Effects And Mechanism Of M2 Macrophages On Pulmonary Fibrosis Induced By Lipopolysaccharide

Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are common causes of morbidity and mortality in ICU.Most of ALI/ARDS are caused by viruses,bacteria and other microorganisms,which cause severe pulmonary or even systemic inflammation.The prognosis of ALI/ARDS usually depends on the degree of structural remodeling and fibrosis after lung injury.When severe pulmonary fibrosis occurs and develops,the patient's lung function decreases,even respiratory failure,which is life-threatening.It has been suggested that M2 macrophages are activated during the regression and healing stages of ALI/ARDS inflammation and play an important role in the regulation of pulmonary interstitial synthesis and fibrosis.The differentiation of myofibroblasts is an important link in the development of pulmonary fibrosis.Axin2~+Myofibrogenic Progenitor cells in the lung stroma have the potential to differentiate into myofibroblasts,which may participate in the occurrence and development of pulmonary fibrosis.As an important carrier of intercellular signal transmission,exosomes are involved in many pathophysiological processes and can also be used as a target for disease treatment,which has received much attention in recent years.After the occurrence of ALI,many exosomes,especially macrophage derived exosomes,were found in the bronchoalveolar lavage fluid,which were involved in the inflammatory response,tissue damage and repair process.Therefore,studying the relationship between M2 macrophage exosomes and Axin2+Myofibrogenic Progenitor cells may provide new directions and ideas for the research and treatment of pulmonary fibrosis after ALI.Part?Activation of M2 macrophages after acute lung injury and itseffect on the proliferation and activation of AMPObjective:In order to explore the effect of M2 type macrophages on the inflammatory response of Acute lung injury(ALI),we established a mouse model of Acute lung injury(ALI),and tested the effect of macrophages on the development of lung fibers after Acute lung injury through elimination of macrophages by drug treatment.Methods:1.Thirty-six Specific pathogen-free(SPF)C57BL/6 mice,aged 6-8 weeks and weiged 20-22 g.They were randomly divided into two groups.The experimental group(ALI group)intraperitoneal injection of Lipopolysaccharide(LPS),injection volume:15 mg/kg.The control group(group N)was intraperitoneally injected with saline.Six mice were randomly sacrificed at day 1,3,and 7,respectively.The alveoli were lavaged with PBS for three times,and the Bronchoalveolar lavage fluid(BALF)was collected.The ratio of M1 macrophages and M2 macrophages was detected by flow cytometry.2.Thirty ALI mouse models were established and randomly divided into 2groups.Clodronate Liposomes were administered to the experimental group(ALI+CL group)for a week continuously,and inhaled into nasal cavity.The ALI group was injected with normal saline,and the mice were killed at 1 week.The lung specimens were dissected and the expression levels of?-SMA and collagen I protein were detected by Western blot.3.Transforming growth factor-?1(TGF-?1),Platelet derived factor(PDGF)and insulin-like growth factor(IGF-1)were added to incubate AMP cells for 4 days,and the proliferation rate of AMP cells was detected by CCK-8.In the experimental group,ALI mouse model was established in the same way as method 1,and the control group was injected with normal saline.Balf was collected in one week.CD206~+/CD163~+M2 macrophages(ALI-M?group)of the experimental group and alveolar CD68~+macrophages(N-M?)of the control group were obtained by flow cytometry,which were co-cultured with Axin2+Myofibrogenic Progenitor cells(AMP)cells,respectively.Flow analysis to detect AMP cell proliferation and Western blot detection of vascular smooth muscle protein(alpha smooth muscle actin,?-SMA),Collagen?expression level.Results:1.Compared with the 1st and 3rd day after ALI,the number of CD68~+/i NOS~+M1 macrophages decreased on the 7th day after ALI,while the number of CD206~+/CD163~+M2 macrophages increased,with significant differences(P<0.001).2.Compared with the control group,clodronate liposomes can improve the lung tissue inflammatory overflow and reduce pulmonary fibrosis in mice with ALI.At the same time,the protein levels of?-SMA and collagen I in the lung tissue were significantly decreased(P<0.01).3.AMP cells were cultured with TGF-?1,PDGF and IGF-1 for 4 days,and there was no significant change in AMP cell proliferation level.Compared with the N-M?group,the proliferation level of AMP cells in the ALI-M group was increased.At the same time,the morphology of AMP cells changed from stem cell like mass to fibroblast like spindle,and the expression of?-SMA was up-regulated.The content of collagen I in the supernatant of ALI-M?group was also significantly higher than that of N-M?group(P<0.01).Conclusion:1.In the late ALI stage,macrophages migrated to the M2 subtype.2.The results showed that 1 week after LPS induced ALI,clodronate liposomes could significantly reduce pulmonary fibrosis in mice.3.M2-type macrophages isolated from BALF of ALI 1 week mice could significantly promote the proliferation of AMP,differentiation of myofibroblasts and secretion of collagen.Part?The mechanism of M2-type macrophage exosomesregulating PF after acute lung injuryObjective:This research was aimed to study the role of AMP in alleviating Ali and the specific mechanism of M2 macrophage regulating pulmonary fibrosis by establishing Ali mouse model.Methods:1.twelve Specific pathogen-free(SPF)C57BL/6 mice,aged 6-8 weeks and weiged 20-22 g,were randomly divided into two groups.The experimental group(ALI-M?-Exo group)was intraperitoneally injected with lipopolysaccharide,and the control group(N-M?-Exo group)was intraperitoneally injected with saline.Lung samples were collected at 1 week for ALI and pulmonary fibrosis evaluation.CD206~+/CD163~+M2 macrophages were isolated from BALF by flow cytometry in ALI-M?-Exo group;CD68~+macrophages were isolated from BALF by flow cytometry in N-M?-Exo group.Secreting exosomes from alveolar macrophages of ALI mice(ALI-M?-Exo)and normal mice(N-M?-Exo)were isolated and co-cultured with AMP,respectively.Cell counting kit-8(CCK-8)was used to detect the proliferation level of AMP.Western blot was used to detect the expression level of collagen I.immunofluorescence staining was used to observe the morphology of AMP and the expression of?-SMA.2.The methylation level of N~6-methyl-adenosine(m~6A)in AMP total RNA was detected by colorimetry.The expression level of METTL3 protein in AMP was detected by Western blot.The m RNA level of METTL3 in AMP was detected by q PCR.3.The secretion of exosomes from alveolar macrophages of ALI mice(ALI-M?-Exo)and normal mice(N-M?-Exo)were collected.The size and morphology of exosomes were observed by transmission electron microscope.Western blot was used to detect the expression of METTL3 protein in exosomes.Results:1.Compared with N-M?-Exo group,AMP proliferation level was significantly increased in ALI-M?-Exo group.At the same time,the morphology of AMP changed from stem cell like mass to fibroblast like spindle,and the expression of?-SMA was up-regulated.AMP medium supernatant Collagen?levels rose significantly(P<0.01).2.Compared with the control group,the m~6A level and METTL3 protein expression level in AMP total RNA were significantly increased after stimulation by ALI-M?-Exo,while the METTL3 m RNA level in AMP was not significantly different.3.Western blot showed that METTL3 protein was highly expressed in the exosomes of M2 macrophages(P<0.01).Conclusion:1.These results indicated that the exosomes secreted by M2-type macrophages in BALF of mice after 1 week of ALI could significantly promote AMP proliferation,myofibroblast differentiation and collagen secretion.2.METTL3 promotes AMP cell proliferation by regulating m~6A level.3.In ALI mice,the METTL3 protein carried in exosomes secreted by M2macrophages promoted AMP activation and fibrous proliferation through the modification of m~6A.