Adipocyte Fatty Acid Binding Protein In Palmitate-induced Macrophage Apoptosis:Role And Mechanisms

Objective:To study the role of adipocyte fatty acid binding protein (A-FABP) in palmitate-induced macrophage apoptosis.Methods:Primary human monocytic leukemia cell line (Thp-1cell) was cultured and differentiated into macrophages after treatment with100nM phorbol(PMA) for72h. Macrophages were treated with different concentrations of palmitate or at various time points, then quantitative Real-Time PCR was used to determine the expression of A-FABP mRNA and Western blot analysis was used to detect the expression of A-FABP protein. Cell apoptosis was examined by ELISA and Western blot. SiRNA targeting the A-FABP mRNA was transfected into macrophages, and the influence of A-FABP siRNA on palmitate-induced apoptosis in macrophages was also observed.Results:Palmitate treatment led to an increased expression of A-FABP mRNA and protein in a time-and dose-dependent manner in macrophages. Macrophages treated with palmitate showed time-and dose-dependent increases in DNA fragmentation examined by Cell Death Detection Elisaplus kit, and the DNA fragmentation could be markedly reduced by A-FABP siRNA. Consistent with the ELISA results, the expression of pro-apoptotic protein Bax was greatly increased in palmitate-treated macrophages compared with control group, whereas the expression of anti-apoptotic protein Bcl-2was reduced in palmitate-treated macrophages compared with control group. The transfection of A-FABP siRNA remarkably reduced the expression of Bax and increased the expression of Bcl-2.Conclusion:Palmitate induces macrophage apoptosis, which was mediated by A-FABP. Objective:①To observe the impact of palmitate on mitochondrial function and mitochondrial apoptosis pathway in macrophages;②To explore the effects of A-FABP on palmitate-induced mitochondrial dysfunction and activation of mitochondrial apoptosis pathway in macrophages.Methods:Cellular total reactive oxygen species (ROS) were detected by fluorescence staining. Mitochondrial transmembrane potential was examined by JC-1staining. ATP content was tested by ATP detection kit. Malondialdehyde (MDA) content was tested by MDA detection kit. Caspase3activity was tested by caspase3activity detection kit. Caspase9activity was examined by caspase9activity detection kit. Succinnate dehydrogenase (SDH) activity was tested by SDH activity detection kit. Mitochondrial respiratory chain complex IV activity was examined by mitochondrial respiratory chain complex IV activity detection kit. The expression of cytochrome c was examined by Western blot.Results:Palmitate caused impaired respiratory chain activity, decreased ATP content, increased production of ROS and enhanced lipid peroxidation, indicating mitochondrial dysfunction in palmitate-treated macrophages. In addition, palmitate also reduced mitochondrial membrane potential, increased the release of mitochondrial cytochrome c from mitochondria into cytosol, enhanced the activity of caspase9and caspase3, indicating the activation of mitochondrial apoptosis pathway in palmitate-treated macrophages. The transfection of A-FABP siRNA decreased the ROS levels, increased mitochondrial membrane potential, reduced the release of cytochrome c, down-regulated the activity of caspase3and caspase9, restored the respiratory chain activity, elevated the ATP level and alleviated lipid peroxidation, indicating A-FABP gene silencing partially reversed mitochondrial dysfunction and apoptosis.Conclusion:A-FABP mediates palmitate-induced mitochondrial dysfunction and activation of mitochondrial apoptosis pathway in macrophages. Objective:①To study the impact of palmitate on mitochondrial biogenesis and antioxidant ability in macrophages;②To explore the effects of A-FABP on palmitate-induced mitochondrial biogenesis and antioxidant ability.Methods:Mitochondrial DNA(mtDNA) copy numbers were examined by Real-Time PCR. The expression of mRNA and proteins involved in mitochondrial biogenesis were examined by quantitative Real-Time PCR and Western blot, respectively. The mRNA expression of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (Cu/ZnSOD) and uncoupling protein2UCP2(UCP2) were examined by quantitative Real-Time PCR. Mito-Tracker Green staining was used to detect mitochondrial numbers.Results:We examined the expression of critical molecules involved in mitochondrial biogenesis and antioxidases. Treatment of palmitate decreased mRNA levels of molecules involved in mitochondrial biogenesis such as peroxisome proliferator activated receptor gama coactivator1alpha (PGC-1α), oestrogen-related receptor a (ESRRA) and nuclear respiratory factor1(NRF1), palmitate treatment also down-regulated the mRNA levels of antioxidase including MnSOD, Cu/ZnSOD and UCP2. Mitochondrial numbers detected with Mito-Tracker Green were decreased in palmitate-treated macrophages compared with untreated cells. However, palmitate had no effect on both mtDNA copies and mRNA level of mitochondrial transcription factor A(TFAM) which was a critical molecular regulating mitochondria DNA replication/translation. The transfection of A-FABP siRNA increased mitochondrial numbers, the mRNA level of these antioxidases and the mRNA level of molecules involved in mitochondrial biogenesis, but had no effect on mtDNA copies and TFAM mRNA level.Conclusion:Gene silencing of A-FABP might reverse the palmitiate-induced mitochondrial dysfunction through alleviating the palmitate-induced damage of mitochondrial biogenesis and antioxidant ability.