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Objective To investigate the protective effects of lipoxin A4 (LXA4) on the lung epithelial cells (MLE-12) in mice with hyperoxia injury.Methods MLE-12 cells were cultured in vitro and divided into air group,air + LXA4 group,hyperoxia group and hyperoxia + LXA4 group.The receptor of LXA4 (ALX) was verified by using reverse transcription-polymerase chain reaction (RT-PCR).MLE-12 cells were exposed to hyperoxia (＞ 850 mL/L oxygen concentration) for 12 h followed by pretreatment of 1 nmol/L,10 nmol/L and 50 nmol/L LXA4 for 1 h,6 h,12 h and 24 h.Quantitative real-time PCR (qRT-PCR) was applied to analyze the heme oxygenase 1 (HO-1) expression to determine the optimal concentration and the optimal pretreatment time of LXA4.The cell morphology was observed by using inverted microscope.The survival rates and cell viability were determined by using Trypan Blue stain and cell counting kit-8 (CCK-8).The superoxide dismutase (SOD) level was determined by using hydroxylamine method.The expressions of mRNA and protein of HO-1 were measured by using qRT-PCR,western blot and immunofluorescence assay,respectively.The interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined by using enzyme-linked immunosorbent assay.Results ALX was expressed in MLE-12 cells.The optimal intervention concentration and time of LXA4 was 10 nmol/L for 12 h.Compared with air group [(84 ± 5) ％,1.22 ± 0.27,(5.33 ± 1.16) kU/L],the cell survival rate,viability and SOD level of hyperoxia group [(66 ± 8) ％,0.67 ± 0.21,(2.38 ± 0.65) kU/L] decreased,and the differences were significant (t =3.98,2.55,4.86;P =0.01,0.03,0.00);compared with the hyperoxia group,the cell survival rate,viability and SOD level of hyperoxia + LXA4 group [(88 ± 5) ％,1.43 ± 0.05,(6.50 ± 0.19) kU/L] significantly increased,and the differences were significant (t =4.83,3.52,6.78;P =0.01,0.02,0.00).The HO-1 mRNA and protein expression of hyperoxia group (0.57 ± 0.03,1.31 ± 0.11) increased as compared to air group (0.13 ± 0.03,0.24 ± 0.10),and the differences were significant (t =8.00,10.10;all P =0.00);the HO-1 expression of hyperoxia + LXA4 group (0.78 ± 0.08,1.82 ± 0.09) significantly increased as compared to hyperoxia group,and the differences were significant (t =3.94,8.82,all P=0.00).The levels of MCP-1 and IL-6 of hypemxia group [(1 025.18 ±35.51) rig/L,(1 136.65 ±160.01) ng/L] significantly increased as compared to air group [(467.63 ± 13.69) ng/L,(470.03 ± 118.22) ng/L],and the differences were significant (t =16.51,7.48;all P =0.00);the MCP-1 and IL-6 of hyperoxia + LXA4 group [(640.25 ± 61.03) ng/L,(655.48 ± 88.57) ng/L] significantly decreased as compared to hyperoxia group,and thedifferences were significant (t =11.40,5.40,all P =0.00).Conclusions LXA4 can attenuate hyperoxia-induced injury in MLE-12 cells.The protective role of LXA4 in the hyperoxia-induced cell injury is related to the up-regulation of HO-1 expression and down-regulation of IL-6 and MCP-1 levels.
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Objective To study the protective mechanisms of lipoxin A 4 ( LXA4 ) for hyperoxia-induced lung injury through modulation of let-7c/TGF-β1 signal pathway in mice.Method MLE-12 cells was transfected with let-7c mimic, mimic negative control ( NC) , let-7c inhibitor and inhibitor NC.The cells were assigned into hyperoxia group , LXA4 group, let-7c over-expression group, let-7c silence group, let-7c silence+LXA4 group, and all exposed to 85% oxygen.The mRNA level of the extracellular matrixα-smooth muscle actin (α-SMA) and collagen Ⅰ( COL-Ⅰ) , and the expression of related genes in TGF-β1 signaling pathway (Smad 2, Smad 3, Smad 4, TGF-βR1, TGF-βR2) were examined using qPCR.The protein expressions in TGF-β1 signaling pathway was examined using Western blot .Result The mRNA expressions of α-SMA, COL-Ⅰ, Smad 3, Smad 4, TGF-βR1 and TGF-βR2 in LXA4 group [(24.3 ±2.1), (14.6 ±0.2), (17.0 ±0.0), (14.9 ±0.1), (20.8 ±0.1), (9.0 ±0.0) ] and let-7c over-expression group [ ( 12.2 ±0.5 ) , ( 3.0 ±0.0 ) , ( 3.1 ±0.0 ) , ( 9.6 ±0.4 ) , ( 28.5 ±0.2 ) , ( 7.6 ± 0.1)] were decreased comparing with the hyperoxia group [(51.4 ±0.5), (32.0 ±0.1), (40.6 ±0.2), (16.3 ±0.1), (89.1 ±1.1), (19.3 ±0.2)].These expressions were increased in both let-7c silence group [(87.3 ±7.0), (38.5 ±0.3), (48.0 ±0.2), (56.5 ±0.2), (126.0 ±0.9), (33.1 ±1.0)] and let-7c silence +LXA4 group [(144.5 ±12.9), (86.3 ±3.0), (91.5 ±4.7), (86.5 ±3.3), (109.0 ±4.5), (45.6 ±1.6)].The protein levels of Smad 2, Smad 3, Smad 4, p-Smad 2, p-Smad 3 and TGF-βR1 of LXA4 group and let-7c over-expression group were decreased comparing with the hyperoxia group, while p-Smad 2, p-Smad 3 of let-7c silence+LXA4 group were increased(P<0.05).Conclusion LXA4 may play a protective role through let-7c /TGF-β1 signal pathway of lung epithelial cells for hyperoxia-induced lung injury in mice .
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Objective To identify the important regulatory elements in the promoter of human CD2 associated protein(CD2AP) by conserved sequence analysis among different species and luciferase functional detection. Methods The promoter sequences of CD2AP from different species were analyzed by BLAST. Plasmids containing different length of deletion mutations of human CD2AP promoter were constructed. Pro-moter activities were tested in 3 kinds of cells from different species by luciferase analysis and were tested in HEK-293 cells treated with all-trans-retinoic acid. Results Homologous sequence comparison in CD2AP promoter area among human, cattle and pig showed that putative specific protein 1 (Sp1) sites and down-stream promoter element (DPE) were highly evolutional]y conserved. Progressive deletion luciferase analysis of DNA fragments revealed similar promoter activity style among 3 different cell lines from 3 different spe-cies, HEK-293, BHK-21 and Vero cells. One basic promoter activity located within 500 bp upstream of ATG. Fragments of further upstream 100 bp or more had drastically 10 times increased promoter activity. Two putative Sp1 sites were in this 100 bp region. All-trans-retinoic acid decreased the luciferase activity of CD2AP promoter. Conclusion Putative Spl sites and DPE have important functions in the promoter activity of CD2AP.
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ObjectiveTo investigate the change of lipoxin A4 (LXA4), leuotriene B4(LTB4) in blood and urine and leukocyte 15-lipoxygenase (15-LO) of the children with acute poststreptococcal glomendonephritis (APSGN) and to evaluate its significance. MethodsBlood and urinary levels of LXA4 and LTB4 were measured with ELISA within 3 days (acute phase), 10 to 14 days (early resolution phase) and 6 to 8 weeks (late resolution phase) respectively after onset of APSGN in 22 patients. In 8 children with APSGN, expression level of leukocyte 15-LO mRNA was examined with RT-PCR. Leukocyte LTB4 synthesis was assessed with ELISA. Chemotactic effect of LTB4, LXA4 and 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) on neutrophils was determined by in vitro chemotaxis assay. Twenty-two healthy children were served as control. ResultsBlood and urinary levels of LXA4 and leukocyte 15-LO mRNA were up-regnlated in acute phase, further increased in early resolution phase, and decreased in late resolution phase of APSGN, which were stir higher than those in the controls (P＜0.01). Blood and urinary levels of LTB4 were increased in acute phase (P＜0.01) and then were decreased in early resolution phase and hte resolution phase of APSGN, which were still higher than those in the controls (P＜0.01). Administration of 15-S-HETE or LXA4 in vitro inhibited LTB4-induced chemotactic effect on neutrophils of the patients,and inhibited the production of leukocyte LTB4. ConclusionsChanges of blood and urinary levels of LXA4 and LTB4 in early resolution phase of APSGN are contrary. 15-S-HETE and LXA4may play a role in anti-inflammation and resolution of APSGN via inhibiting LTB4.
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Objective To investigate the role of vascular endothelial growth factor-C (VEGF-C) and its receptors in the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. Methods By the domestic and overseas literatures review,the expressions of VEGF-C and its receptors in gastric cancer,their role in tumor lymphatic metastasis and prospect in treatment of gastric cancer were summarized. Results There was a significant correlation between VEGF-C and its receptors and the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. VEGF-C high expression might be an early event in lymphatic metastasis and could be considered as an independent predictive factor of lymphaticmicrometastasis. By inhibition of gastric cancer cell from secrete VEGF-C or blockage of the interaction of VEGF-C with VEGFR-3,it was possible to inhibit tumor angiogenesis and the invasion and distant spread of cancer cells,thereby decreased mortality and improve survival. Conclusion VEGF-C and its receptors may promote the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. It may be an effective way to gastric cancer for the treatments against VEGF-C and its receptors.
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Objective To examine whether lipoxin A4(LXA4) has an antagonistic effect on interleukin (IL)-1?-induced synthesis of IL-6 in glomerular mesangial cells, and to explore its mechanism. Methods Cultured glomerular mesangial cells (GMCs) of rat were treated with IL-1?, with or without preincubation with LXA4. Protein secretion of IL-6 in supernatants was examined analyzed by enzyme-linked immunosorbent assay (ELISA). Expression of IL-6 mRNA was determined by RT-PCR. The expression of Src homology 2 (SH2) containing protein-tyrosine phosphatase 2 (SHP-2) was assessed by immunoblotting. Activities of DNA-binding of nuclear factor-kappa B (NF-?B) were measured by electrophoretic mobility shift assay (EMSA). Results The secretion of protein and expression of mRNA of IL-6 in GMCs stimulated by IL-1? were inhibited by LXA4 in a dose-dependent manner. LXA4 reduced the phosphorylation of SHP-2 and activities of NF-?B. Pretreatmnet of GMCs with NF-?B inhibitor pyrrolidine dithio-carbamate (PDTC) blocked both the secretion of IL-6 protein and activation of NF-?B induced by IL-13- Conclusion LXA4 antagonists IL-1?-induced synthesis of IL-6 in GMCs through the pathway of SHP-2/NF-?B signal transduction.
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Objective To explore the inhibitory effect of mutant influenza A viruses to the activation of interferon regulatory factor 3 (IRF-3). Methods HEK293 cells were infected with A/FM/1/47,A/HK/1/68, A/HK/1/68-MA20, A/HK/1/68-MA20C and positive control Sendai virus (SV). Whether the slowly moved phosphorylation form Ⅲ and Ⅳ of IRF-3 appeared or not was compared by Western blot in cells infected with these viruses. Wild type of NS1 from A/HK/1/68 and mutant NS1 from A/HK/1/68-MA20 were subcloned into pcDNA3.1-flag respectively. They were transfected in HEK 293 cells respectively. At 16 hours posttransfection, cells were infected with Sendai virus for 8 hours. Whole cell extracts were analyzed by Western blot and then probed with monoclonal flag antibody to check the expression of NS1, or with anti-IRF-3 to observe the inhibitory effects of the wild and mutant NS1 to the activated IRF-3. Luciferase assay was carried out by co-transfection with reporter plasmid, pGL2B with interferon ? promoter, and wild or mutant NS1 cDNA expression plasmid. SV was used to infect these cells after the co-transfection. Results Only less virulent A/HK/1/68-MA20 and positive control SV can activate IRF-3. Activated form Ⅲ and Ⅳ of IRF-3 began to appear 9 hours post infection (h.p.i), and most significant activated IRF-3 appeared 23 and 26 h.p.i. Sequence analysis of NS1 of MA20 revealed that nucleotide position number 94 is mutated from T to C, and amino acid at position number 23 is changed from valine to alanine. Co-transfected with wild type NS1 made form Ⅲ and Ⅳ of IRF-3 almost disappear, but not mutant NS1. In the luciferase functional analysis, wild type NS1 can inhibit the luciferase activity of IFN-? promoter, which was induced by SV, to around 1/10. Again no inhibitory effects was observed of mutant NS1 in the luciferase assay. Conclusion The mechanism that A/HK/1/68-MA20 can activate IRF-3 is that point mutant NS1 abolished the inhibitory function of NS1.
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Objective Interferon regulatory factors 3 (IRF-3) is a key transcription factor to regulate gene expression of interferon after virus infection. This study aims to look for new spliced isoforms of IRF-3 and to investigate their structures and functions. Methods RNA extracts from human embryonic kidney 293 cells were amplified by RACE and RT-PCR. New sequences were compared with published sequences of IRF-3 and murine EST database using bioinformatics method. A new sequence, IRF-3c, was subcloned into pcDNA3.1-flag. The IRF-3c/pcDNA3.1-flag plasmid was transfected in HEK 293 cells. Whole cell extract was analysed by Western blot and then probed with monoclonal Flag antibody. Luciferase assay was carried out by co-transfection with reporter plasmid, pGL2B with interferon ? promoter, and IRF-3c cDNA expression plasmid. At 16 hours posttransfection, cells were infected with Sendai virus for 8 hours. Cells were collected and assayed for luciferase activity. Results A novel spliced isoform of IRF-3, named IRF-3c was discovered. The new isoform is almost the same as IRF-3, except for the utilization of the 180 bp bases in intron 6 adjacent to exon 6. The first 2,3 and 4 bases are a stop codon, which may produce a protein with a truncated C-terminal stoped at amino acids 327. Western blot analysis confirmed an expected 44 kDa strong band. The new inserted bases can be found in murine EST database, suggesting a conservative function in evolution. The functional luciferase assay showed that IRF-3c inhibited the IFN? promoter activity to (around) 40%～50% as that of control after Sendai virus infection. Conclusions The discovery of a new isoform of IRF-3 provides a new insight into the functional regulation of IRF-3 family. It is a dominant-negative inhibitor for interferon ? promoter activity in the virus infection pathway, provides a mechanism for the fine-tuning of the virus-induced activation of the interferon response, and prevents interferon ? from its overexpression and its toxic effects. It is worthwhile to explore the role of IRF-3c in the pathogenesis of human diseases using IRF-3c’s specific sequence.
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Objective To examine whether lipoxin A4(LXA4) induces apoptosis of rat renal interstitial fibroblasts and explore the mechanism concerned.Methods Rat renal interstitial fibroblasts (NRK 49F cells)were incubated in RPMI 1640 medium supplemented with 5%fetal calf serum and exposed to LXA4 at the concentration of 10 nmol/L, 100 nmol/L or 1 ?mol/L for 24 hours. Prior to experiment,some NRK 49F cells were transfected with Smac antisense oligodeoxynucleotide. Apoptosis of NRK 49F cells was recognized by double staining using fluorescent dye acridine orange and ethidium bromide,and observed under laser scanning confocal microscopy and counted by flow cytometry following propidium iodide and annexin staining. Activity of caspase 3 was measured by colorimetric assay. The expression of Smac was determined by Western blotting analysis.Results LXA4 at the concentration of 100 nmol/L or 1 ?mol/L induced apoptosis of 9 83%or 33 82%of NRK 49F cells respectively, and reduced the cells of S and G2～M phase and increased the cells of G0～G1 phase in a dose dependent manner. Treatment of NRK 49F cells with LXA4 up regulated the expression of Smac protein and increased the activity of caspase 3. The transfection with Smac antisense oligodeoxynucleotide inhibited the LXA4 induced apoptosis and expression of Smac in NRK 49F cells. Conclusion LXA4 at high concentration can induce apoptosis of rat renal interstitial fibroblasts via the up regulation of Smac expression.
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AIM: To find whether lipoxin A_4 (LXA_4) inhibits cell proliferation induced by TNF-? in rat mesangial cells, and to explore the molecular mechanisms of signal pathways of LXA_4 actions. METHODS: Cultured rat mesangial cells were growth-arrested and exposed to TNF-? with or without preincubation with LXA_4. Proliferation of mesangial cells was measured by MTT methods. Activities of STAT_3 were analyzed by electrophoretic mobility shift assay. Expression of cyclin E mRNA was assessed by RT-PCR. Cyclin E proteins were determined by Western blotting analysis. RESULTS: TNF-?-induced proliferation and increased mRNA and protein expression of cyclin E in mesangial cells were inhibited by LXA_4 in a dose-dependant manner. TNF-?-stimulation of the STAT_3-binding activities in mesangial cells was down-regulated by lipoxin A_4. CONCLUSION: Inhibitory effect of LXA_4 on TNF-?-induced mesangial cell proliferation is mediated by Jak_1/STAT_3 signal pathway.
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AIM: To examine whether lipoxin A_4 (LXA_4) induces apoptosis of rat renal interstitial fibroblasts and explore the mechanisms. METHODS: Rat renal interstitial fibroblasts (NRK-49F cells) were incubated in RPMI-1640 medium supplemented with 5% fetal calf serum and exposed to LXA_4 at the concentration of 10 nmol/L, 100 nmol/L or 1 ?mol/L for 24 hours. Prior to experiment, some cells were transfected with calpain 10 antisense oligodeoxynucleotide. Apoptosis of cells was recognized by double staining using fluorescent dye acridine orange and ethidium bromide, observed under laser scanning confocal microscopy and counted by flow cytometry following propidium iodide and annexin staining. Activity of caspase-3 was measured by colorimetric assay. The expression of calpain 10 mRNA was determined by RT-PCR. RESULTS: LXA_4 at the concentration of 100 nmol/L or 1 ?mol/L induced 9.83% or 33.82% apoptosis of cells, respectively. Treatment of cells with LXA_4 up-regulated the expression of calpain 10 mRNA and increased the activity of caspase-3. The transfection of the cells with calpain 10 antisense oligodeoxynucleotide inhibited the LXA_4-induced apoptosis, activity of caspase-3 and expression of calpain 10. CONCLUSION: LXA_4 at high concentration induceds apoptosis in rat renal interstitial fibroblasts via up-regulating of calpain 10 mRNA expression.