Effects And Mechanisms Of Peroxisome Proliferator-activated Receptor-γ (PPAR-γ) Ligands On Inflammatory Response Induced By Oxidative Stress In Rat Cardiac Myocytes

Objectives Part one:Hydrogen peroxide(H₂O₂) is a useful tool for studying the inflammatory response induced by reactive oxygen species(ROS) in myocardium.The current study aimed at investigating the properties of primary rat cardiac ventricular myocytes using cell biology technology to record their spontaneous pulsation under time-lapse video microscopy,and inspecting the role of H₂O₂ in cultured cardiomyocytes. Part two:On the basis of part one,this part was designed to evaluate the effects of two distinct ligands of PPAR-γin regulating the inflammatory response induced by oxidative stress,and to examine the potential molecular mechanisms of their cardioprotective effects.Methods Part one:(1) Neonatal rat cardiomyocytes were isolated with mechanical dissection and enzyme digestion,then purified by means of differential attachment technique and chemical drug inhibition.Morphological changes of cardiomyocytes were observed dynamically with phase-contrast microscopy.The ratio of survival cells was identified by trypan blue staining.The purity of cardiomyocytes was evaluated by immunofluorescence staining with antibody to cardiacα-sarcomeric actin.MTT method was carried out to assay the cell viability when treated with different concentration of H₂O₂.The rhythmic pulsation of the cardiomyocytes was followed microscopically using a time-lapse recorder 8050 before and at various time points during treatment.To count the beating rate,the culture dishes were maintained on the microscope bench.Part two: On the basis of part one,we investigated the effects of a certain concentration of H₂O₂ (100μmol/L) on expression of the proinflammatory cytokine in cardiac myocytes.The expression of TNF-αand LIX mRNA were detected by RT-PCR;the levels of TNF-αin the supematants were measured with commercially available enzyme-linked immunosorbent assay kit;Wwestern blot analysis was performed to detect the LIX and IκBαprotein in the cytoplasm.To investigate whether two chemically unrelated PPAR-γligands,15d-PGJ₂ and pioglitazone could regulate the expression of TNF-αand LIX,the gel electrophoresis mobility shift assay(EMSA) was used to detect the transcriptor factor NF-κB activity under the challenge of H₂O₂ by cardiomyocytes;Western blot analysis was used to detect the effects of 15d-PGJ₂ and pioglitazone on H₂O₂-induced IκBαdegradation;EMSA was performed to determine the effects of PPAR-γligands on the induction of NF-κB DNA binding activity in response to H₂O₂;the effect of 15d-PGJ₂ and pioglitazone or 100μmol/L H₂O₂ on the DNA binding of HSF1,and the relationship between NF-κB and HSF1 signaling pathway were also determined by EMSA.Results Part one:(1) Primary cultures of neonatal rat cardiac myocytes were prepared from ventricles of Wistar rats.The ratio of survival cells was above 95%. Cardiomyocytes were isolated and purified by means of differential attachment technique. Morphological changes of cardiomyocytes were observed dynamically with phase-contrast microscopy,and the cell shape changed from circles to polygons or fusiform shape after attach along the flask.About 8 hours after plantation,partly cardiomyocytes began to beat spontaneously;after 24 hours all cardiomyocytes had attached and above 90%of them beated.Cardiomyocytes got together and beat synchronously in clusters after 48 hours.The purity of cardiomyocytes was above 95% and cross striation could be observed clearly.(2) Under normal conditions,the beating rate of the cells is no statistical difference at various time points within two weeks.(3) Incubation of cardiomyocytes with various concentrations of H₂O₂ for 48h,MTT analysis showed that there is no decrease in cell viability at concentrations of 10 and 50μmol/L;however,cardiomyocyte viability decreased slightly but not significant at a concentration of 100μmol/L(P＞0.05);200μmol/L H₂O₂ inhibited cell viability significantly(P＜0.05).(4) We recorded the action state of the selected cells using a time-lapse recorder 8050,and analyzed the data.The results indicated that 100μmol/L H₂O₂ has no effect on the beating rate of cell within 48h.After treatment cells with 200μmol/L H₂O₂,the beating rate of cell declined rapidly within 4 hours(P＜0.05),and decreased significantly in a time-dependent manner(P＜0.01)。Part two:(1)The levels of TNF-αand LIX mRNA were induced within 30 minutes after stimulation with 100μmol/L H₂O₂,remained elevated for 4 hours,and decreased thereafter.The NF-κB-blocking peptide SN50 peptide could inhibit H₂O₂-inducd TNF-αand LIX mRNA expression.(2) Stimulation of cells with H₂O₂(100μmol/L) led to an increase in NF-κB binding activity reaching a plateau at 4 hours and at this time,the amount of IκBαprotein was notably reduced.(3) Both 15d-PGJ₂ and pioglitazone markedly decreased H₂O₂-induced TNF-αand LIX(mRNA and protein) expression in a concentration-dependent manner.(4) Myocytes were pretreated either 30 minutes or 3 hours with 5μmol/L 15d-PGJ₂ or 10μmol/L pioglitazone.Neither 15d-PGJ₂ nor pioglitazone could inhibit H₂O₂-induced IκBαdegradation after a 30-min preincubation, while H₂O₂-induced IκBαdegradation was attenuated if cells were preincubated for 3 hours with 15d-PGJ₂ or pioglitazone.(5) PPAR-γligands prevented the induction of NF-κB DNA binding activity in response to H₂O₂ in a dose-dependent manner.However, pioglitazone is less effective than 15d-PGJ₂ at the same concentration(10μmol/L). Treatment with the PPAR-γantagonist GW9662 abolished the inhibitory effect on NF-κB activation afforded by pioglitazone,while it did not affect the inhibitory effect afforded by 15d-PGJ₂.(6) 5μmol/L 15d-PGJ₂ stimulated the time-dependent activation of HSF-1 by cardiomyocytes that was first apparent at 3 hours,whereas pioglitazone or H₂O₂ failed to modify the DNA binding ability of HSF-1.(7) H₂O₂-induced IκBαdegradation was attenuated if cardiomyocytes were preincubated for 3 hours with 15d-PGJ₂,or under conditions in which this PPAR-γagonist activated the DNA-binding activity of HSF1.15d-PGJ₂ failed to inhibit H₂O₂-induced IκBαdegradation after a 30-min preincubation when this PPAR-γagonist failed to activate HSF1.(8) 15d-PGJ₂ activated HSF1,and prevented the induction of NF-κB DNA binding activity in response to H₂O₂ in a dose-dependent manner.Treatment with the PPAR-γantagonist GW9662 failed to abolish the effect of 15d-PGJ₂ on HSF1 and NF-κB binding.Conclusions:(1)Hydrogen peroxide(H₂O₂) as an extracellular source of oxidative stress induced TNF-αand LIX expression via activation of NF-κB in rat cardiac myocytes.(2) Pretreatment of myocytes with PPAR-γligands decreased H₂O₂-induced TNF-αand LIX production(mRNA and protein) in a concentration-dependent manner.(3) 15d-PGJ₂ or pioglitazone inhibits TNF-αand LIX expression through regulation of the NF-κB signaling pathway.(4) The anti-inflammatory effects of 15d-PGJ₂,but not pioglitazone, may be related to the enhance of the heat shock response which,in turn,renders cardiomyocytes unresponsive to stimulation of NF-κB in a PPAR-γ-independent manner.