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Objective To investigate the effect of pretreatment with U75302,antagonist of leukotriene B4 receptor 1 (BLT1),on cisplatin induced acute kidney injury in mice and its immunoregulatory mechanism.Methods Healthy C57BL/6 mice were randomized into four subgroups:1.healthy control group;2.cisplatin group;3.U75302 control group;4.cisplatin + U75302 group,n=6.Group 2 and 4 received intraperitoneal injection of cisplatin (20 mg/kg) on day 0,group 3 and 4received intraperitoneal injection of U75302 (5 μg/mouse) on day 0 and day 2.Mice were sacrificed on the 3rd day and blood and kidney were collected.Renal function and histological changes were estimated,the infiltration of immune cells were determined by flow cytometry,the level of peroxidase (MPO) in kidney were determined by colorimetry,relative expression of TNF-α,IL-1β,CXCL1,CXCL2 were detected by Real-time PCR.Results Compared with healthy control group,levels of BUN,Scr were higher in cisplatin group with serious tubular structural damage.There were more neutrophils,macrophages,CD4+ T lymphocytes,CD8+ T lymphocytes in kidneys of cisplatin group,the level of MPO and relative expression of TNF-α,IL-1β,CXCL1,CXCL2 were also higher in cisplatin group.Compared with cisplatin group,lower BUN [(17.75±1.80) mmol/L vs (42.6±6.66) mmol/L,P ＜0.05],Scr were found in cisplatin+ U75302 group with less tubular structural damage.Meanwhile,U75302 reduced infiltration of neutrophils [(146±13)×103/g vs (296±66) ×103/g,P ＜ 0.05],macrophages [(245± 13)× 103/g vs (420±78)× 103/g,P ＜ 0.05] in the kidney.Levels of MPO [(1.756±0.283) U/g vs (3.308±0.577) U/g,P＜0.05] and relative expression of TNF-α,IL-1β,CXCL1,CXCL2 were also lower.Conclusions BLT1 antagonist U75302 protects mice against AKI induced by cisplatin,and the mechanism is associated with reduced infiltration of inflammatory cells in kidney and the inhibition of kidney inflammation.
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Objective To detect the expression and function of cysteinyl leukotriene receptors (CysLTRs)in keratinocytes.Methods Human keratinocytes were isolated from the tissue of foreskin by digestion with dispase Ⅱ and trypsin,and subjected to primary culture.By using confocal laser scanning microscopy and reverse transcriptase PCR,the localization and expression of CysLTRs were studied in kemtinocytes.respectively.Some primarily cultured keratinocytes were pretreated with leukotriene D4 (30 nmoi/L),MK571(300 nmol/L),and BAYu9773 for 5 minutes followed by the detection of intmcellular calcium level using the Ca2+ indicator dye Fura-2/AM as well as cell proliferation bv MTT assay.Results The expressions of CysLTR1 and CysLTR2 were observed in cultured keratinocytes,and they were mainly located on cell membrane,partly in cytoplasm and nuclei.Compared with non.stimulated cells,a significant increase Was noted in the expression of CysLTRs,especially in the nuclei of keratinocytes stimulated by LTD4(P＜0.05),together with an elevation in intracellular calcium level(42.27±3.00 mmol/L,P＜0.01)and acceleration in cell proliferation (P＜0.01).However,both MK571 and BAYu9773 could completely block the effect of LTD4 on intmcellular calcium level and cell proliferation.and there was no significant difference in the blocking effect between MK571 and BAYu9773.Conclusions Functional CysLTRs are expressed in human keratinocytes.and they carl increase the intracellular calcium level in,and cell proliferation of,keratinocytes.
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Objective To investigate the effect of iuvenile rheumatoid arthritis(JRA) serum (leukotriene B4,LTB4) LTB4-BLT2 on mice DCs.Methods Bone marrow(BM)-derived DCs from healthy mouse were purified.and induced by cytokine IL-4 and GM-CSF to immature DCs and then differentiated to mature DCs under the stimulation of LPS in vitro.DCs were evaluated by light microscope and flow cvtometry.The concentrations of LTB4 in DCs supernatant,normal serum,active JRA,and that of the co-cuItured with BLT2 antagonist LY255283 were detected by ELISA.The expression of BLT2 protein and mRNA in DCs was examined by immunocytochemistry,immunofluorescence and RT-PCR.Meanwhile,the expression of BLT2 in DCs after 18 h co-cultured with normal serum,in serum of active JRA and that of BLT2 antagonist (LY255283)group was assayed by flow cytometry respectively.Results LTB4-BLT2 was expressed by DCs.Not only BLT2 mRNA but also its protein was expressed in DCs.The concentration of LTB4 Was(17±3)pg/ml,(82±20)pg/ml,(82±20)pg/ml and(24±6)pg/ml,(115±20)pg/ml,(91±11)pg/ml in normal serum group,active JRA group and LY255283 group before and after 1 8 h,respectively.The expression Was higher in serum of active JRA group than that of normal sertlm group(P<0.01)and there was a tendency to be higher when compared with LY255283 group(P<0.05).The DC BLT2 expression was 27.7±2.9,46.3±8.7 and 30.3±5.5 in normal serum group,serum of active JRA group and LY255283 grbup after 1 8h respectively.The expression was stronger in active JRA group than other groups(P<0.05).Conclusion DC can develop a LTB4-BLT2 signal pathway by BLT2 with autocrine and/or extrinsic LTB4.The overexpression of this pathway may be involved in the initiating and activation of JRA.
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Objective To determine whether LTB4 could indirectly stimulate human osteoclast differentiation in RA through increasing RANKL expression of RAFLs. Methods We utilize the coculture model of RAFLs and monocyte which were stimulated in the presence of 2.5 ng/ml M-CSF in the control group, 2.5 ng/ml M-CSF +10-8 mol/L LTB4 in the experimental group A, 2.5 ng/ml M-CSF+10-8 mol/L LTB4+100 ng/ml OPG in the experimental group B. After culture for 3 weeks, through TRAP staining we counted the number of multinucleated TRAP staining positive osteoclast-like cells stained with TRAP to evaluate the differentiation effect in each group. Results There was almost no osteoclast-like cell in the control group and the experimental group B. Whereas there were many osteoclast-like cells in the experimental group A. Conclusion LTB4 can indirectly stimulate human osteoclast differentiation in RA through increasing RANKL expression of RAFLs.
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Specific receptors for leukotriene C4 LTC4 have been identified on intact smooth muscle cells derived from bovine cerebral microvasculatures. Specific pHJLTC4, binding at a fixed input at 25 ℃ was rapid , reaching the maximum at 20min With incremental inputs of radioligand and a constant cell number, specific [3H]LTC4 binding reached a plateau indicative of a saturable binding site. Analysis of Scatchard plots demonstrated a single high affinity binding site with a dissociation constant (Kd) of 2.01?0.4 nmol/L and Bmax = 156.6?13.1 fmol/106 cells. The specific [3H]LTC4 binding could be inhibited by unlabted LTC4, LTD4 and FPL-55712 with an inhibitory rate of 96.9%, 73.9% and 44.9% at 10-5 mol/L, respectively.