The Role And Mechanism Of Inhibiting PTBP1 In Regulating The M1 To M2 Polarization Of Macrophages

A variety of diseases caused by central nervous system(CNS)injury play an important role in disabling and fatal diseases worldwide.They seriously endanger people’s lives and health and bring great pressure to the society.When the injury occurs,the oxygen and glucose supply in the central area of the injury is insufficient,the function of tissue cells is dysfunctional,and the electrophysiological disorder leads to acute or long-term chronic neuroinflammation.Among them,a large number of inflammatory cell infiltration and cytokine expression constitute the pathological basis of neuroinflammation.If there is no blood supply and damage repair for a long time,the damage and necrosis area of CNS will gradually expand,and eventually cause apoptosis and necrosis of nerve cells,leading to continuous nerve dysfunction.In the acute and subacute stages of CNS injury,there is always an inflammatory reaction characterized by the activation of CNS microglia and blood-derived macrophages at the injury site.Macrophages derived from microglia and blood-derived monocytes belong to macrophages.It is difficult to distinguish the two types of cells functionally,and they both display M1 and M2 phenotypes.Moreover,these two types of macrophages play an important role in the inflammatory response caused by CNS injury by phenotypic transformation between pro-inflammatory M1 and anti-inflammatory M2.In M1 type,it can phagocytize necrotic cells,clear tissue fragments,secrete pro-inflammatory factors,and aggravate further damage.Polarization to M2 type will release many chemokines and anti-inflammatory substances,thus offsetting some early inflammatory cascade reactions,avoiding excessive damage to the body,and promoting wound healing.The time point and function of the two kinds of cells are different after CNS injury.Take the spinal cord injury as an example,microglia were activated as M2 type in the early stage of injury(day 1-3)to make positive reactions such as secretion of inflammatory factors and phagocytic fragments to the injury,but a large number of blood-derived macrophages began to appear in the subacute stage of injury(day 3-14),mainly polarized as M1 type,which has adverse effects on promoting inflammatory reaction and expanding the area of injury.Therefore,"inhibiting M1 type polarization of blood-derived macrophages at the injury site" is one of the important strategies for CNS injury treatment.Among the alternative splicing factors,polypyrimidine tract-binding protein(PTB)PTBP1 is a kind of protein that interacts with inhibitory elements.Many studies have proved that PTBP1 can participate in the regulation of cell differentiation,apoptosis,cell movement,cell metabolism and immune response as a splicing factor,and play a role in various diseases.Some researchers found that PTBP1 can regulate the age-related secretory phenotype of pro-inflammatory and promote the polarization of macrophages to M1 type by up-regulating the relevant inflammatory pathway;In addition,PTBP1,as an important regulator of glycolysis pathway,can promote the expression of pyruvate kinase M2(PKM2),leading to the transformation of cell metabolic mode from oxidative phosphorylation to glycolysis,thus metabolizing and reprogramming M1 type polarization of macrophages.However,the role of PTBP1 in the polarization of macrophages is rarely discussed.In this study,we explored how PTBP1 affects the polarization of macrophages and its potential molecular mechanism.Purpose:Exploring the differential expression changes of PTBP1 between the injured and uninjured CNS tissues,clarifying the effect of PTBP1 on mouse macrophage polarization,and further exploring the molecular mechanism of its anti-inflammatory effect not only helps to gain a deeper understanding of the regulation of PTBP1 in macrophage inflammatory response,but also provides treatment strategies for improving CNS injury related diseases.Methods:(1)LPS treated polarized macrophages into M1 type and determined the effect on PTBP1: After 500ng/m L LPS treatment,the mouse RAW264.7 macrophage cell line was cultured for 24 hours,its polarization phenotype was identified by i NOS,and the expression of PTBP1 and its intracellular distribution were detected by immunofluorescence and Western blot.(2)Determine the siRNA concentration that inhibits the expression of PTBP1: treat RAW264.7 cells with PTBP1 siRNA of different concentrations of 50 n M,75 n M and100 n M,and determine the best siRNA treatment concentration by qRT-PCR and Western blot.(3)To analyze the effect of interference inhibition PTBP1 on the phenotype of M1 type macrophages: the experimental design was divided into four groups:(1)Negative control group(NC);(2)si-m-PTBP1 knockdown group(SI);(3)LPS treatment group(LPS);(4)si-m-PTBP1 knockdown and LPS treatment group(LPS+SI).qRT-PCR and Western blot were used to detect the macrophage polarization markers(i NOS and ARG1)and pro-inflammatory factor(TNFα and IL6),anti-inflammatory factor(IL10 and TGFβ1)expression.(4)Analyze the effect of interference inhibition of PTBP1 on ROS and ATP production of M1 macrophages: use ROS detection kit to detect the change of ROS expression before and after PTBP1 knockdown;ATP detection kit was used to detect the change of ATP expression before and after PTBP1 knockdown.(5)High throughput sequencing analysis of the m RNA expression profile of M1 type macrophages after inhibition of PTBP1: m RNA high throughput sequencing and bioinformatics analysis were performed on LPS+NC and LPS+SI cells,and the results of the differential expression genes of the two groups were analyzed.The role of PTBP1 was analyzed through the analysis results of thermal map,GO and KEGG pathway,and GSEA.(6)To explore the molecular mechanism that PTBP1 knockdown regulates RAW264.7 to polarize into M2 type and play an anti-inflammatory role: qRT-PCR and Western blot were used to verify the changes in the expression levels of PTBP1 targeted binding sites miR-124,p38 MAPK and PKM2 before and after PTBP1 knockdown.(7)Analyze the effect of inhibiting PTBP1 on the protein expression profile of M1 type macrophages : differential protein screening and subcellular localization function analysis were performed on LPS+NC and LPS+SI cells through quantitative proteomic sequencing,and the differential protein interaction network was visualized.Results:(1)After LPS treatment,RAW264.7 was induced into M1 type macrophages.The total expression of PTBP1 was unchanged,but the amount of PTBP1 protein in the nucleus was significantly increased.Compared with the control group,the expression of i NOS in RAW264.7 was significantly increased after LPS treatment at500ng/m L(p<0.001);Western blot showed that the protein expression of total PTBP1 did not change after LPS treatment,but PTBP1 in the nucleus was significantly up-regulated(p<0.05);Cellular immunofluorescence confirmed that the nucleus became larger after LPS treatment and PTBP1 was widely distributed in the nucleus.(2)Inhibition of PTBP1 expression promotes the polarization of M1 type macrophages to M2 type macrophages: the best concentration of si-m-PTBP1 transfection of RAW264.7 is 50 n M;qRT-PCR and Western blot showed that 50 n M siRNA significantly inhibited the expression of PTBP1 in RAW264.7 cells;Compared with other control groups,siRNA inhibits the expression of PTBP1 and down-regulates i NOS and TNF α And IL6 gene and protein expression;Increase ARG1,IL10 and TGFβ1 gene and protein expression;After inhibiting PTBP1,ROS production decreased,PKM2 expression decreased significantly(p<0.05),and ATP production increased significantly(p<0.05).(3)The m RNA expression profile of macrophages that inhibit the expression of PTBP1 was significantly changed,and the MAPK pathway was down-regulated:compared with LPS group,244 genes in LPS+SI group were significantly up-regulated(more than 2 times,p<0.05),and 205 genes were significantly down-regulated(more than2 times,p<0.05);Among them,some pro-inflammatory factors and related inflammatory pathway member genes were significantly down-regulated(such as Ccl5,Cd83 and Tnfrsf12 a,etc.),while genes with anti-inflammatory and neuroprotective effects were significantly up-regulated(such as Cish,Psrc1 and Mx1,etc.).KEGG enrichment、GSEA analysis showed that after PTBP1 knockdown,many differentially expressed genes(such as Map3k13,Map2k1 and Mapk3,etc.)were enriched in the MAPK pathway and down-regulated the expression of the pathway.(4)Interference with PTBP1 inhibits the activation of MAPK pathway by regulating the expression of miR-124 and down-regulating the expression of p38 MAPK protein:after inhibiting the expression of PTBP1 with siRNA,it significantly up-regulates the expression of miR-124 mature body(p<0.01),p38 MAPK is the target gene of miR-124;After interfering with PTBP1,the total p38 protein expression did not change significantly(p>0.05).The protein expression of p-p38 was significantly decreased(p<0.01),which was reversed by the antagonist of miR-124.(5)TNF may be another key protein connecting PTBP1 and MAPK pathway.After PTBP1 knockdown,the expression of TNF protein was significantly down-regulated,and the expression of TAK1 and MSK1/2 in MAPK signal pathway was down-regulated,thereby reducing the inflammatory factor TNF α、CCL5 and CXCL2 secrete and play an anti-inflammatory role.Conclusion:1.By inhibiting the expression of PTBP1,it can promote the polarization of RAW264.7 from M1 to M2,reduce the expression of proinflammatory factors,increase the expression of anti-inflammatory factors,and transform part of the metabolic model from aerobic glycolysis to oxidative phosphorylation.2.Inhibiting PTBP1 expression downregulates the MAPK pathway by regulating the expression of miR-124 and TNF,thereby promoting RAW264.7 polarization towards M2 type and exerting anti-inflammatory protective effects.