The Role Of Fpr And TLR9 In The Production Of Inflammatory Cytokines IL-6 And IL-8 In Vascular Smooth Muscle Cells During Hypoxia

Background and objective:Pulmonary arterial hypertension(PAH)is a common clinical syndrome.It is an important pathological basis for the formation of various types of chronic heart and lung diseases,such as chronic obstructive pulmonary disease,high altitude heart disease and so on.Among them,hypoxia leads to the steady-state destruction of cardiovascular and respiratory functions,and the resulting pulmonary vasoconstriction and pulmonary vascular remodeling are the major factors in the formation of pulmonary hypertension.The inflammatory response plays an important role in hypoxic pulmonary vascular remodeling.IL-6 and IL-8 are important inflammatory factors in the formation of pulmonary vascular remodeling [1].In hypoxia,in addition to inflammatory cells such as macrophages,whether vascular smooth muscle cells(VSMCs)are also important sources of IL-6 and IL-8 has not been reported.In hypoxia,the tissue "oxygen supply" decreases.On the other hand,the pulmonary vascular smooth muscle cells(PVSMC)undergoes intense contraction,requiring mitochondria to produce a large amount of ATP,"oxygen demand" increases,and "balance of oxygen supply and demand." "break in.Therefore,partial mitochondrial dysfunction in PVSMC is eliminated,and some mitochondria are destroyed due to oxidative stress damage caused by breaking the “oxygen supply and demand balance”.Insufficiently functioning mitochondria and damaged mitochondria are autophagy or released outside the cell.The mitochondria contain damageassociated molecular patterns(DAMPs)such as formylated peptides similar to bacteria and DNA containing CpG islands.Formylated peptides and mitochondrial DNA(mtDNA)trigger various cellular events through the formyl peptide receptor and TLR9,respectively.Formyl peptide receptor(FPR)is a G protein-coupled receptor that is mainly expressed on the cell membrane and can be expressed in various cells,such as smooth muscle cells [2].The binding of FPR to its ligand can activate the corresponding signaling pathway,resulting in the release of inflammatory mediators and cell chemotaxis.TLR9(Toll-like Receptor 9)is an innate immune system pattern recognition receptor that recognizes and responds to DAMPs.Activated TLR9 activates inflammation-related signaling pathways and promotes the secretion of cytokines.Are hypoxic vascular smooth muscle cells also an important source of increased IL-6 and IL-8? Whether the activation of FPR and TLR9 by mitochondrial components is an important mechanism for the increase of IL-6 and IL-8 in vascular smooth muscle cells during hypoxia? No report has been reported.Therefore,we have studied this.Purpose:The characteristics and mechanism of inflammatory factors produced by vascular smooth muscle cells during hypoxiaMethod:Rat vascular smooth muscle cells were randomly divided into 0h,12 h,and 24 h groups according to the hypoxia time.Each group of cells was placed in a hypoxic incubator(1% O2,5% CO2,94% N2)and cultured for a corresponding period of time.The protein levels of IL-6 and IL-8 in smooth muscle cells were detected by blotting and mRNA levels were detected by RT-PCR.The mitochondria of vascular smooth muscle cells were extracted and diluted to different concentrations(100%,75%,50%,25%)and applied to vascular smooth muscle cells.mRNA levels of IL-6 and IL-8 were detected by RT-PCR.The mitochondrial DNA in culture supernatant of vascular smooth muscle cells was extracted at different time(0h,12 h,18h,24 h,48h)in hypoxia group,and the expression of mtDNA was detected by RT-PCR in different hypoxia time.After treatment of vascular smooth muscle cells with FPR/TLR9 agonist fMLP/ODN,the protein expression levels of IL-6 and IL-8 were detected by Western blot.Vascular smooth muscle cells were treated with FBO/TLR9 antagonist t BOC/CQ.The levels of IL-6 and IL-8 were detected after hypoxia culture at different time(0h,12 h,24h).The supernatants of 24 h vascular smooth muscle cells cultured in hypoxia were used to treat normoxic cultured cells,and the expression levels of IL-6 and IL-8 were detected.Further,FPR/TLR9 antagonists were used to pretreat normoxic cultured cells in hypoxia.After cultured for 24 hours,the supernatant of vascular smooth muscle cells was used to detect the expression of IL-6 and IL-8 in vascular smooth muscle cells.fMLP/ODN was used to stimulate p38 inhibitor and ERK inhibitor pretreated vascular smooth muscle cells to detect the expression levels of IL-6 and IL-8,so as to observe the dependence of inflammatory factor expression on the p38 pathway after FPR/TLR9 activation..Result:1.Changes of IL-6 and IL-8 in vascular smooth muscle cells under hypoxiaUnder hypoxia,the expression of IL-6 and IL-8 in vascular smooth muscle cells increased with the increase of hypoxic time.Compared with the control group,mRNA and protein levels increased significantly(p<0.05)at 12 h after hypoxia.Protein levels were significantly increased at 24 h oxygen(p<0.05).2.The expression of IL-6 and IL-8 in vascular smooth muscle cells by mitochondriaAfter the mitochondrial components acted on vascular smooth muscle cells,the expression of IL-6 and IL-8 mRNA increased with the increase of mitochondrial concentrations.Compared with the control group(wireless granules),the mitochondrial concentrations were 75% and 100% with significant differences(p<0.05).3.Changes in mtDNA in the hypoxic supernatant of vascular smooth muscle cells.Compared with the normoxic supernatant control group,the mtDNA content in the hypoxic supernatant increased,and when the hypoxia time increased,the COX1-DNA fragment content in the supernatant at 18 h and 24 h was significantly increased,and compared with the control group.The difference was statistically significant(p<0.05).The levels of COX2-DNA and NADH-DNA were significantly increased in hypoxic 24 h and 48 h supernatants,and there was a statistically significant difference compared with the control group(p<0.05).4.The relationship between the activation of FPR and TLR9 and the production of inflammatory factors in vascular smooth muscle cellsCompared with the unstimulated group(0 nM),as the concentration of fMLP increased,the levels of IL-6 and IL-8 protein and mRNA increased gradually,and the fMLP concentration in the 100 nM and 1000 nM groups increased significantly(p<0.05).IL-6 expression was significantly higher in the 10 n M fMLP group than in the 0nM group(p<0.05).Compared with the control group(0?M),the IL-6 protein level was gradually increased with increasing ODN concentration,and it was significantly higher at the ODN concentration of 3?M and 5?M group(p<0.05);the IL-8 level also increased with the ODN concentration.The increase was significant and was significantly higher in the ODN concentration 5 ?M group(p<0.05).5.Relationship between inflammatory factor production and FPR/TLR9 in vascular smooth muscle cells under hypoxiaCompared with tBOC0 nM,in the hypoxic 12 h and 24 h groups,as the t BOC/CQ concentration increased,the mRNA expression levels of IL-6 and IL-8 decreased gradually,and the tBOC100 nM and 1000 nM groups decreased significantly,and the difference was statistically significant.Significance(p<0.05).In addition,as the time of hypoxia increased,the protein expression levels of IL-6 and IL-8 increased,compared with 0h at 12 h,24h,respectively,increased by 40%,45%,and the difference was statistically significant.When tBOC was added,this effect was weakened,and the expression levels of IL-6 and IL-8 at 12 h and 24 h were reduced by 30% and 45% compared with those without tBOC(p<0.05);Compared with CQ,it decreased by 25% and 35%(p<0.05).With the increase of CQ concentration,compared with CQ 0?M,the mRNA expression levels of IL-6 and IL-8 decreased gradually in hypoxic 12 h and 24 h groups,and decreased significantly in CQ concentrations of 5?M and 10?M groups.Statistical significance(p<0.05).Compared with CQ 0?M,the expression levels of IL-6 and IL-8 were significantly decreased after the cells were added with CQ,and were decreased by 25% and 35% compared with those without tBOC(p<0.05).6.Hypoxia FPR/TLR9 agonists activate FPR/TLR9 via the extracellular pathway to induce IL-6 and IL-8 production in smooth muscle cells.The supernatants of the hypoxic cells were treated with the cells pretreated with normoxic cells and FPR antagonists,respectively.Compared with the normoxic group,the expression of inflammatory factor proteins was increased in the A10 cells,and the difference was statistically significant.Significance(p<0.05).Second,the expression of inflammatory factor protein was significantly reduced in the hypoxic group after addition of FPR antagonists compared with the normoxic supernatant group,and the difference was statistically significant(p<0.05).Compared with the normoxic supernatant group,the content of IL-6 and IL-8 in the hypoxic supernatant group increased,and the difference was statistically significant(p<0.05).In the hypoxic groups,the expression levels of IL-6 and IL-8 were significantly decreased in the CQ-treated group compared with the untreated group(p<0.05),ie,after the CQ treatment,the degree of increase was lower.The untreated group decreased significantly.The expression levels of IL-6 and IL-8 in the hypoxic group were higher than those in the normoxic group,but the difference was not statistically significant.7.Activated FPR/TLR9 induces the expression of IL-6 and IL-8 in vascular smooth muscle cells through p38The p38 protein level of phosphorylated A10 cells was significantly lower in the cells treated with p38 inhibitor SB203580 pretreated with fMLP than that of cells added with fMLP alone;the expression levels of IL-6 and IL-8 in A10 cells were significantly lower than those of cells added with fMLP alone.fMLP group(p<0.05).Choosing CpG-ODN 5?M as the optimal concentration of ODN stimulated vascular smooth muscle cells and caused a significant increase in the expression of IL-6 and IL-8 protein compared with the group without Cp G-ODN(p<0.05).Pretreatment with the p38 inhibitor SB203580 significantly down-regulated this effect.Compared with the untreated group,the p38 inhibitor-treated group also showed a significant decrease in the expression of IL-6 and IL-8(p<0.05).In conclusion:FPR and TLR9 mediate the upregulation of IL-6 and IL-8 in vascular smooth muscle cells through hypoxia.The activation of FPR and TLR9 may be related to the release of mitochondrial components from vascular smooth muscle cells.Therefore,inhibiting the activation of FPR/TLR9 or timely regulating the expression of FPR/TLR9,controlling the production of inflammatory factors,and thus reducing pulmonary vascular remodeling may become a new measure to prevent the formation of pulmonary hypertension.