced by LPS as shown in Figs. 3A and 4A, and reported by us before [34]. However, according to present results, there is a significant contribution of COX-1 to PGE2 formation by microglia upon LPS challenge. This is supported by a previous study performed in LPS-stimulated human adult microglial cells, in which selective inhibition of COX-1 was also very effective in reducing PGE2 production [62]. Results from control experiments using other highly selective COX-1 inhibitors (SC-560 and VAS), in addition to resveratrol, indicate that COX-1 isoform is not only important in LPS-induced PGE2 synthesis, but it is also a key source of free radicals in microglia. This is an unexpected observation, and represents the first evidence that microglial COX-1 activity is a significant source of free radicals during neuroinflammation.
In summary, we are proposing here that significant attenuation of PGE2 and free radical production by activated microglia might contribute to the neuroprotective effects of resveratrol. This study gives further support to the potential use of resveratrol as a therapeutic agent to reduce microglial activation following different types of brain injury.
The ISO 11064 standard establishes principles, recommendations and requirements to be applied in the design of control centers. This standard proposes general-purpose aspects as well as aspects for its specific application in industrial control rooms. Ergonomics is principally used as physical ergonomics. This standard is divided into 5 basic parts that are listed below:
Also, besides the application of the GEDIS guide, international standards have been taken into account, such as ISO 11064 and 191 EEMUA [10]. The evaluation of the performance of BMS Alarm Systems has yielded positive results. These results include the fact that the number of alarms that may occur in 10 minutes is less than 2, and, as such, the system is manageable for operators. The number of alarms that may occur during the first 10 minutes after the first critical alarm that occurred is dependent on equipment fails. In the system that was analyzed there are 7 alarms that could trigger. Therefore, this is a lower number than the value established by the EEMUA 191 standard, which has a 10-alarm maximum. So, the management system is predictable.
The D-ALIX supervisors need to know about the consumption of the different equipment located at the NAP. Tostudy these statistics, the ITER uses "Pentaho", which is a suite composed by a set of free programs to generate business intelligence, including built-in tools to generate reports, data mining, etc. Due to the fact that "Pentaho" is running on a different server and in addition to the latency of the graphics of consumption display, the need to employ a web technology was identified in the draft D-Alix, in order to integrate these graphs in the BMS. 2ff7e9595c
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