Study On The Mechanism Of Eucalyptol Inhibiting Pulmonary Fibrosis By Down-regulating The Polarization Of M2 Macrophages

BackgroundIdiopathic pulmonary fibrosis(IPF)is a chronic and progressive interstitial lung disease with progressive decline in lung function and even respiratory failure.In recent years,the incidence of IPF has been on the rise,which is difficult to cure,and its morbidity and mortality have been on the rise year by year.At present,due to the wide application of anti-fibrosis drugs pirfenidone and nintedanib,the quality of life of patients has been improved,but idiopathic pulmonary fibrosis is still incurable.At present,precise treatment of pulmonary fibrosis has become a hot topic for scholars at home and abroad.Therefore,it is great significance for the prevention and treatment of idiopathic pulmonary fibrosis to explore the pathological mechanism and molecular mechanism of idiopathic pulmonary fibrosis and seek more effective therapeutic targets.Inflammatory response and immune system regulation play a key role in the formation and development of IPF,in which a variety of immune cells and cytokines are involved,among which M2 macrophage polarization plays a key role.Therefore,starting from the aspects of inflammation and M2 macrophage polarization,it is very important to explore the pathogenesis of IPF and its potential new targets for drug therapy.As an intermediate drug,eucalyptol has been shown to have anti-inflammatory,anti-oxidation,and anti-fibrosis effects on some diseases in many studies,but there are no studies on the effect of eucalyptol on pulmonary fibrosis.In view of this,this study firstly observed the expression levels and clinical significance of inflammatory factors and pro-fibrosis factors in peripheral blood of clinical IPF patients.Secondly,an intervention study was conducted by establishing a bleomycin-induced fibrosis mouse model to explore the effects of eucalyptol on bleomycin-induced pulmonary inflammation and fibrosis in mice,and to observe the effects of eucalyptol on the polarization of M2 macrophages in the lung tissue of mice.Finally,the molecular mechanism of the antifibrotic effect of eucalyptol was explored through the induction and differentiation of IL-13 induced by mouse bone marrow derived macrophages and intervention.In conclusion,this study focuses on exploring the effect of eucalyptol on pulmonary fibrosis and the possible mechanism of action,so as to provide a new theoretical basis and therapeutic direction for clinical treatment of IPF patients.Chapter 1 The expression and clinical significance of serum IL-13,IL-6,TNF-α and TGF-β1 in patients with idiopathic pulmonary fibrosisObjectiveTo observe the levels and clinical significance of IL-13,IL-6,TNF-α,TGF-β1,CD163 and CD206 in peripheral blood of patients with IPF.Methods1.40 IPF patients admitted to our department from October 2020 to October 2022 were included as the observation group,and 40 healthy physical examination patients during the same period were included as the control group.2.Combined with the patient’s gender,age,leukocytes,neutrophils and other clinical baseline data,the differences were observed.3.The expressions of IL-13,IL-6,TNF-α,TGF-β1,CD163 and CD206 in peripheral blood of the two groups were detected by ELISA.4.Routine pulmonary function tests were conducted to detect FVC%and DLCO%in IPF patients.5.Correlation analysis was used to analyze the correlation of IL-13,IL-6,TNF-α and TGF-β1 with CD163 and CD206.6.Correlation analysis was used to analyze the correlation of IL-13,IL-6,TNF-α and TGF-β1 with FVC%and DLCO%,and to analyze the expression and clinical significance of cytokines in IPF patients.Results1.The expression of IL-13,IL-6,TNF-α,CD163 and CD206 in IPF patients were higher than those in health people(P<0.05).The level of TGF-β1 in IPF patients was slightly higher than that in normal control group,but the difference was no significant(P>0.05).2.The expression of CD 163 and CD206 in IPF patients were higher than those in health people(P<0.05).2.Spearman correlation analysis showed that IL-13,IL-6 and TNF-α were positively correlated with CD163 and CD206(P<0.05),and that IL-13,IL-6 and TNF-α were negatively correlated with FVC%and DLCO%;TGF-β1 has no correlation with FVC%and DLCO%(P>0.05).ConclusionThe expression of IL-13,IL-6,TNF-α,CD 163 and CD206 in serum of IPF patients were increased,M2 macrophage polarization is involved in the formation and development of IPF.The expression of IL-13,IL-6 and TNF-α were positively correlated with CD 163 and CD206 and were negatively correlated with FVC%and DLCO%,which could serve as a reference index for clinical evaluation of the severity of IPF patients.Chapter 2 The effect of eucalyptol on bleomycin-induced pulmonary fibrosis in mice Section 1 The effections of different doses of ecuclyptol on blemycin-induced pulmonary fibrosisObjectiveTo observe the effects of different doses of eucalyptol on bleomycin-induced pulmonary fibrosis in mice.Methods1.C57BL/6 male mice aged 6-8weeks were randomly divided into 5 groups:blank control group,IPF model group,25mg/kg eucalyptol group,50mg/kg eucalyptol group and 100mg/kg eucalyptol group.After the establishment of pulmonary fibrosis mouse model,the mice were sacrificed on the 21st day of continuous intervention,and lung tissues were collected for further detection.2.HE staining and Masson staining were used to observe the pathological characteristics of lung tissue and the infiltration of inflammatory cells.3.The content of hydroxyproline in mouse lung tissue was detected.4.The expressions of α-SMA and fibronectin in mouse lung tissue were detected by real-time PCR.Results1.HE staining and Masson staining of lung tissue showed that the mouse pulmonary fibrosis model was successfully established.Eucalyptol reduced the inflammatory cell infiltration and collagen deposition in the lung tissue of bleomycin-induced pulmonary fibrosis mouse model in a dose-dependent manner.2.Eucalyptol reduced the expression of hydroxyproline in lung tissue of mice in a dose-dependent manner,with statistical significance(P<0.01).3.Eucalyptol reduced the expressions of α-SMA and fibronectin in lung tissue in a dose-dependent manner,with statistical significance(P<0.01).ConclusionEucalyptol reduced the expression of HYP,α-SMA,and fibronectin in the lung tissue of BLM-induced pulmonary fibrosis model mice,and inhibited pulmonary fibrosis,which has a dose-dependent effect.The best effective dose of eucalyptol is 50mg/kg,which provides the experimental basis for subsequent experiments.Section 2 The effections of ecuclyptol on blemycin-induced pulmonary inflammatory,fibrosis and M2 macrophages polarrizationObjectiveTo observe the effects of eucalyptol on bleomycin-induced pneumonia in mice and the polarization of M2 macrophages.MethodsAccording to the analysis of the results after the intervention of different doses of eucalyptol in the second part,considering the effectiveness and side effects of the drug,50mg/kg was selected as the effective dose of eucalyptol with small side effects for the following experiment.1.C57BL/6 male mice aged 6-8 weeks were randomly divided into blank control group,50mg/kg eucalyptol control group,pulmonary fibrosis model group and 50mg/kg eucalyptol treatment group,with 10 mice in each group.The mouse model of pulmonary fibrosis was established and treated continuously for 7 days,then the mice were killed on the 7th and 21st day respectively,and lung tissue or BALF was collected for detection.2.HE staining and Masson staining were used to observe the pathological characteristics of lung tissue and the infiltration of inflammatory cells.The content of hydroxyproline in mouse lung tissue was detected.3.The expressions of α-SMA and fibronectin in mouse lung tissue and mRNA expressions of M2 macrophage markers Arginase-1,Ym-1,IL-13 and TGF-β1 were detected by real-time PCR.4.MPO activity in lung tissues of mice in each group was detected with MPO kit.5.The-expressions of pro-inflammatory factors IL-13,TNF-α,IL-6 and pro-fibrosis factor TGF-β1 in BALF were detected by ELISA.6.The activity of TGF-β1 in mouse BALF was detected by luciferase reporting system;7.The total number of inflammatory cells,the number of neutrophils and the number of macrophages in BALF of mice in each group were detected by cell counting plate with fine ball.8.BCA protein concentration detection method was used to detect the total protein concentration in BALF.Results1.HE staining and Masson staining of mouse lung tissue showed that inflammatory cell infiltration and collagen deposition were reduced in the eucalyptol treated group compared with the fibrosis model group.2.Test results of pulmonary inflammation indicators:Compared with the fibrosis model group,the infiltration of inflammatory cells,the activity of MPO and the wet-dry weight ratio of lung tissue were decreased significantly in the eucalyptol treatment group(P<0.05),and the total number of cells,neutrophil number,total protein concentration,TNF-α and IL-6 in BALF were decreased significantly(P<0.01).3.Pulmonary fibrosis index detection results:compared with the fibrosis model group,the expression levels of fibrosis-promoting factors IL-13 and TGF-β1 in lung tissue and BALF of mice in the eucalyptol treatment group were significantly decreased,and the activity of TGF-β1 was also significantly decreased(P<0.01).The content of HYP,α-SMA and fibronectin decreased significantly(P<0.01).4.Detection results of M2 macrophage markers:Compared with the fibrosis model group,the expressions of Arginase-1 and Ym-1 in lung tissue of mice in eucalyptol treatment group were significantly decreased(P<0.01).Conclusion1.Eucalyptol can inhibit lung inflammation by decreasing inflammatory cell infiltration and expression of inflammatory factors;Eucalyptol can reduce the content of HYP,α-SMA,fibronectin and pro-fibrosis factor expression in lung tissue and inhibit pulmonary fibrosis.2.Eucalyptol can inhibit the polarization of M2 macrophages and the expression of TGF-β1 secreted,thus inhibiting pulmonary fibrosis.Section 3 Effect of eucalyptol on reducing pulmonary fibrosis by inhibiting IL-13ObjectiveIn this study,the BLM-induced pulmonary fibrosis model of mice was established to investigate whether eucalyptol alleviates pulmonary fibrosis by inhibiting IL-13.Methods1.C57BL/6 male mice aged 6-8 weeks were randomly divided into blank control group,IPF model group,50mg/kg eucalyptol group and 50mg/kg eucalyptol combined with IL-13 group,with 5 mice in each group.After the establishment of a mouse model of pulmonary fibrosis,the mice were treated with 10μg/kg IL-13 on the day of modeling,day 2 and day 4,respectively,and the mice were sacrificed on day 21,and lung tissues were collected for further detection.2.HE staining and Masson staining were used to observe the pathological characteristics of lung tissue and the infiltration of inflammatory cells.3.The content of hydroxyproline in mouse lung tissue was detected.The expressions of α-SMA and fibronectin in mouse lung tissue were detected by real-time PCR.Results1.HE staining and Masson staining of lung tissues showed that eucalyptol treatment inhibited inflammatory cell infiltration and collagen deposition in lung tissues of mice,and IL-13 treatment significantly increased inflammatory cell infiltration and collagen deposition in lung tissues of mice.2.Fibrosis index test results:hydroxyproline content in lung tissue fibrosis model group mice increased significantly,α-SMA,fibronectin expression significantly increased;eucalyptol treatment group reduced hydroxyproline content,α-SMA,fibronectin expression significantly decreased(P<0.01).Compared with eucalyptol treatment group,eucalyptol and IL-13 intervention group of hydroxyproline content in the lung tissue of mice(P<0.05),α-SMA,fibronectin expression significantly increased(P<0.01).ConclusionEucalyptol can play an anti-fibrosis role by inhibiting the pro-fibrosis factor IL-13.Chapter 3 Study on the mechanism of eucalyptol inhibiting pulmonary fibrosisObjectiveto clarify the molecular mechanism of inhibiting pulmonary fibrosis by eucalyptol,Macrophages from mouse bone marrow were obtained,cultured and interfered in vitro.Methods1.Mouse bone marrow derived macrophages(BMDMs)were collected for culture.2.Different doses of eucalyptol(0,5,15,45μM)were used to intervene for 15 min,and then 15ng/mL IL-13 was used to induce BMDMs.3.The expression of TGF-β1 in cell fluid was detected by ELISA.4.The expressions of M2 macrophage markers Arginase-1 and Ym-1 in BMDMs were detected by Western blotting.Phosphorylation of STAT6 and p38 MAPK and the expression of transcription factors KLF4 and PPAR were detected by Western blotting.Results1.The detection results of M2 macrophage markers in BMDMs showed that compared with IL-13-induced group,the expressions of Ym-1 and Arginase-1 in BMDMs decreased significantly(P<0.001)in the eucalyptol intervention group in a dose-dependent manner.2.The results of TGF-β1 in the cell fluid after the intervention of BMDMs showed that compared with IL-13-induced group,the expression of TGF-β1 in cell fluid was significantly decreased in the eucalyptol intervention group in a dose-dependent manner(P<0.01).3.The phosphorylation levels of STAT6 and p38 MAPK showed that compared with IL-13-induced group,the phosphorylation levels of STAT6 and p38 MAPK in BMDMs were significantly increased(P<0.001)in the eucalyptol intervention group in a dose-dependent manner.4.The expression of transcription factors KLF4 and PPAR-y showed that the expression levels of KLF4 and PPAR-y in BMDMs induced by IL-13 were greatly increased.After eucalyptol intervention,the expression levels of KLF4 and PPAR-γ in BMDMs were greatly increased(P<0.001),and the expression levels were significantly decreased in a dose-dependent manner.Conclusion1.Eucalyptol inhibited the polarization of M2 macrophages induced by IL-13 and reduced the production of TGF-β1 secreted by M2 macrophages.2.Eucalyptol can inhibit the polarization of M2 macrophages by down-regulating the activation of STAT6 and p38 MAPK and the expression of downstream transcription factors KLF4 and PPAR-y,thus inhibiting pulmonary fibrosis.In summary,this study confirmed the mechanism of eucalyptol inhibiting pulmonary fibrosis and M2 macrophage polarization by down-regulating STAT6/p38MAPK and KLF4/PPAR-y signaling pathway through preliminary detection of peripheral blood of clinical IPF patients,in vivo mouse pulmonary fibrosis model study and in vitro BMDMs exploration.