Effect Of Ppar Delta Agonist Gw501516 On Cell Apoptosis And Lipid Accumulation Induced By Oxldl In Thp-1 Derived Macrophages

BACKGROUND:PPARδis one of the three isoforms of peroxisome proliferator-activated receptors (PPARs) which is expressed ubiquitously. PPARδcan enhance fatty acid catabolism and energy uncoupling in adipose tissue and muscles, suppress macrophage-induced inflammation, decrease atherosclerosis and is involved in the progression of many diseases. PPARδligands are divided into two kinds, natural ligands and synthetic ligands. GW501516 is a synthetic ligand which belongs to the phenoxy acetic acid derivative. When it binds to PPARδ, PPARδis activated and heterodimers will be formed together with retinoid X receptors (RXRs). The heterodimer binds to peroxisome proliferator response element (PPRE) to modulate transcription and translation of the target genes downstream and regulate biological functions such as controlling weight gain, enhancing physical endurance, and improving insulin sensitivity. We investigated the effect of oxLDL on the PPARδexpression in THP-1 derived macrophage and observed the effect of PPARδagonist GW501516 on macrophage proliferation, apoptosis and foam cells formation.PartⅠ. Effect of oxLDL on PPARδExpression Induced by oxLDL in THP-1 Derived MacrophagesObjective:To identify the effect of oxLDL on PPARδexpression in THP-1-derived macrophages.Method:1. THP-1 derived macrophages were incubated with different concentrations (0 mg/L,25 mg/L,50 mg/L,100 mg/L) of oxLDL for 24 hours. RT-PCR and Western blotting were applied to detect PPAR 8 mRNA and protein expression, respectively.2. THP-1 derived macrophages were treated with 50mg/L oxLDL in different time (Ohour, 12 hours,24 hours h,48 hours). RT-PCR and Western blotting were used to detect PPARδmRNA and protein expression in cellsResults:Different concentrations of oxLDL increased the mRNA and protein expression of PPARδgene in THP-1 derived macrophage. With increasing in the concentration of oxLDL the mRNA and protein expression of PPARδwas gradually increased with statistical significance At the concentration of 50mg/L oxLDL the expression level of PPARδmRNA was peaked (P<0.05). The time-course experiments showed that after 50mg/L oxLDL incubation with THP-1 derived macrophages for 24 hours, the expression levels of PPARδboth mRNA and protein significantluy increased, (P<0.05). In the time duration of 48 hours incubation, the expression of PPARδdecreased, but there was no significant difference compared to that in 24 hours.Conclusions:oxLDL may increase THP-1 derived macrophages PPARδexpression.PartⅡ. Effect of PPARδAgonist GW501516 on Oxidized LDL-induced Cell Proliferation and Apoptosis of THP-1 Derived MacrophagesObjective:To explore the effect of PPARδagonist GW501516 on proliferation and apoptosis induced by oxLDL in THP-1 derived macrophages.Methods:THP-1 monocytes were stimulated with PMA to differentiate into macrophage. Then the cells were incubated with 50mg/L oxLDL for 24 hours, also designed as the positive control group. Other groups were co-incubated 50mg/L of oxLDL and different concentrations of PPARδagonists GW501516 (1,10, 100nmol and 1μmol, respectively) for 24h. MTT assay was used to determine THP-1 derived macrophage proliferation activity. The dye Hoechst33258 staining, Annexin V/PI double staining and flow cytometer were applied to detect cell apoptosis in control group, oxLDL group and lumol GW501516 group of cells, Spectrophotometry determined caspase 3 activity in cells. Results:PPARδagonist GW501516 down-regulated oxLDL inhibition effect on proliferation of THP-1 derived macrophage, and reduced the oxLDL-induced THP-1 derived macrophage apoptosis. MTT and Hoechst33258 staining results showed that 100nmol GW501516 was effective and in 1μmol such effect was more significantly (P <0.05). Flow cytometry analysis results also showed that 1μmol GW501516 significantly inhibited oxLDL-induced THP-1 derived macrophage apoptosis (P<0.05). Spectrophotometry results showed GW501516 reduced the activity of apoptotic executive enzyme, caspase 3, in a concentration-dependent manner (P<0.05).Conclusion:PPAR 8 activation may reduce the activity of caspase 3 and inhibit oxLDL induced apoptosis of THP-1 derived macrophage.PartⅢ. Effect of PPARδAgonist GW501516 on lipid accumulation in THP-1 Derived MacrophagesObjective:To observe the effect of PPARδagonist GW501516 on the lipid accumulation in THP-1 derived macrophage induced by treatment of oxidized LDL.Methods:THP-1 derived macrophages were from monocytes pretreated with PMA. Then, THP-1-derived macrophages were incubated with 50mg/L of oxLDL for 24 hours, that was assigned as the positive control. In other experiments, the cells were co-incubated with 50mg/L of oxLDL and different concentrations of PPARδagonists GW501516 (1,10,100 nmol and 1μmol, respectively) for same time as in control. The harvested cells were stained with oil red to show the lipid droplets under microscope. High performancel iquid chromatography (HPLC) was applied to analyze and evaluate the contents of intracellular cholesterol and cholestryl ester.Results:Oil red O staining showed that PPARδagonist GW501516 increased lipid accumulation in THP-1 derived macrophages. 100nmol of GW501516 significantly increased intracellular lipid droplets both in size and in number (P<0.05). HPLC data demonstrated that the contents of cholesterol and cholesteryl ester in cells were also significantly increased. the data showed in positive group, total cholesterol was 483.10±12.70, cholesterol ester/total cholesterol(%) was 49.60%, and in 1,10,100 nmol and 1μmol GW501516 treatment groups, the total cholesterol levels were 501.53±15.73, 497.69±14.25,647.42±18.62 and 696.55±20.35 respectively; cholesterol ester/total cholesterol(%)respectively,56.7%,53.90%,64.48% and 66.26%. The cholesterol ester/total cholesterol(%) in 100nmol and 1μmol GW501516 group were both significantly increased compared with the positive control group (P<0.05)Conclusion:PPARδagonist GW501516 increases the THP-1 derived macrophage lipid accumulation.Summary(1) oxLDL may up-regulate both mRNA and protein expression in THP-1 derived macrophages.(2) PPARδagonist GW501516 may inhibit oxLDL-induced cell apoptosis of THP-1 derived macrophages, and reduce the activity of caspase 3.(3) PPARδagonist GW501516 may increase the lipid accumulation of THP-1 derived macrophages.