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Objective To investigate the effects of 12-lipoxygenase (12-LO) and angiotensin Ⅱ (Ang Ⅱ) on the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs) p21,p27 and p57 related to cell hypertrophy.Methods Mesangial cells were treated with high glucose for 24 hours and 48 hours respectively.12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and Ang Ⅱ were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively.Rats fed high fat diet were received low dose streptozotocin (STZ) to make type 2 diabetes (DN).The rats were divided into normal control group,DN group,DN+Ang Ⅱ type 1 receptor blocker (ARB) group or 12-LO inhibitor (CDC) group.DN+ARB rats were treated by losartan for 6 weeks,and DN+CDC rats were treated for 8 weeks.Urine albumin and protein expressions of p21,p27 and p57 were detected by ELISA and Western blotting respectively.Glomeruli injury and expressions of p21 and p27 were detected by PAS staining and immunohistochemistry respectively.Results High glucose increased p21 and p27 protein expression in mesangial cells significantly compared with the relative control (all P ＜ 0.05),but had no effect on p57.Ang Ⅱ increased p27 protein expression in gloneruli significantly (P ＜ 0.05),but had no effect on p21 and p57 protein expression.12(S)-HETE increased both p21 and p27 protein expression in glomeruli significantly (all P ＜ 0.05),but had no effect on p57 protein expression.Blood glucose,kidney/body weight,urinary protein,and glomerular p21 and p27 protein expressions were increased in DN group (all P ＜ 0.05) compared with those in control group,with little change of p57 protein expression (P ＜ 0.05).Moreover,glomerular hypertrophy and extra cellular matrix accumulation were observed in DN group.However,urine protein,kidney/body weight,renal injury,but not blood glucose,were decreased in DN+ARB group and DN+CDC group compared with DN group respectively (P＜ 0.05).Further DN+CDC rats had decreased both p21 and p27 protein expressions in glomeruli,but DN+ ARB rats only had decreased p27 protein expression (all P ＜ 0.05).Conclusions 12-LO may induce both p21 and p27 protein expression in DN glomeruli,but Ang Ⅱ may induce only p27 expression.
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ObjectivesTo study the expression of lipoxygenase-12 (12-LOX) in human pancreatic adenocarcinoma and its relationship with the clinicopathological parameters.MethodsExpression of 12-LOX in pancreatic carcinoma tissue,pancreatic cancer cell lines SW1990 and PANC1,and adjacent normal pancreatic tissue was detected by using immunohistochemistry,RT-PCR,and Western blot,respectively.The relationship between 12-LOX expression and clinicopathological parameters was analyzed.ResultsExpression of 12-LOX was negative in 8 cases,weak positive in 7 cases and strong positive in 15 cases of pancreatic carcinoma.The overall positive rate in pancreatic carcinoma was 73.3％.The expressions of 12-LOX mRNA and protein were positive in pancreatic cancer cell lines SW1990 and PANC1,and negative in adjacent normal pancreatic tissue.The strong positive expression of 12-LOX in pancreatic cancer was associated with TNM stage,pathological grade,lymph node metastasis (P ＜ 0.05 ).ConclusionsThe expression of 12-LOX was up-regulated in pancreatic cancer and is correlated well to malignant behaviors of tumor.
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Angiotensin II (Ang II) plays an important role in vascular hypertension. The role of the chemokine CCL5 on Ang II-induced activities in vascular smooth muscle cells (VSMCs) has not been studied. In this study, we elucidated the effect of CCL5 on Ang II-induced 12-lipoxygenase (LO) expression and cell proliferation in spontaneously hypertensive rats (SHR) VSMCs. CCL5 decreased Ang II-induced 12-LO mRNA expression and protein production, and it increased Ang II type 2 (AT2) receptor expression in SHR VSMCs. The inhibitory effect of CCL5 on Ang II-induced 12-LO mRNA expression was mediated through the AT2 receptor. Although treatment of CCL5 alone induced SHR VSMCs proliferation, CCL5 inhibited Ang II-induced VSMCs proliferation and PD123,319, an AT2 receptor antagonist, blocked the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation. Phosphorylation of p38 was detected in VSMCs treated with Ang II or CCL5 alone. But, decrease of p38 phosphorylation was detected in VSMCs treated with Ang II and CCL5 simultaneously (Ang II/CCL5) and PD123,319 increased p38 phosphorylation in VSMCs treated with Ang II/CCL5. Therefore, these results suggest that the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation is mediated by the AT2 receptor via p38 inactivation, and CCL5 may play a beneficial role in Ang II-induced vascular hypertension.
Subject(s)
Angiotensin II , Angiotensins , Arachidonate 12-Lipoxygenase , Cell Proliferation , Chemokine CCL5 , Down-Regulation , Hypertension , Muscle, Smooth, Vascular , Phosphorylation , Rats, Inbred SHR , Receptor, Angiotensin, Type 2 , RNA, MessengerABSTRACT
BACKGROUND: We previously demonstrated remarkable differences in the expression of IL-8/CXCL8 in aortic tissues and vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) compared to VSMC from normotensive Wistar-Kyoto rats (WKY). In the present study, we investigated the direct effect of IL-8/CXCL8 on expression of 12-lipoxygenase (LO), a hypertensive modulator, in SHR VSMC. METHODS: Cultured aortic VSMC from SHR and WKY were used. Expression of 12-LO mRNA was determined by real-time polymerase chain reaction. Phosphorlyation of ERK1/2 and production of 12-LO and angiotensin II subtype 1 (AT1) receptor were assessed by Western blots. IL-8/CXCL8-stimulated DNA synthesis was determined by measuring incorporation of [3H]-thymidine. And effect of IL-8/CXCL8 on vascular tone was determined by phenylephrine-induced contraction of thoracic aortic rings. RESULTS: Treatment with IL-8/CXCL8 greatly increased 12-LO mRNA expression and protein production compared to treatment with angiotensin II. IL-8/CXCL8 also increased the expression of the AT1 receptor. The increase in 12-LO induced by IL-8/CXCL8 was inhibited by treatment with an AT1 receptor antagonist. The induction of 12-LO mRNA production and the proliferation of SHR VSMC by IL-8/CXCL8 was mediated by the ERK pathway. The proliferation of SHR VSMC and the vascular contraction in the thoracic aortic ring, both of which were induced by IL-8/CXCL8, were inhibited by baicalein, a 12-LO inhibitor. CONCLUSION: These results suggest that the potential role of IL-8/CXCL8 in hypertensive processes is likely mediated through the 12-LO pathway.
Subject(s)
Animals , Rats , Angiotensin II , Arachidonate 12-Lipoxygenase , Blotting, Western , Contracts , DNA , Flavanones , MAP Kinase Signaling System , Muscle, Smooth, Vascular , Rats, Inbred SHR , Real-Time Polymerase Chain Reaction , RNA, MessengerABSTRACT
Endothelin-1 (ET-1) has been characterized as a potent vasoconstrictor secreted by the endothelium, and play a major role in the regulation of vascular tone. It has been also known to participate in inflammatory reactions. The production of ET-1 by macrophages during infection and inflammation is related to tissue perfusion and leukocyte extravasation. The aim of this study is to investigate the role of IL-8/CXCL8, as a major inflammatory chemokine, for ET-1 expression in macrophges. Expression of ET-1 mRNA in mouse peritoneal macrophages (PeM phi) was weaker than that in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). However, expression of IL-8/CXCL8-induced ET-1 mRNA in PeM phi was much more stronger than that in SHR and WKY VSMCs. Maximum expression of ET-1 mRNA was observed at 50 ng/ml dose of IL-8/CXCL8 and occurred at 2 h after addition of IL-8/CXCL8. Expression of ET-1 by IL-8/CXCL8 was dependent on NF-kappaB activation and ERK1/2 phosphorylation. Baicalein, a 12-lipoxygenase (LO) inhibitor, inhibited the expression of IL-8/CXCL8-induced ET-1 mRNA. This inhibitory action of baicalein was mediated via ERK1/2 inactivation. Induction of 12-LO mRNA by IL-8/CXCL8 and expression of ET-1 mRNA by 12-LO metabolite, 12(S)-HETE were also detected. The expression of IL-8/CXCL8-induced ET-1 mRNA was not detected in PeM phi transfected with 12-LO siRNA. These results suggest that IL-8/CXCL8 can act as one of main inducers of ET-1 in vascular inflammatory reactions, and ET-1 expression by IL-8/CXCL8 is related to 12-LO pathway in PeM phi.
Subject(s)
Animals , Mice , Rats , Arachidonate 12-Lipoxygenase , Endothelin-1 , Endothelium , Flavanones , Inflammation , Leukocytes , Macrophages , Macrophages, Peritoneal , Muscle, Smooth, Vascular , NF-kappa B , Perfusion , Phosphorylation , Rats, Inbred SHR , RNA, Messenger , RNA, Small InterferingABSTRACT
Objective To investigate the effect of 12-lipoxygenase(12-LOX) inhibitor inducing the apoptosis of human hepatocellular carcinoma cell line HepG2 on the expression of survivin gene.Methods HepG2 cells were cultivated in RPMI-1640 medium.12-LOX mRNA was analyzed by reverse transcription polymerase chain reaction(RT-PCR).The effect of baicalein on the proliferation of the cells was detected by thiazolyl blue tetrazoliumbromide(MTT) method.The subcellular structures were observed under electron microscope.DNA ladder pattern on agarose gel electrophores was used to evaluate the apoptotic index of hepG2 cells.The expression of survivin mRNA was analyzed by RT-PCR.Results 12-LOX mRNA was expressed in human HepG2 cells.At concentrations from 20 to 80 ?mol?L-1,baicalein inhibited the proliferation of HepG2 cells in a concentration-and time-dependent manner from 24 to 72 h.There were significant differences between any other groups(P
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BACKGROUND AND OBJECTIVES: We determined the localization of leukocyte-type 12-lipoxygenase (L-12-LO) in murine nasal mucosa and to investigate the expression of L-12-LO according to the development of murine nasal mucosa. MATERIALS AND METHOD: Immunohistochemical staining was done on the nasal mucosa of mice at gestational days 16, 17, 18, and mice at postnatal days 1, 3, 7, 14, and adult mice. Alcian blue (pH 2.5)-periodic acid Schiff staining on murine nasal mucosa was performed. RESULTS: In murine nasal respiratory mucosa, the expression of L-12-LO was noted in ciliated epithelial cells, basal cells, serous acini, and secretory ducts, but it was not found in the mucous acini and goblet cells. In olfactory mucosa, the expression of L-12-LO was noted in the olfactory receptor cells, supporting cells, and basal cells. The expression in respiratory mucosa according to the development was strongly noticed from the gestational day 16 through postnatal day 7. The expression in postnatal day 14 and adult mice was weaker than in the previous time point. The expression in olfactory mucosa showed no difference throughout the developmental stage. CONCLUSION: As a result of this study, we found the exact localization of L-12-LO in murine nasal mucosa, and we also found the different expression of L-12-LO between the respiratory and olfactory mucosa. This fact suggests the possible involvement of L-12-LO in the development of murine respiratory mucosa.