This study examined in vitro effect of lipoxin A 4 (LXA 4) on interleukin-1β (IL-1β) production of monocytes and its possible mechanism in severe preeclampsia (PE).Peripheral venous blood was drawn from 15 patients ...This study examined in vitro effect of lipoxin A 4 (LXA 4) on interleukin-1β (IL-1β) production of monocytes and its possible mechanism in severe preeclampsia (PE).Peripheral venous blood was drawn from 15 patients with severe preeclampsia (PE group) and 20 normal pregnant women (control group) to prepare monocytes which were then treated with LXA 4 at different concentrations of 0,10,100 nmol/L respectively.IL-1β level in the supernatant of monocytes was detected by enzyme linked immunoassay.The [Ca 2+ ] i of monocytes was measured by laser scanning confocal microscopy.The results showed that the IL-1β level and the [Ca 2+ ] i of monocytes in the PE group were significantly higher than those in the control group.LXA 4 significantly decreased the generation of IL-1β in a dose-dependent manner in the PE group.After treatment with 100-nmol/L LXA 4,in the PE group,the [Ca 2+ ] i concentration of monocytes was significantly reduced.It was concluded that LXA 4 may inhibit the IL-1β production of monocytes from severe preeclampsia women by inhibiting extracellular calcium influx.展开更多
In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and ...In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and the modulatory effect of lipoxin A4 （ LXA4 ） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4, cultured rat mesangial cells were treated with CTGF, with or without preincubation with LXA4. Expression of mRNA was analyzed by RT-PCR. Protein of RANTES in the supematants was determined by ELISA. Monocyte transmigration was assessed by in vitro chemotaxis assay. Expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （ PI3- K） and protein kinase B （PKB） was assessed by Western blotting. DNA-binding activity of nuclear factor-roB （NF-kB） was determined by electrophoretic mobility shift assay （EMSA）. To observe whether transfection of LXA4 receptor homologue gene （LRHg） into mesangial cells intensified these modulatory effects of LXA4, mesangial cells were transfected with pcDNA3.1/LRHG vector. The results showed that CTGF enhanced the mRNA expression and protein release of RANTES, and the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-kB. P-p42/44 MAPK blockade inhibited the CTgF-induced expression of P-p42/44 MAPK and partially decreased the level of RANTES in supematants. P- PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-kB, and partially decreased the release of RANTES. NF-kB blockade abrogated the CTGF-activated NF-kB and partially decreased the secretion of RANTES. LXA4 dose-dependently inhibited the CTGF-stimulated above action. Transfection of LRHG into mesangial cells intensified these inhibitory effects of LXA4 on CTGFinduced release of RANTES and expression of the P-p42/44 MAPK. In conclusion, LXA4 inhibits CTGFinduced production of RANTES via PI3-K/PKB/NF-kB and p42/44 MAPK-dependent signal pathway, which is mediated by LRHG in rat mesangial cells.展开更多
AIM:Excessive dissolve of corneal tissue induced by MMPs which were activated by cytokins and chemokines will lead to corneal ulcer. The molecular mechanism of Lipoxin A4 (LXA4) on corneal collagen degradation in thre...AIM:Excessive dissolve of corneal tissue induced by MMPs which were activated by cytokins and chemokines will lead to corneal ulcer. The molecular mechanism of Lipoxin A4 (LXA4) on corneal collagen degradation in three dimensions was investigated. ·METHODS:Rabbit corneal fibroblasts were harvested and suspended in serum -free MEM. Type I collagen, DMEM, collagen reconstitution buffer and corneal fibroblast suspension were mixed on ice. The resultant mixture solidified in an incubator, after which test reagents and plasminogen was overlaid and the cultures were returned to the incubator. The supernatants from collagen gel incubations were collected and the amount of hydroxyproline in the hydrolysate was measured. Immunoblot analysis of MMP-1,-3 and TMMP-1,-2 was performed. MMP-2, -9 was detected by the method of Gelatin zymography. Cytotoxicity assay was measured. RESULTS:LXA4 inhibited corneal collagen degradation in a dose and time manner. LXA4 inhibited the IL -1β induced increases in the pro-MMP-1, -2, -3, -9 and active MMP -1,-2,-3,-9 in a concentration dependent manner. LXA4 also inhibited the IL-1β induced increases in TIMP-1, -2. CONCLUSION:As a potent anti-inflammation reagent, LXA4 can inhibit corneal collagen degradation induced by IL-1β in corneal fibroblasts thus inhibiting corneal dissolving pathology process.展开更多
Background： Lipoxin A4 （LXA4） can alleviate lipopolysaccharide （LPS）-induced acute lung injury （ALl） and acute respiratory distress syndrome through promoting epithelial sodium channel （ENaC） expression in lu...Background： Lipoxin A4 （LXA4） can alleviate lipopolysaccharide （LPS）-induced acute lung injury （ALl） and acute respiratory distress syndrome through promoting epithelial sodium channel （ENaC） expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4. Methods： A549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction （qRT-PCR） of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing ofA549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis ofA549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor （ALX） inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase （PI3K） inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression. Results： The A549 cell models of ALl were constrticted and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream- regulated gent- 1 （NDRG 1 ） was validated by real-time-PCR and Western blot. NDRG 1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG 1 expression induced by LXA4. NDRG I siRNA suppressed viability of LPS-treated A549 cells （treatment vs. control, 0.605± 0.063 vs. 0.878 ± 0.083, P = 0.040） and ENaC-α expression （treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008）. LY294002 inhibited NDRG 1 （treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P 0.001 ） and ENaC-α （treatment vs. control, 0.236 ± 0.021 vs. 0.814 ±0.025, P 〈 0.001 ） expressions and serum- and glucocorticoid-inducible kinase I phosphorylation （treatment vs. control, 0.442± 0.024 vs. 1.046 ± 0.082, P = 0.002）, indicating the PI3K signaling pathway was involved in regulating NDRG 1 expression induced by LXA4. Conclusion： Our research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.展开更多
目的探讨脂氧素A4(LXA4)对脂多糖诱导肥胖大鼠感染脓毒症的保护作用及其对肝脏Toll样受体4(TLR4)、肿瘤坏死因子受体相关因子6(TRAF6)表达的影响。方法选取3周龄雄性SpragueDawley大鼠60只随机分为普通组和肥胖组(n=30),建立高脂饮食诱...目的探讨脂氧素A4(LXA4)对脂多糖诱导肥胖大鼠感染脓毒症的保护作用及其对肝脏Toll样受体4(TLR4)、肿瘤坏死因子受体相关因子6(TRAF6)表达的影响。方法选取3周龄雄性SpragueDawley大鼠60只随机分为普通组和肥胖组(n=30),建立高脂饮食诱导的肥胖大鼠模型,将两组大鼠再随机分成对照组、脓毒症组、脂氧素组,每组各取8只大鼠。对照组、脓毒症组、脂氧素组分别予生理盐水、脂多糖、脂氧素A4+脂多糖腹腔注射,观察12 h后心脏采血并采集肝脏组织。ELISA法检测血清白细胞介素6(IL-6)及肿瘤坏死因子-α(TNF-α)水平;蛋白免疫印迹法检测肝脏组织TLR4、TRAF6蛋白水平;实时荧光定量PCR法检测TLR4 m RNA、TRAF6 m RNA的表达水平。结果高脂饮食喂养6周后,肥胖组大鼠的平均体重、Lee's指数明显高于普通组(P<0.05)。与普通组比较,肥胖组血清IL-6、TNF-α水平明显增高(P<0.05);同一饮食组内,脓毒症组较对照组血清IL-6、TNF-α水平明显增高(P<0.05),脂氧素组较脓毒症组血清IL-6、TNF-α水平显著降低(P<0.05)。与普通组比较,肥胖组大鼠肝脏组织中TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6m RNA表达水平上调(P<0.05);同一饮食组内,脓毒症组较对照组TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6 m RNA表达量明显增高(P<0.05),而与脓毒症组比较,脂氧素组TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6 m RNA表达量显著降低(P<0.05)。结论 LXA4能降低血清IL-6、TNF-α水平,减轻炎症反应。LXA4能抑制TLR4、TRAF6在脓毒症大鼠肝脏中的表达,可能是通过抑制TLR4的信号传导途径来实现的。展开更多
文摘This study examined in vitro effect of lipoxin A 4 (LXA 4) on interleukin-1β (IL-1β) production of monocytes and its possible mechanism in severe preeclampsia (PE).Peripheral venous blood was drawn from 15 patients with severe preeclampsia (PE group) and 20 normal pregnant women (control group) to prepare monocytes which were then treated with LXA 4 at different concentrations of 0,10,100 nmol/L respectively.IL-1β level in the supernatant of monocytes was detected by enzyme linked immunoassay.The [Ca 2+ ] i of monocytes was measured by laser scanning confocal microscopy.The results showed that the IL-1β level and the [Ca 2+ ] i of monocytes in the PE group were significantly higher than those in the control group.LXA 4 significantly decreased the generation of IL-1β in a dose-dependent manner in the PE group.After treatment with 100-nmol/L LXA 4,in the PE group,the [Ca 2+ ] i concentration of monocytes was significantly reduced.It was concluded that LXA 4 may inhibit the IL-1β production of monocytes from severe preeclampsia women by inhibiting extracellular calcium influx.
文摘In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and the modulatory effect of lipoxin A4 （ LXA4 ） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4, cultured rat mesangial cells were treated with CTGF, with or without preincubation with LXA4. Expression of mRNA was analyzed by RT-PCR. Protein of RANTES in the supematants was determined by ELISA. Monocyte transmigration was assessed by in vitro chemotaxis assay. Expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （ PI3- K） and protein kinase B （PKB） was assessed by Western blotting. DNA-binding activity of nuclear factor-roB （NF-kB） was determined by electrophoretic mobility shift assay （EMSA）. To observe whether transfection of LXA4 receptor homologue gene （LRHg） into mesangial cells intensified these modulatory effects of LXA4, mesangial cells were transfected with pcDNA3.1/LRHG vector. The results showed that CTGF enhanced the mRNA expression and protein release of RANTES, and the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-kB. P-p42/44 MAPK blockade inhibited the CTgF-induced expression of P-p42/44 MAPK and partially decreased the level of RANTES in supematants. P- PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-kB, and partially decreased the release of RANTES. NF-kB blockade abrogated the CTGF-activated NF-kB and partially decreased the secretion of RANTES. LXA4 dose-dependently inhibited the CTGF-stimulated above action. Transfection of LRHG into mesangial cells intensified these inhibitory effects of LXA4 on CTGFinduced release of RANTES and expression of the P-p42/44 MAPK. In conclusion, LXA4 inhibits CTGFinduced production of RANTES via PI3-K/PKB/NF-kB and p42/44 MAPK-dependent signal pathway, which is mediated by LRHG in rat mesangial cells.
基金Jilin University Basic Scientific Research Operating Expenses Fund, China (Research Fund of the Bethune B Plan of Jilin University, 2012 No.2012230)Research Fund of Jilin Provincial Science and Technology Department, China (international cooperation item, No.20120726)
文摘AIM:Excessive dissolve of corneal tissue induced by MMPs which were activated by cytokins and chemokines will lead to corneal ulcer. The molecular mechanism of Lipoxin A4 (LXA4) on corneal collagen degradation in three dimensions was investigated. ·METHODS:Rabbit corneal fibroblasts were harvested and suspended in serum -free MEM. Type I collagen, DMEM, collagen reconstitution buffer and corneal fibroblast suspension were mixed on ice. The resultant mixture solidified in an incubator, after which test reagents and plasminogen was overlaid and the cultures were returned to the incubator. The supernatants from collagen gel incubations were collected and the amount of hydroxyproline in the hydrolysate was measured. Immunoblot analysis of MMP-1,-3 and TMMP-1,-2 was performed. MMP-2, -9 was detected by the method of Gelatin zymography. Cytotoxicity assay was measured. RESULTS:LXA4 inhibited corneal collagen degradation in a dose and time manner. LXA4 inhibited the IL -1β induced increases in the pro-MMP-1, -2, -3, -9 and active MMP -1,-2,-3,-9 in a concentration dependent manner. LXA4 also inhibited the IL-1β induced increases in TIMP-1, -2. CONCLUSION:As a potent anti-inflammation reagent, LXA4 can inhibit corneal collagen degradation induced by IL-1β in corneal fibroblasts thus inhibiting corneal dissolving pathology process.
文摘Background： Lipoxin A4 （LXA4） can alleviate lipopolysaccharide （LPS）-induced acute lung injury （ALl） and acute respiratory distress syndrome through promoting epithelial sodium channel （ENaC） expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4. Methods： A549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction （qRT-PCR） of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing ofA549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis ofA549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor （ALX） inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase （PI3K） inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression. Results： The A549 cell models of ALl were constrticted and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream- regulated gent- 1 （NDRG 1 ） was validated by real-time-PCR and Western blot. NDRG 1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG 1 expression induced by LXA4. NDRG I siRNA suppressed viability of LPS-treated A549 cells （treatment vs. control, 0.605± 0.063 vs. 0.878 ± 0.083, P = 0.040） and ENaC-α expression （treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008）. LY294002 inhibited NDRG 1 （treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P 0.001 ） and ENaC-α （treatment vs. control, 0.236 ± 0.021 vs. 0.814 ±0.025, P 〈 0.001 ） expressions and serum- and glucocorticoid-inducible kinase I phosphorylation （treatment vs. control, 0.442± 0.024 vs. 1.046 ± 0.082, P = 0.002）, indicating the PI3K signaling pathway was involved in regulating NDRG 1 expression induced by LXA4. Conclusion： Our research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.
文摘目的探讨脂氧素A4(LXA4)对脂多糖诱导肥胖大鼠感染脓毒症的保护作用及其对肝脏Toll样受体4(TLR4)、肿瘤坏死因子受体相关因子6(TRAF6)表达的影响。方法选取3周龄雄性SpragueDawley大鼠60只随机分为普通组和肥胖组(n=30),建立高脂饮食诱导的肥胖大鼠模型,将两组大鼠再随机分成对照组、脓毒症组、脂氧素组,每组各取8只大鼠。对照组、脓毒症组、脂氧素组分别予生理盐水、脂多糖、脂氧素A4+脂多糖腹腔注射,观察12 h后心脏采血并采集肝脏组织。ELISA法检测血清白细胞介素6(IL-6)及肿瘤坏死因子-α(TNF-α)水平;蛋白免疫印迹法检测肝脏组织TLR4、TRAF6蛋白水平;实时荧光定量PCR法检测TLR4 m RNA、TRAF6 m RNA的表达水平。结果高脂饮食喂养6周后,肥胖组大鼠的平均体重、Lee's指数明显高于普通组(P<0.05)。与普通组比较,肥胖组血清IL-6、TNF-α水平明显增高(P<0.05);同一饮食组内,脓毒症组较对照组血清IL-6、TNF-α水平明显增高(P<0.05),脂氧素组较脓毒症组血清IL-6、TNF-α水平显著降低(P<0.05)。与普通组比较,肥胖组大鼠肝脏组织中TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6m RNA表达水平上调(P<0.05);同一饮食组内,脓毒症组较对照组TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6 m RNA表达量明显增高(P<0.05),而与脓毒症组比较,脂氧素组TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6 m RNA表达量显著降低(P<0.05)。结论 LXA4能降低血清IL-6、TNF-α水平,减轻炎症反应。LXA4能抑制TLR4、TRAF6在脓毒症大鼠肝脏中的表达,可能是通过抑制TLR4的信号传导途径来实现的。
