Objective: To examine whether lipoxin A 4 (LXA 4) induces apoptosis of renal interstitial fibroblasts and explore the mechanisms of signal pathway of LXA 4. Methods: Rat renal inte rstitial fibroblasts (NRK-49F ...Objective: To examine whether lipoxin A 4 (LXA 4) induces apoptosis of renal interstitial fibroblasts and explore the mechanisms of signal pathway of LXA 4. Methods: Rat renal inte rstitial fibroblasts (NRK-49F cells) were exposed to LXA 4 at different concen trations. Prior to the experiment, the cells were transfected with Smac or calpa in 10 antisense oligodeoxynucleotide (ODN), or treated with calcium channel inhi bitor SK&F96365. Apoptosis of cells was recognized by double staining using acri dine orange and ethidium bromide, observed in laser scanning confocal microscope , and counted by a flow cytometer. Caspase-3 activities were measured by colori metric assay. The levels of free cytosolic calcium ( i) were anal yzed in fura-2-loaded cells by laser scanning confocal microscopy. Expression of calpain 10 mRNA was determined by RT-PCR. Expres sions of Smac protein and threonine phosphorylated Akt 1 proteins at 308 site w ere determined by a Western blotting analysis. Activity of signal transducers an d activators of transcription-3 (STAT 3) was determined by electrophoretic mob ility shift assay. Results: LXA 4 at the concentrations of 0.1 and 1 μmol/L induced 9.83% and 33.82% apoptosis of NRK-49F cel ls respectively, reduced at S and G 2-M phase and increased the cells at G 0 -G 1 phase in a dose-dependent manner. Treatment of the cells with LXA 4 inc reased the expressions of calpain 10 and Smac, the levels of i a nd activity of caspase-3. It also down-regulated the DNA-binding activity of STAT 3 and expression of threonine phosphorylated Akt 1. Transfection of the c ells with calpain 10 antisense ODN inhibited the LXA 4-induced apoptosis, acti vity of caspase-3 and expression of calpain 10, and ameliorated the decreased a ctivity of STAT 3. Transfection of the cells with Smac antisense ODN inhibited the LXA 4-induced apoptosis, activity of caspase-3 and expression of Smac. Pr etreatment of the cells with SK & F96365 inhibited the LXA 4-induced apoptosis , levels of i, expression of calpain 10 and Smac. Conclu sion: LXA 4 at high concentration induced apoptosis of rat renal inters titial fibroblasts via i-dependent up-regulation of calpain 10 and Smac expressions.展开更多
基金135 Medical Emphasis grant from Government of Jiangsu Province (2002 45)
文摘Objective: To examine whether lipoxin A 4 (LXA 4) induces apoptosis of renal interstitial fibroblasts and explore the mechanisms of signal pathway of LXA 4. Methods: Rat renal inte rstitial fibroblasts (NRK-49F cells) were exposed to LXA 4 at different concen trations. Prior to the experiment, the cells were transfected with Smac or calpa in 10 antisense oligodeoxynucleotide (ODN), or treated with calcium channel inhi bitor SK&F96365. Apoptosis of cells was recognized by double staining using acri dine orange and ethidium bromide, observed in laser scanning confocal microscope , and counted by a flow cytometer. Caspase-3 activities were measured by colori metric assay. The levels of free cytosolic calcium ( i) were anal yzed in fura-2-loaded cells by laser scanning confocal microscopy. Expression of calpain 10 mRNA was determined by RT-PCR. Expres sions of Smac protein and threonine phosphorylated Akt 1 proteins at 308 site w ere determined by a Western blotting analysis. Activity of signal transducers an d activators of transcription-3 (STAT 3) was determined by electrophoretic mob ility shift assay. Results: LXA 4 at the concentrations of 0.1 and 1 μmol/L induced 9.83% and 33.82% apoptosis of NRK-49F cel ls respectively, reduced at S and G 2-M phase and increased the cells at G 0 -G 1 phase in a dose-dependent manner. Treatment of the cells with LXA 4 inc reased the expressions of calpain 10 and Smac, the levels of i a nd activity of caspase-3. It also down-regulated the DNA-binding activity of STAT 3 and expression of threonine phosphorylated Akt 1. Transfection of the c ells with calpain 10 antisense ODN inhibited the LXA 4-induced apoptosis, acti vity of caspase-3 and expression of calpain 10, and ameliorated the decreased a ctivity of STAT 3. Transfection of the cells with Smac antisense ODN inhibited the LXA 4-induced apoptosis, activity of caspase-3 and expression of Smac. Pr etreatment of the cells with SK & F96365 inhibited the LXA 4-induced apoptosis , levels of i, expression of calpain 10 and Smac. Conclu sion: LXA 4 at high concentration induced apoptosis of rat renal inters titial fibroblasts via i-dependent up-regulation of calpain 10 and Smac expressions.
