Chlorogenic Acid Regulates M1 Macrophage Polarization Via PP2Ac Methylation

Objective and significance: Chlorogenic acid(CGA)is a phenolic compound containing caffeic acid and quinic acid.It has important immunomodulatory functions,including inhibition of inflammation,reduction of oxidative damage of cells and tissues,and anti-liver fibrosis.Chlorogenic acid can inhibit M1 macrophage pro-inflammatory factors by regulating inflammatory signaling pathways such as NF-κB.Macrophages are one of the essential cells that mediate immune response and are involved in initiating inflammation and tissue repair.Glycolysis and oxidative phosphorylation are the pivotal glycometabolism pathways in cells.Glycolysis is the main energy supply pathway of M1 macrophages and participates in the polarization reprogramming of macrophages.Mitophagy is a sticking point to maintain the balance of mitochondrial number and function.studies have shown that glycometabolism and mitophagy are the main driving forces for the polarization of M1 macrophages.Our results show that the polarization of M1 macrophages is regulated by PP2 Ac methylation.At present,The effect and molecular mechanism of chlorogenic acid regulates glycometabolism and mitophagy through PP2 Ac methylation interferes with the polarization of M1 macrophages have not been reported in detail.In this study,THP-1 cells and PBMC were used to construct a polarization model of human M1 macrophages in vitro,through the design specific inhibition PP2 Ac methylation.To study the mechanism of chlorogenic acid regulating glucose metabolism and mitophagy intervening in the polarization of M1 macrophages through PP2 Ac methylation and provide a new scientific theoretical basis for the treatment of chlorogenic acid in the treatment of inflammation-related diseases.Methods:1.Chlorogenic acid is involved in regulating the polarization of M1 macrophages.(1)Polarization and grouping of macrophages.In the M0 macrophage group,THP-1 cells were polarized into M0 macrophages after inducing by 100 n M PMA for 48 h or PBMC was stimulated by 10 ng/ml rh GM-CSF for 7 days to polarize into M0 macrophages;M1macrophages group: M0 macrophages were treated with 10 ng/m L lipopolysaccharide(LPS)and 20 ng/m L interferon-γ(IFN-γ)combined stimulation 48 h to polarize into M1 macrophages;chlorogenic acid group:co-treatment with different concentrations of chlorogenic acid while polarizing M1 type macrophages 48 h.(2)CCK-8 detects the effect of chlorogenic acid on the activity of M1 macrophages.On the basis of polarization of M1 macrophages,25,50,100 and 150μg/m L chlorogenic acid were added at the same time for 48 h.After that,10 μL of CCK-8 was added to each well and incubated at 37℃ for 2 h,The absorbance values of each group were measured at 450 nm by microplate reader.(3)The effect of chlorogenic acid on the polarization of M1 macrophages.The inverted microscope was used to observe the morphological changes of the polarization of M1 macrophages after treating with chlorogenic acid.Real-time fluorescence quantitative PCR was used to detect the m RNA expression levels of M1 polarization-related gene markers interleukin 6(IL-6)and tumor necrosis factor alpha(TNF-α),cyclooxygenase-2(COX-2),CXC chemokine ligand 10(CXCL-10),CD14(bacterial lipopolysaccharide,co-receptor for LPS)and M2 polarization markers mannose Receptor(MRC-1),interleukin 10(IL-10),chemokine 22(CCL-22),and CD16(Ig G low-affinity type III Fc receptor).The effect of chlorogenic acid on the phagocytic function of M1 macrophages was detected by neutral red method.2.The mechanisms of chlorogenic acid regulating the polarization of M1 macrophages.(1)The effect of chlorogenic acid on the glycolysis and oxidized phosphoric acid levels of polarized M1 macrophages.The changes of glycolytic metabolic enzymes hexokinase(HK),pyruvate kinase(PK),lactate dehydrogenase(LDH)and oxidative phosphorylase pyruvate dehydrogenase(PDH)activities of M1 macrophages after chlorogenic acid intervention were detected by microassay,and the levels of intracellular ATP were detected by ATP kit.(2)The effect of chlorogenic acid on mitochondria and mitophagy in M1 macrophage polarization.In the chlorogenic acid groups: the levels of mitochondrial membrane potential and intracellular ROS in M1 macrophages were detected by fluorescence microscope and multifunctional enzyme plate analyzer,and the changes of the number of mitochondria and lysosomes were detected.Laser confocal microscopy was used to observe the degree of fluorescence co-localization of lysosomes and mitochondria in M1 type macrophages.The expressions of Beclin-1,p62,LC3II/I,PINK1,Parkin and VDAC1 were detected by western blot.(3)Chlorogenic acid regulates the polarization of M1 macrophages through PP2 Ac methylation.WB was used to detect the expression of PP2 Ac methylation-related protein in M1 macrophages after the intervention of chlorogenic acid.In the ABL127 combined with chlorogenic acid group,the expression of PP2 Ac methylation-related protein and mitophagy-related protein were detected by western blot,real-time fluorescence quantitative PCR was used to detect the expression of M1 polarization related markers in M1-macrophages.The changes in the activities of glycolytic metabolic enzymes hexokinase(HK),pyruvate kinase(PK),lactate dehydrogenase(LDH)and oxidative phosphorylase pyruvate dehydrogenase(PDH)and the intracellular ATP level of M1 macrophages with ABL127 increased PP2 Ac methylation intervention on chlorogenic acid were detected by microassay.Results: 1.Chlorogenic acid is involved in regulating the polarization of M1 macrophages.(1)Chlorogenic acid protected the activity of M1-type macrophages in a dose-dependent manner at 25 μg /m L-100 μg/m L,and the effect was most significant at 100 μg/m L.(2)Most of the macrophages in the M1 group were gray and opaque,some were omelet-like and irregular in shape,and the m RNA expression levels of related gene markers TNF-α,IL-6,CXCL-10,COX-2 and CD14 were increased,while in the chlorogenic acid intervention group,the number of spindle-like cells was significantly decreased,and the expression of M1-related gene markers was decreased.M2-type polarization related markers MRC-1,IL-10,CD16 m RNA expression levels increased,and macrophage phagocytosis was inhibited in a dose-dependent manner,the difference was statistically significant(P<0.05).2.The mechanisms of chlorogenic acid regulating the polarization of M1 macrophages.(1)Chlorogenic acid interferes with glycolysis and oxidative phosphorylation to regulate polarization of M1 macrophages.The activities of HK,PK and LDH in M1 macrophages were increased,while the activities of PDH and ATP were decreased.On the contrary,those activities were decreased in the chlorogenic acid treatment group,with statistical significance(P<0.05).(2)The Effect of chlorogenic acid on mitochondria and mitochondrial autophagy in polarization of M1 macrophages.The number of mitochondria,mitochondrial membrane potential,ROS and lysosome of macrophages in the M1 model group decreased.the fluorescence co-localization of lysosome and mitochondria also decreased The expressions of Beclin-1,LC3 II/LC3 I,PINK1 and VDAC1 were increased,while the expressions of Parkin and P62 were decreased,but those evident changes on the above were reversed in the 2-DG treatment group.The difference was statistically significant(P<0.05).(3)Chlorogenic acid regulates the polarization of M1 macrophages through PP2 Ac methylation.Compared with the M1 group,there was no significant difference in the total PP2 Ac protein expression level in the chlorogenic acid intervention group,while PP2 Ac demethylation and PPME-1 expression increased,PP2 Ac methylation and LCMT1 expression decreased in a dose-dependent manner,with statistical significance(P<0.05).ABL127 increased PP2 Ac methylation,decreased PP2 Ac demethylation,and promoted the expression of M1polarization-related genes intervened by chlorogenic acid.the activities of HK,PK,LDH increase and PDH activity and ATP level decreased at the same time,the difference was statistically significant(P<0.05).The expression of mitochondrial autophagy related protein P62 was decreased,LC3 II/LC3 I,PINK1 and VDAC1 were increased in a dose-dependent manner,and the difference was statistically significant(P<0.05).Conclusions: 1.CGA inhibited the m RNA expression of TNF-α,CXCL-10,COX-2,IL-6 and CD14 in M1 macrophages,decreased the phagocytic function of cells,and increased The expression of M2 polarization-related gene markers MRC-1,IL-10,and CD16 m RNA induces the transformation of M1 macrophages to the M2 phenotype.2.CGA reduces the levels of ROS,and reduces mitophagy of the PINK-VDAC1 pathway through PP2 Ac methylation.Mitophagy regulates the glycolysis and oxidative phosphorylation of macrophages,and then regulates the polarization level of M1 macrophages.