Arachidonic acid Alox15/12-HETE signaling inhibits vascular calcification / 生理学报

This study aims to explore the effects of arachidonic acid lipoxygenase metabolism in vascular calcification. We used 5/6 nephrectomy and high-phosphorus feeding to establish a model of vascular calcification in mice. Six weeks after nephrectomy surgery, vascular calcium content was measured, and Alizarin Red S and Von Kossa staining were applied to detect calcium deposition in aortic arch. Control aortas and calcified aortas were collected for mass spectrometry detection of arachidonic acid metabolites, and active molecules in lipoxygenase pathway were analyzed. Real-time quantitative PCR was used to detect changes in the expression of lipoxygenase in calcified aortas. Lipoxygenase inhibitor was used to clarify the effect of lipoxygenase metabolic pathways on vascular calcification. The results showed that 6 weeks after nephrectomy surgery, the aortic calcium content of the surgery group was significantly higher than that of the sham group (P < 0.05). Alizarin Red S staining and Von Kossa staining showed obvious calcium deposition in aortic arch from surgery group, indicating formation of vascular calcification. Nine arachidonic acid lipoxygenase metabolites were quantitated using liquid chromatography/mass spectrometry (LC-MS) analysis. The content of multiple metabolites (12-HETE, 11-HETE, 15-HETE, etc.) was significantly increased in calcified aortas, and the most abundant and up-regulated metabolite was 12-HETE. Furthermore, we examined the mRNA levels of metabolic enzymes that produce 12-HETE in calcified blood vessels and found the expression of arachidonate lipoxygenase-15 (Alox15) was increased. Blocking Alox15/12-HETE by Alox15 specific inhibitor PD146176 significantly decreased the plasma 12-HETE content, promoted calcium deposition in aortic arch and increased vascular calcium content. These results suggest that the metabolism of arachidonic acid lipoxygenase is activated in calcified aorta, and the Alox15/12-HETE signaling pathway may play a protective role in vascular calcification.

Low-dose UVB irradiation stimulates matrix metalloproteinase-1 expression via a BLT2-linked pathway in HaCaT cells

Skin exposure to low-dose ultraviolet B (UVB) light up-regulates the expression of matrix metalloproteinase-1 (MMP-1), thus contributing to premature skin aging (photo-aging). Although cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), have been associated with UVB-induced signaling to MMP expression, very little are known about the roles of lipoxygenases and their products, especially leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), in MMP-1 expression in skin keratinocytes. In the present study, we demonstrate that BLT2, a cell surface receptor for LTB4 and 12(S)-HETE, plays a critical role in UVB-mediated MMP-1 upregulation in human HaCaT keratinocytes. Moreover, our results demonstrated that BLT2-mediated MMP-1 upregulation occurs through a signaling pathway dependent on reactive oxygen species (ROS) production and the subsequent stimulation of ERK. Blockage of BLT2 via siRNA knockdown or with the BLT2-antagonist LY255283 completely abolished the up-regulated expression of MMP-1 induced by low-dose UVB irradiation. Finally, when HaCaT cells were transiently transfected with a BLT2 expression plasmid, MMP-1 expression was significantly enhanced, along with ERK phosphorylation, suggesting that BLT2 overexpression alone is sufficient for MMP-1 up-regulation. Together, our results suggest that the BLT2-ROS-ERK-linked cascade is a novel signaling mechanism for MMP-1 upregulation in low-dose UVB-irradiated keratinocytes and thus potentially contributes to photo-aging.

Metabolism of arachidonic acid in sheep uterus: in vitro studies.

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins - PGF2alpha and prostacyclin derivative 6-keto PGF1alpha. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.

The Difference between the Concentrations of Arachidonic Acid Metabolites in the Cholesterol Granuloma and Granulation Tissues in Chronic Otitis Media / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Both the granulation tissue and cholesterol granuloma can erode the surrounding bone and ossicles. However, the etiology of bone resorption in the granulation tissue and cholesterol granuloma has not been evident. The aim of this study was to assay the concentrations of arachidonic acid metabolites (AAMs) in cholesterol granuloma and the mastoid granulation tissue in order to better understand the possible role of AAMs in the pathophysiology of cholesterol granuloma in comparison with the granulation tissue. MATERIALS AND METHOD: Cholesterol granuloma tissues were obtained from eight patients who had underwent tympanomastoidectomy. Granulation tissues, which served for comparison, were taken from 12 patients who had underwent tympanomastoidectomy. Tissue concentrations of prostagladin (PG)E2, 6-keto-PGF1alpha, leukotriene (LT)C4, LTD4, LTE4, 15-hydro xyeicosatetraenoic acid (HETE), 12-HETE, 5-HETE, and thromboxane (TXB)2 were calculated using high performance liquid chromatography and compared between cholesterol granuloma and granulation tissue. RESULT: The level of 12-HETE was higher in cholesterol granuloma than in the granulation tissue. Among the PGs, the tissue concentration of PGE2 was particularly high in cholesterol granuloma than in the granulation tissue. LTD4 was the only LT detectable in cholesterol granuloma. In comparison to the AAMs in cholesterol granuloma, the lipoxygenase pathway products such as 12-HETE, 15-HETE, and 5-HETE were present in lower concentrations in the granulation tissue. LT was undetectable in the granulation tissues. CONCLUSION: Our results suggest that cholesterol granuloma and the granulation tissue is different not only in terms of histology, but also in terms of biochemical properties such as arachidonic acid metabolism.

Arachidonic Acid Metabolites in Antrochoanal Polyp and the Nasal Polyp Associated with Chronic Paranasal Sinusitis / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: It is known that various inflammatory mediators released from the eosinophils and mast cells play important roles in the pathogenesis of nasal polyp. Among those mediators, the arachidonic acid has particular importance as a precursor of other mediators. By assaying the tissue concentration of the6-keto-PGF(1alpha), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETE) in the nasal polyp, we aimed to investigate the role of arachidonic acid metabolite in the pathogenesis of antrochoanal polyp and nasal polyp associated with chronic paranasal sinusitis. MATERIALS AND METHODS:Three turbinate tissues taken during the septoplasty were served as the control. The experimental group consisted of 3 antrochoanal polyps and 7 inflammatory polyps. The tissue level of the 6-keto-PGF(1alpha), LTC(4), LTD(4), LTE(4), 15-HETE, and 12-HETE were measured using high performance liquid chromatography. RESULTS: The level of 6-keto-PGF(1alpha), LTC4, 15-HETE, 12-HETE were significantly lower in antrochoanal polyp than in the control turbinate. In the inflammatory polyp, the levels of 6-keto-PGF(1alpha) and LTC(4) were lower than the control. However, in the inflammatory polyp, LTD(4) and LTE(4) were detectable, which were not detected in the control turbinate and antrochoanal polyp. CONCLUSION: The results of this study indicate that the decreased arachidonic acid metabolism may underlie the pathogenesis of the antrochoanal polyp. However, in the pathogenesis of inflammatory polyp, the increased production of LTD(4) and LTE(4) may have an important role.

Simultaneous HPLC analysis of arachidonic acid metabolites in biological samples with simple solid phase extraction

A reversed-phase high-performance liquid chromatography (RP-HPLC) has been developed to analyze the metabolites of arachidonic acid based on the specificities of ultraviolet absorption of these various metabolites and is sensitive to the nanogram level. This procedure makes it possible to extract complex mixtures of eicosanoids efficiently with a single step and to analyze them simultaneously by RP-HPLC from biological samples using octadesylsilyl silica extraction column and PGB2 as an internal standard. The cyclooxygenase, products (prostaglandin (PG)D2, PGE1, PGE2, PGF1alpha, PGF2alpha, 6-keto-PGF1alpha, and thromboxane B2 (TXB2)) and lipid peroxidation product, isoprostanes, of arachidonic acid were monitored by one isocratic HPLC system at 195 nm wavelength. The lipoxygenase products (leukotriene(LT)B4, LTC4, LTD4, and 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, 15-HETE) were measured by another isocratic HPLC system at 280 nm for LTs and 235 nm for HETEs. This method provides a simple and reliable way to extract and assess quantitatively the final arachidonic acid metabolites.