Specific Histone Deacetylase 8 Inhibitor Regulates Galectin-3 To Inhibit Polarization Of M2 Macrophages In The Treatment Of Asthma And Its Mechanisms

Background:Bronchial asthma is one of the common chronic diseases that seriously affect people’s health.The pathological changes are:airway inflammation,airway remodeling,and airway hyperresponsiveness.At present,the treatment of bronchial asthma is still focused on relieving symptoms and controlling inflammation.Glucocorticoids are still the most effective drugs for treating asthma.Histone deacetylases（HDACs）are one of the key enzymes to maintain the balance of histone acetylation in the nucleosome of the basic unit of the chromosome,and are closely related to many life processes in the cell,such as inflammation gene transcription,DNA damage repair,Cell proliferation and apoptosis,tissue fibrosis,etc.In the previous research work,this research group confirmed that broad-spectrum HDAC inhibitor（Givinostat）,HDAC6 inhibitor（Tubastatin A Hcl）,and HDAC8 inhibitor（PCI-34051）can alleviate chronic disease by establishing acute and chronic OVA asthma mouse models.Airway inflammation,airway hyperresponsiveness,and airway remodeling in asthmatic mice have therapeutic effects on bronchial asthmatic mice.Among them,the anti-inflammatory effect of HDAC8 inhibitors in asthma is obviously better than that of broad-spectrum HDAC inhibitors and HDAC6 inhibitors.It is shown that in asthma,HDAC8 inhibitors can better alleviate airway inflammation and airway hyperresponsiveness.Macrophages can be polarized into classic activated macrophages（M1 type）and alternative activated macrophages（M2 type）under different circumstances.Studies have shown that M2 polarization of macrophages plays an important role in the pathogenesis of asthma.Galectin-3 participates in the process of many inflammatory reactions and plays an important role in the polarization of M2macrophages.Objective:To study the effect of PCI-34051,a specific inhibitor of HDAC8,on M2 polarization of macrophages in acute asthmatic mice,and to study its possible mechanism through in vitro experiments.Methods:This study is divided into three parts.The first part studies the effects of PCI-34051 on the M2 polarization of macrophages in lung tissue of acute asthmatic mice.In the second part,PCI-34051 was used to intervene in asthmatic mice,and the effect and possible mechanism of PCI-34051 on Galecitn-3 were evaluated.In the third part,Raw264.7 mouse peritoneal macrophages were selected to investigate the effect of PCI-34051 on Galecitn-3 and the mechanism of PCI-34051 on macrophage M2polarization by simulating M2 macrophage polarization in vitro.1.Effects of HDAC8-specific inhibitor PCI-34051 on the M2 polarization of macrophages in lung tissues of acute asthmatic mice1.1 Grouping of animal models:24 female SPF BALB/C mice of 6-8 weeks were selected.The random number table method was divided into normal group（NS group）,asthma group（OVA group）,budesonide hormone group（OVA/BUD group）,and PCI-34051 group（OVA/PCI group）.On days 0,7,and 14 of the experiment,mice in the OVA group,the OVA/BUD group,and the OVA/PCI group were injected with an sensitizing solution（OVA+Al（OH）₃）intraperitoneally.One week after the last sensitization,an ultrasonic nebulizer was used to stimulate the solution（2%OVA）on the 21st to 27th days for 7 consecutive days,once a day,30 minutes/time.Budesonide（0.04 mg/ml,nebulization）was used in the BUD group,and PCI-34051（0.5 mg/kg,intragastrically）was used in the PCI group,which were administered 30 minutes before each challenge test.In the normal group,OVA and drugs were replaced with normal saline.1.2 Pulmonary function challenge test:24 hours after the last challenge test,the plethysmography method was used to detect the airway enhanced expiratory interval of each group of mice（Penh）.1.3 Cell count and differential count in alveolar lavage fluid（BALF）:BALF was collected from mice,and the precipitated cells were collected by centrifugation.After resuspending the cells,Swiss-Gimsa staining was performed to count the total number of inflammatory cells and the number of cells in BALF.1.4 ELISA test:Blood was collected from mouse eyeballs,and the serum was obtained by centrifugation.The serum levels of IL-4,IFN-γand IgE were detected by enzyme-linked immunosorbent assay（ELISA）.1.5 Preparation and staining of pathological specimens:The left lung tissues of mice were extracted,paraffin-embedded and sectioned,and the left lung tissue sections of the mice were stained with HE and AB-PAS to observe the infiltration of inflammatory cells in the lung tissue of mice in each group And airway mucus secretion.1.6 Flow cytometry:A single cell suspension of mouse lung tissue was extracted,and the expression of macrophage phenotype F4/80,M1 type CD86 and M2 type CD206 were detected by flow cytometry.1.7 Immunohistochemistry:SABC method was used to stain the left lung tissue sections of mice,and the expression of CD68,CD86,and CD163 in lung tissue was detected.1.8 Q-PCR:The mRNA of lung tissue of mice in each group was extracted,and the levels of Arg1 mRNA and iNOS mRNA in the lung tissue were detected.1.9 Western Blot:The lung tissue protein of each group of mice was extracted,and the expression levels of Arg1 and iNOS in the lung tissue were detected.2.Effect of PCI-34051 on Galecitn-3 in lung tissue of acute asthmatic mice2.1 Preparation and grouping of animal models:6-8 weeks SPF female BALB/C female mice were randomly divided into normal groups（NS group）,Asthma group（OVA group）,budesonide hormone group（OVA/BUD group）,PCI-34051 group（OVA/PCI group）.The modeling method is as before.2.2 Immunohistochemistry:The SABC method was used to stain the left lung tissue section of the mouse to detect Galectin-3 and HDAC8 localization in the lung tissue.2.3 Western Blot:The lung tissue protein of each group of mice was extracted,and Galectin-3 and HDAC8 expression levels in the lung tissue were detected.2.4 Immunofluorescence double staining technique:Immunofluorescence double staining was performed on the left lung tissue section of the mouse to detect the expression positions of Galectin-3 and HDAC8 in the lung tissue of the mouse.3.Effect and mechanism of PCI-34051 on M2 polarization of Raw264.7 cells3.1 Cell culture:Add mouse mononuclear macrophage cell Raw264.7 to DMED containing 10%fetal bovine serum and no endotoxin The cells were cultured in a high-sugar liquid medium;the cells were all placed under 5%CO₂ and cultured at 37°C.3.2 Cell processing and grouping:Routine subculture,the culture medium uses DMEM high glucose containing 10%fetal bovine serum.Divided into Con group,IL-4 group and IL-4/PCI group.The Con group was cultured normally.The IL-4 group was stimulated with recombinant IL-4 protein（R&D Systems）to stimulate macrophages（concentration:20ng/ml）.The IL-4/PCI group was stimulated with PCI-34051（concentration:10μM）.After phagocytes,macrophages were stimulated with recombinant IL-4 protein（concentration:20ng/ml）.3.3 Q-PCR detection:The mRNA of each group of cells was extracted,and the levels of Arg1 mRNA and iNOS mRNA in the cells were detected.3.4 Western Blot:Extract intracellular proteins from each group and detect the expression levels of Arg1,iNOS,Galectin-3,and HDAC8 in the cells.3.5 Immunofluorescence double staining technique:Perform immunofluorescence double staining on cells of each group to detect the expression positions of Galectin-3and HDAC8 in macrophages.3.6 Interfering with Galectin-3 expression through shRNA technology.Western Blot detected the expression level of Arg1 protein after Galectin-3 silencing and IL-4induction.3.7 The co-immunoprecipitation test（Co-IP）was used to detect the effects of HDAC8 and Galecitn-3 protein in each group of cells.Results:1.The effect of PCI-34051,a specific inhibitor of HDAC8,on the M2 polarization of macrophages in lung tissue of acute asthmatic mice1.1 Compared with the OVA group,the lung tissue in the OVA/BUD group and the OVA/PCI group.The inflammatory cell infiltration and mucus secretion around the airway were significantly reduced,and the airway hyperresponsiveness was significantly reduced.1.2 Compared with the OVA group,IL-4 and IgE in the serum of the mice were significantly higher in the OVA/BUD group and the OVA/PCI group.1.3 Compared with the OVA group,the total number of inflammatory cells and the number of macrophages in BALF in the OVA/BUD group and the OVA/PCI group were significantly reduced.1.4 The results of flow cytometry showed that compared with the NS group,the expression of CD206/F4-80 of M2 macrophage polarization levels in the lung tissue of mice in the OVA group increased significantly,while the levels of the OVA/BUD group and the OVA/PCI group decreased.1.5 The results of immunohistochemistry showed that compared with the NS group,CD163 in the lung tissue of the OVA group was significantly increased,while CD86 increased slightly.After intervention with budesonide and PCI-34051,CD163 in lung tissue of asthmatic mice was significantly reduced.1.6 Q-PCR results showed that compared with the NS group,the M2 macrophage marker Arg1 mRNA in the lung tissue of the OVA group was significantly increased,while the Arg1 mRNA of the OVA/BUD group and the OVA/PCI group was significantly reduced.1.7 Western Blot results showed that compared with the NS group,the M2macrophage marker Arg1 protein level in the lung tissue of the OVA group mice was significantly increased,while the OVA/BUD group and the OVA/PCI group Arg1protein levels were significantly reduced.2.Effect of PCI-34051 on Galecitn-3 in lung tissue of acute asthmatic mice2.1 Immunohistochemical results showed that Galectin-3 was significantly increased in lung tissue of OVA group compared with NS group,while that of OVA/BUD group and The expression of Galectin-3 in the lung tissue of mice in the OVA/PCI group was significantly reduced.In the lung tissue of mice in the OVA group,HDAC8 was significantly increased,while the expression of HDAC8 in the lung tissue of the mice in the OVA/BUD group and the OVA/PCI group was significantly reduced.2.2 Western Blot showed that compared with the OVA group,the expression of Galectin-3 and HDAC8 protein in the lung tissue of the OVA/BUD group and the OVA/PCI group were significantly reduced.2.3 The results of immunofluorescence showed that Galectin-3 and HDAC8 had no significant co-expression in the NS group.In the OVA group,part of Galecitn-3 was expressed intranuclearly,and HDAC8 was co-expressed.In the OVA/BUD group and the OVA/PCI group,co-expression of Galectin-3 and HDAC8 was significantly reduced.3.PCI-34051’s effect on M2 type polarization of Raw264.7 cells and its mechanism3.1 Q-PCR results showed that compared with the Con group,IL-4 promoted the expression of Arg1 mRNA and inhibited the expression of iNOS mRNA.Compared with the IL-4 group,the IL-4/PCI group inhibited the expression of Arg1 mRNA.3.2 Western Blot results showed that PCI-34051 inhibited the increase of HDAC8and Galecn-3 expression induced by IL-4.3.3 After Galectin-3 silencing,the expression of Arg1 protein induced by IL-4stimulation was significantly reduced.3.4 Immunofluorescence results showed that Galecitn-3 was expressed in the cytoplasm and nucleus after IL-4 stimulation,and co-expression of HDAC8 and Galecitn-3 was significantly increased.PCI-34051 not only inhibits the expression of Galectin-3 and HDAC8,but also suppresses its co-expression.3.5 The results of co-immunoprecipitation（Co-IP）showed that compared with the Con group,the interaction between Galectin-3 and HDAC8 in the IL-4 group was significantly increased.After PCI-34051 treatment,the interaction between Galectin-3and HDAC8 was significantly reduced.Conclusions:1.HDAC8-specific inhibitor PCI-34051 can inhibit airway inflammation and airway hyperresponsiveness in acute asthmatic mice.2.PCI-34051 can inhibit M2 polarization of macrophages in mice with asthma.3.Budesonide can simultaneously inhibit M1 type M2 polarization of macrophages in asthmatic mice,while PCI-34051 can only inhibit M2 type polarization of macrophages in asthmatic mice.4.Galectin-3 plays an important role in M2 polarization of macrophages.5.PCI-34051 can inhibit M2 polarization of macrophages by inhibiting the interaction between Galectin-3 and HDAC8.