Intervention Effect And Mechanism Of Quercetin Regulating Macrophage Polarization On Tracheal Stenosis After Tracheal Injury

Tracheal intubation is an important means to rescue critical patients and airway management.While saving the lives of a large number of patients,it may lead to the occurrence of tracheal stenosis.The incidence of tracheal stenosis in tracheal intubation or tracheotomy is high.In patients treated with mechanical ventilation,tracheal stenosis often occurs,which has become a common disease in respiratory department.Tracheal stenosis will lead to patients with ventilation difficulties,and even directly endanger the safety of patients’lives,although after repeated treatment,the effect is still poor,and it is a difficult problem of respiratory interventional therapy.The current treatment of tracheal stenosis after intubation and the main methods of benign tracheal stenosis with respiratory medicine intervention such as thermal ablation,balloon expansion,freezing treatment and carotid stenting,and surgical excision narrow section of trachea and end to end anastomosis,but both medical interventional therapy and surgery,were secondary damage,still can cause postoperative tracheal stenosis.Therefore,interventional therapy for the treatment of tracheal stenosis of immediate effect,but the intervention as a secondary injury,postoperative restenosis has become a thorny problem.For the treatment of tracheal stenosis,take effective measures to prevent tracheal injury aggravated and pathological repair,to prevention and treatment of tracheal stenosis is needed in the early stages of tracheal injury.So,research and reveal the pathogenesis of tracheal stenosis after injury and optimize treatment became popular.Inflammation is an important reason for the occurrence and development of tracheal stenosis after injury,while macrophages,as both inherent immune cells and important inflammatory cells,can regulate the activation of fibroblasts and the synthesis of collagen,playing an important role in post-injury repair,and may be the key cells in post-injury repair of tracheal.Macrophages have different phenotypes and their functions in injury repair are also different.Therefore,this study will take the M1/M2 macrophages polarization as the breakthrough point of the research.First,we detected the macrophages expression in patients with stenosis after endotracheal intubation.At the same time,detect the expression of M1/M2and the relationship of airway inflammation in tracheal stenosis rabbit model tracheal mucosa cells.After the intervention of quercetin,detect the changes of tracheal stenosis degree,inflammatory factors and cytokines,and their relationships.Finally,study whether quercetin and other drugs can affect the inflammatory response of the trachea by regulating the expression of macrophage M1/M2,so as to reduce the stenosis of the trachea after injury,and the possible mechanism of regulating the polarization of macrophage M1/M2,to explore the possibility of the precise targeted treatment of traumatic tracheal stenosis.Part IThe expression of macrophage M1/M2 polarization in patients with stenosis after intubation and its relationship with airway inflammationObjective:To investigate the role of inflammation and immune imbalance in tracheal stenosis after injury,especially to study the expression of M1/M2polarization of macrophages in tracheal stenosis,and the effects of inflammatory factors related to the expression level M1/M2 polarization of macrophages.Methods:(1)The experimental group was divided into three groups according to the following,with 10-20 cases in each group:(1)Normal control group;(2)Stenosis group after tracheal injury;(3)group recovered after interventional treatment.(2)Specimen collection:venous blood was collected preoperatively,and tracheal granulation tissue from patients with tracheal stenosis and tracheal stenosis after interventional therapy was collected intraoperatively.(3)Total RNA was extracted from the tracheal granulation tissues of each group,and the m RNA expressions of HDAC2,i NOS,CD206,CD163,Arg1 and MMP9 in the tracheal granulation tissues were detected by real-time PCR.Total RNA was extracted from venous blood mononuclear cells,and m RNA expressions of CD14,CD11b,i NOS,CD206 and CD163 in peripheral blood were detected by Real-time PCR.Serum was extracted and ELISA was used to detect the protein concentrations of?-interferon and IL-4 in serum of each group.Results:(1)Compared with control group,the expression of HDAC2m RNA and CD163 m RNA in tracheal granulation tissue of patients with tracheal stenosis was down-regulated(P<0.05);Compared with the control group,the expressions of i NOS m RNA,CD206 m RNA,Arg1 m RNA and MMP9 m RNA in tracheal granulation tissue of patients with tracheal stenosis were up-regulated(P<0.05).(2)Compared with control group,the expression of CD14 m RNA was up-regulated in mononuclear cells extracted from blood of tracheal stenosis group(P>0.05);Compared with the stenosis group,CD14m RNA was up-regulated(P<0.05).Compared with the control group,CD11b m RNA expression was down-regulated in the tracheal stenosis group(P<0.05);CD11b m RNA expression was up-regulated in the cured group compared with the stenosis group after intervention(P<0.05).Compared with the control group,the m RNA expression of i NOS was down-regulated in the tracheal stenosis group(P>0.05);Compared with the stenosis group,the m RNA expression of i NOS was up-regulated in the cured group after intervention(P<0.05).Compared with the control group,the expression of CD163 m RNA and CD206m RNA were up-regulated in tracheal stenosis group after tracheal injury.Compared with the stenosis group,the expression of CD206 m RNA was down-regulated in the cured group after intervention(P<0.05),the expression of CD163 m RNA was down-regulated(P>0.05).(3)Serum concentrations of IL-4and?-IFN decreased in patients with tracheal stenosis,compared with the control group(P<0.05).Conclusion:In patients with tracheal stenosis after injury,the expression of M1 macrophages and M2 macrophages in the local granulation tissue of the trachea is up-regulated,and M1/M2 polarization is closely related to traumatic tracheal stenosis.Part IIThe animal experiment study of promoting phenotypic transformation of macrophages and improving tracheal stenosis after injuryObjective:To study whether quercetin can prevent and treat tracheal stenosis by promoting M1/M2 polarization of macrophages.Methods:(1)A rabbit model of tracheal stenosis after tracheal injury was established by mechanical injury method.The rabbits were randomly divided into 5 groups with 12 rabbits in each group.(1)The blank control group,(2)the mechanical injury group,(3)the mechanical injury+quercetin group,(4)the mechanical injury+budesonide group,(5)the mechanical injury+quercetin+budesonide group,were hereinafter referred to as group C,group M,group B,group Q and group QB.The blank control group was normal rabbits without modeling,and the other groups were given drug intervention for 10 days after modeling.Three rabbits in the M group were sacrificed 1,4,7 and 10 days after surgery,respectively,and venous blood was extracted.Rabbits in the other groups were sacrificed 10 days after surgery,and tracheal specimens of the stenosis segment were removed for subsequent experiments.(2)Measure the degree of tracheal stenosis:(1)cut the stenosis section of the trachea,measure the area of the tracheal cartilage and the area of the airway cavity,calculate the degree of tracheal stenosis(S)as a measure of the degree of tracheal stenosis of the macro index.(2)H&E staining was used to observe the structure of tracheal tissue in each group,and the total thickness of mucosal layer and submucosal layer of tracheal tissue was measured under microscope.(3)Masson staining was used to observe the distribution of collagen fibers in the tracheal epithelium and lamina propria.(3)The m RNA relative expressions of i NOS,CD206,and CD163 in blood mononuclear cells were detected by real-time PCR,and the m RNA relative expressions of i NOS,CD206,CD163,and Arg1 in tracheal granulation tissues were measured by real-time PCR.(4)The expression of CD206 and IL-4 in tracheal tissue were detected by immunohistochemistry.(5)The expressions of a-SMA,M1 macrophages(CD11b~+i NOS~+)and M2macrophages(CD11b~+CD206~+)in tracheal tissues were detected by immunofluorescence.Results:(1)Compared with control group C,the degree of tracheal stenosis in model group M was increased(P<0.05);Compared with group M,the degree of tracheal stenosis in group B,group Q and group QB was reduced,and the decrease in group QB was the most obvious,compared with group Q and group B(P(29)0.05),compared with group M(P<0.05).(2)Compared with group C,the total thickness of mucosa and submucosa increased in group M;Compared with group M,the total thickness of group B,Q and QB decreased,and the decrease of group QB was the most obvious(P<0.05).(3)The results of H&E and Masson staining showed that the tracheal cavity was unobstructed and the mucosal structure was clear and complete in the control group.In the model group,tracheal lumen stenosis was obvious,with a large number of disorderly arranged fibroblasts,deposition of a large number of collagen fibers,and increased neovascularization.Compared with the model group,the epithelial structure of quercetin group and budesonide group was more complete,the structure of tracheal tissue was more distinct,and the fibroblasts and collagen fibers were reduced.In the quercetin combined with budesonide group,the epithelial structure was more intact,and the fibroblasts and collagen fibers were reduced.(4)M1/M2 markers were detected in rabbit blood mononuclear cells.The results showed that after tracheal injury,the expression of i NOS in M1 type macrophages was up-regulated on the first day after tracheal injury,peaked on the fourth day,and then decreased.The expression of i NOS was decreased on the 10th day compared with the day of operation.The expression of i NOS in granulation tissue,was up-regulated on the first day after injury,reached the peak on the fourth day,and then decreased.However,M2 marker CD163gradually increased until the 10th day after surgery,CD206 gradually increased on the 1st day after surgery,then gradually decreased,and then increased again on the 10th day after surgery.(5)The m RNA expressions of i NOS,CD206,CD163 and Arg1 in tracheal granulation tissues of quercetin group were all up-regulated,and the up-regulated m RNA expressions of i NOS,CD206,CD163and Arg1 in quercetin combined with budesonide group were more obvious,while the m RNA expressions of i NOS,CD206,CD163 and Arg1 in budesonide group were down-regulated.(6)In the control group,a small amount of M1macrophages and M2 macrophages were expressed in the tracheal tissue,while the number of M1 macrophages and M2 macrophages increased in the tracheal tissue of the mechanical injury group M.(7)Compared with control group C,the expression of IL-4 in mucosa of tracheal stenosis model group M was up-regulated(P<0.05);The expression of the treatment group Q,B and QB was down-regulated,and the down-regulated expression of the QB group was the most obvious(P<0.05).The expression of CD206 in tracheal mucosa was up-regulated compared with the control tracheal stenosis model group(P>0.05);Compared with the model group,the expression of the treatment group Q and B was down-regulated(P>0.05),and the expression of QB group was up-regulated(P<0.05).(8)Compared with the control group,the expression of a-SMA in the tracheal mucosa of the tracheal stenosis model group after tracheal injury was up-regulated(P<0.05),down-regulated in treatment groups Q,B and QB(P<0.05).Conclusions:(1)Regulating the polarization balance of M1/M2macrophages can prevent and treat tracheal stenosis after injury.(2)Quercetin can regulate the transformation of macrophages from M1 to M2.Part IIIStudy of quercetin promoting phenotypic transformation of M1macrophages in vitroObjective:To explore if quercetin can change the transformation of macrophages,especially the polarization of M1/M2 in vitro cell experiments.Methods:(1)Detected the suitable concentration and time of quercetin on macrophage RAW264.7 by CCK8.(2)RAW264.7 macrophage cell line was cultured and proliferated to logarithmic stage,and then divided into five groups as follows:(1)Control group:M0;(2)LPS group:+LPS;(3)IL-4 group:+IL-4;(4)Quercetin group:+quercetin;(5)Mixed group:+LPS+quercetin,H&E staining to observe the changes of macrophage morphology in each group.(3)RAW264.7macrophage cell line was cultured and proliferated to logarithmic stage,and then divided into four groups as follows:(1)Control group:M0;(2)LPS group:+LPS;(3)Quercetin group:+quercetin;(4)Mixed group:+LPS+quercetin,the m RNA relative expression levels of F4/80,i NOS,TNF-a,Ccl2,Arg1 and IL-10were detected by real-time PCR.(4)RAW264.7 macrophage cell line was cultured and proliferated to logarithmic stage,and then divided into three groups as follows:(1)Control group:M0;(2)LPS group:+LPS;(3)Quercetin group:+quercetin,detect the proportion of M1 macrophages and M2 type by flow cytometry.Results:(1)Quercetin inhibited RAW264.7 cells in a concentration dependent manner.When the concentration of quercetin reached 10000 ug/m L,the inhibition rate was time dependent.The IC50 of quercetin at 12 hours was38.96 ug/m L,the IC50 at 24 hours was 10.60 ug/m L,the IC50 at 36 hours was28.12 ug/m L,and the IC50 at 48 hours was 11.41 ug/m L.The final quercetin intervention concentration was selected as 1 ug/m L for 48 hours.(2)After quercetin intervention,the results were similar to that of IL-4.After being induced by LPS into M1 type,the macrophages were transformed into M2-like cells by quercetin.(3)LPS can up-regulate the expression of i NOS m RNA(P<0.05).Quercetin can down-regulate the expression of i NOS m RNA and up-regulate the expression of Arg1 m RNA.Quercetin decreased the expression of TNF-αm RNA and increased the expression of IL-10 m RNA(P<0.05).(4)After the intervention of quercetin,the ratio of M2/M1 in macrophages changed,and the ratio was close to 1.0.Conclusion:In vitro,quercetin can change the differentiation of macrophage-related phenotypes and regulate the synthesis of cytokines related to different cell phenotypes,so it can be used as drug to regulate macrophage polarization.Part IVPI3K/Akt signaling pathway regulates the polarization of macrophages and the activation effection of macrophages on fibroblastsObjective:To study the signal pathway of quercetin regulating the polarization of macrophages and the activation of fibroblasts by macrophages through cell experiments.Methods:(1)Tracheal granulation tissues were collected from the stenosis group after tracheal injury and the recovery group after intervention.Real-time PCR was used to detect the m RNA expression of Akt1,Akt2 and Akt3 in PI3K/Akt signaling pathway.(2)RAW264.7 macrophage cell line was cultured and proliferated to logarithmic stage,and then divided into three groups as follows:(1)Control group:M0;(2)LPS group:+LPS;(3)Mixture group:+LPS+queretin.Real-time PCR was used to detect the m RNA relative expression levels of PI3K/Akt signaling pathway Akt1,Akt2,Akt3,TLR4 and NF-k B p65.(3)RAW264.7 macrophage cell line was cultured to logarithmic stage,and was treated with PI3K inhibitor 10μM LY294002 for 12 h,then divided into the following five groups:(1)control group,M0;(2)PI3Ki group,was continued with 10μM LY294002 intervention;(3)PI3Ki+LPS group,was given 10μM LY294002 and LPS intervention;(4)PI3Ki+IL-4 group,was given 10μM LY294002 and IL-4 intervention;(5)PI3Ki+quercetin was given 10μM LY294002 and quercetin,and the total RNA of cells in each group was extracted after 48 h of intervention.Real-time PCR was used to detect the relative expression levels of m RNA of Akt1,Akt2,Akt3 and m RNA of i NOS and Arg1,markers of M1/M2 macrophages.(4)RAW264.7 cells were divided into four groups:M0 group after conventional culture,M1 group after 24h LPS stimulation with 1ug/ml,M2 group after 24h LPS stimulation with 1ug/ml IL-4,and 1/2M1+1/2M2 group after equal proportion of M1 and M2 mixed culture.The macrophages in the above groups were directly co-cultured with fibroblasts:(1)M0+fibroblasts;(2)M1+fibroblasts;(3)M2+fibroblasts;(4)1/2M1+1/2M2+fibroblasts,were extracted 48 hours later,and the m RNA expression of a-SMA was detected by PCR,and the protein expression of a-SMA was detected by immunofluorescence.Results:(1)Expression of Akt1 and Akt3 was up-regulated and Akt2 was down-regulated in the strictured tracheal granulation tissue after tracheal injury(P<0.05).(2)Quercetin can up-regulate the expression of Akt2 and Akt3,and down-regulate the expression of Akt1(P<0.05).(3)Quercetin can up-regulate the expression of Akt2 and Akt3,and down-regulate the expression of Akt1.After using PI3K inhibitor,Akt1 and Akt3 expression were down-regulated,Akt2 expression was up-regulated,and Akt1 expression could not be reduced after quercetin intervention,and Akt2 and Akt3 up-regulation were not affected.After using PI3K inhibitor,the expression of i NOS was up-regulated,and adding quercetin could down-regulate the expression of i NOS;after using PI3K inhibitor,the expression of Arg1 was up-regulated,and after adding quercetin,the expression of Arg1 was up-regulated.It shows that quercetin can promote the expression of Akt1 and then inhibit the polarization of M1,up-regulate the expression of Akt2,and promote the transformation of M1 to M2.(4)Compared with the M0 group,the m RNA and protein expressions of a-SMA were up-regulated after co-culture of M1 and fibroblasts(P<0.05).Compared with M1 group,the m RNA and protein expression of A-SMA were down-regulated in co-culture of M2 and fibroblasts(P<0.05).The expression of a-SMA m RNA and protein in the 1/2M1+1/2M2 group was higher than that in the above two groups,and the expression of a-SMA m RNA and protein in the 1/2M1+1/2M2 group was higher than that in the M0 group(P<0.05).Conclusions:(1)The PI3K/Akt signaling pathway can effectively regulate the M1/M2 polarization of macrophages.(2)Quercetin can balance the M1/M2polarization by regulating the PI3K/Akt signaling pathway.(3)When M1 and M2 were present simultaneously,the activation of fibroblasts was weakened.