| Background:Asthma is a common allergic disease of the respiratory system,in which inflammatory cells(such as eosinophils,T lymphocytes,etc.)and airway structural cells(such as epithelial cells,smooth muscle cells)play an important role in the pathogenesis of asthma.Inflammatory cells release inflammatory mediators leading to persistent chronic airway inflammation,causing changes in airway structure,including loss of epithelial integrity,basement membrane thickening or subepithelial fibrosis,mucous gland and goblet cell hyperplasia,smooth muscle cell hypertrophy,proliferation or migration leading to increased smooth muscle mass,increased airway vascularity,leading to airway remodeling,increased airway smooth muscle mass is a hallmark change of airway remodeling in asthma.Recent studies have shown that transforming growth factor-?1(TGF-?1)can induce mitosis/proliferation of human airway smooth muscle(h ASM)cells.Moreover,in a mouse model induced by ovalbumin(OVA),house dust mites(HDM),lipopolysaccharides(LPS),and toluene diisocyanate sensitization and repeated challenge,airway smooth muscle(ASM)tissue enlargement occurred simultaneously with increased TGF-?1 expression and signal transduction.Autophagy is a process in which eukaryotic cells perform lysosomal degradation and recycling of damaged proteins or organelle encapsulation,with multiple effects to maintain nuclear body homeostasis.Studies have shown that autophagy plays an important role in asthmatic respiratory tract infections.Mitophagy is the selective degradation of mitochondria by autophagy.It typically occurs in mitochondria damaged after exposure to environmental pollutants,allergens,or stress and plays a key role in promoting mitochondrial turnover and preventing dysfunctional mitochondrial accumulation.Mitochondrial dysfunction and increased reactive oxygen species(ROS)production have been implicated in allergic diseases,including compliance,atopic dermatitis,and asthma.TGF?1 is thought to drive airway remodelling by activating smooth muscle cells and subsequently inducing the release of fibrotic extracellular matrix(ECM)proteins.The increase of ASM thickness in human asthmatic lungs was closely related to the high expression of autophagy markers,and the association was not present in non-asthmatic lungs.Recently,it has been shown that autophagy promotes and affects TGF?1-driven airway remodeling.However,the effect of TGF?1 on mitophagy has not been deeply studied,and the regulation of ASMCs proliferation and cellular function by TGF?1 through mitophagy as well as its role in airway remodeling has not been addressed.In this study,we used airway smooth muscle cells to observe the effect of TGF?1 on mitophagy and explored and elaborated its mechanism of action to determine the effect of TGF?1-induced mitophagy on the proliferation and cellular function of ASMCs.In vivo experiments were performed using an asthmatic airway remodeling model to observe the effect of TGF-?1 on TAK1-AMPK? signal transduction in asthmatic airway remodeling model mice in vivo,and whether subsequent PINK1/Parkin activation mediates mitophagy.ObjectiveTo elucidate the mechanism by which TGF-?1 activates the PINK1-Parkin pathway through TAK1-AMPK? to regulate mitophagy mediated airway smooth muscle cell proliferation,and to explore the role of this mechanism in airway remodeling in asthma,providing a new target for the treatment of clinical asthma.Methods Part I: In vitro experiment RASMC cells were resuscitated,and RASMC cells were pretreated with SB505124,TAKinib,3-MA,and CQ,respectively,and si RNA interfered with AMPK?,PINK1,and Parkin expression,followed by stimulation of RASMC cells with TGF-?1 at different concentrations or for different times as needed.Specifically as follows: 1)MTT assay was used to detect the viability of RASMC cells by TGF-?1;2)RT-q PCR was used to detect the contents of TGF-?1,TAK1,PINK1,and Parkin,i.e.The effect of siker-PINK1,si Parkin,and si AMPK? was also verified;3)Small interfering RNA silenced PINK1,Parkin,and AMPK? expression;4)Western blot was used to detect the expression of TGF-?1,p-TAK1,TAK1,p-AMPK?,AMPK?,PINK1,mito PINK1,cyto PINK1,Parkin,mito Parkin,LC3-I/II,Beclin-1,Atg5,Atg7,p62,PCNA,?-sma,MMP9,Drp1,Fis1,Par N2,OPA1,OPTN,and NDP52 proteins in RASMC cells;5)CCK-8 assay was used to detect RASMC cell proliferation;6)flow cytometry was used to detect RASMC cell cycle,mitochondrial membrane potential change,and ROS release;7)Cell staining: AO dye,TMRE reagent,JC-1 reagent,Mitosox,and Mitotrared were used to stain RASMC cells;8)Immunofluorescence was used to detect the expression of K1 and Mitoskin in RASMC cells.Part II: In vivo experiment Thirty-two BALB/C mice were randomly divided into four groups: normal control group(control group),asthmatic airway remodeling model group(OVA group),TGF-?1 inhibitor-treated group(OVA + SB505124 group),and autophagy inhibitor-treated group(OVA + CQ group).Asthma airway remodeling models were established in mice of other groups except the normal group,and each inhibitor group was given the corresponding intervention means.Outcome measures: 1)airway reactivity in mice was detected by Ach respiratory challenge test;2)TGF-?1 content in BALF of mice was detected by ELISA;3)TGF-?1 content in lung tissue was detected by RT-q PCR;4)HE,PAS and Masson staining were performed in lung tissue of mice;5)TGF-?1 content in BALF and correlation between TGF-?1 m RNA level in lung tissue and airway smooth muscle;6)The expression of ?-SMA and MMP-9 in lung tissue was detected by immunohistochemistry;7)TGF-?1,p-TAK1,TAK1,p-AMPK?,AMPK?,PINK1,Parkin,LC3-I/II,Beclin-1,Atg5,Atg7,p62,PCNA,?-SMA,and MMP9 protein expression in lung tissue of mice was detected by western blot.Results:Part I: In vitro experiment1.TGF-?1 could induce the high expression of PINK1 and Parkin in RASMC cells,and the increase of PINK1 and Parkin protein levels and m RNA levels was the most significant when stimulated with 10 ng/ml TGF-?1.Treatment of cells with the TGF-?1 receptor inhibitor SB505124 inhibited TGF-?1-induced high PINK1 and Parkin protein expression and high m RNA levels.2.TGF-?1 can induce PINK1 and Parkin to be highly expressed in mitochondria,but lowly expressed in the cytoplasm in RASMC cells.TGF-?1 receptor inhibitor SB505124 could significantly increase PINK1 and Parkin expression in the cytoplasm and reduce expression in the mitochondria.3.TGF-?1 can induce the decrease of mitochondrial membrane potential in RASMC cells,and the weakening of fluorescence intensity can be observed under a fluorescence microscope,while inducing the increase of total cellular ROS level and mitochondrial ROS level,and the enhancement of fluorescence intensity.The above phenomenon was inhibited after application of the TGF-?1 receptor inhibitor SB505124.4.TGF-?1 can induce the high expression of mitochondrial division-related proteins Drp1 and Fis1 and adaptor molecule proteins OPTN and NDP52 in RASMC cells,but inhibit the expression of mitochondrial fusion-related proteins OPA1 and MFN2.These changes were reversed by the application of SB505124,a TGF-?1 receptor inhibitor.5.TGF-?1 can induce changes in mitochondrial morphology in RASMC cells,and the fibrillar morphology changes to a truncated or even fragmented division phenotype.These changes were reversed by the application of SB505124,a TGF-?1 receptor inhibitor.6.TGF-?1 can induce increased protein expression levels of autophagy-related proteins LC3II/LC3-I,Beclin-1,Atg5 and Atg7 and reduce p62 protein expression,a degraded substrate.Treatment of cells with the TGF-?1 receptor inhibitor SB505124down-regulated TGF-?1-induced high expression of LC3II/LC3 I,Beclin-1,Atg5,and Atg7 proteins and up-regulated p62 protein levels.7.Silencing PINK1 and Parkin gene expression can significantly reverse the increased expression of LC3II/LC3 I,Beclin-1,Atg5 and Atg7 and reduced p62 expression induced by TGF-?1 stimulation.8.autophagy inhibitor CQ was able to significantly enhance TGF-?1 induced expression of autophagy-related proteins LC3I/II,Beclin-1,Atg5 and Atg7.Also TGF-?1 induced p62 degradation was significantly inhibited by CQ,resulting in an increase in the degraded substrate p62.9.TGF-?1 can induce RASMC cell proliferation,and affect the RASMC cell cycle,so that the S phase and G2/M phase are prolonged.The TGF-?1 receptor inhibitor SB505124 and autophagy inhibitor 3-MA could reverse the above changes.10.TGF-?1 can induce the high levels of proliferation-related proteins PCNA,?-sma,and MMP9 in RASMC cells,and the TGF-?1 receptor inhibitor SB505124 and autophagy inhibitor 3-MA can reverse the above changes.11.TGF-?1 can promote the migration ability of RASMC cells,significantly reduce the distance of cell scratch,and increase the migration ability of cells,while TGF-?1receptor inhibitor SB505124 and autophagy inhibitor 3-MA treatment can significantly reduce the migration ability of cells.12.Small interfering RNA silencing of PINK1 and Parkin significantly decreased cell proliferation,PCNA,a-sma and MMP-9 protein expression in ASMCs,while reducing the percentage of S phase and G2/M phase.13.TGF-?1 could not induce the expression of TAK1 protein,but could significantly increase the expression of p-TAK1,and TGF-?1 receptor inhibitor SB505124 also had no effect on TAK1,but could inhibit the expression of p-TAK1.14.TGF-?1 can induce AMPK? phosphorylation levels in RASMC cells while inducing high PINK1 and Parkin expression,but the elevated phosphorylated AMPK?is blocked by TGF-?1 receptor inhibitors or TAK1 inhibitors.Similarly,TGF-?1induced highly expressed PINK1 and Parkin to be decreased by TAK1 inhibitor.Part II: In vitro experiment1.TGF-?1 was highly expressed in the lung tissue of asthmatic airway remodeling model mice,and the phosphorylation levels of TAK1 and AMPK? were significantly increased.These protein levels were suppressed in the TGF-?1 receptor inhibitor group.2.PINK1 and Parkin were highly expressed in the lung tissue of asthmatic airway remodeling model mice,but the protein level was significantly decreased in the inhibitor group.3.The m RNA levels of TGF-?1 in BALF and lung tissue were increased in asthmatic airway remodeling model mice,and their levels were positively correlated with tracheal smooth muscle thickness..4.TGF-?1 inhibitor group alleviated the infiltration of inflammatory cells in the airway,destroyed the tissue structure in the lung,and promoted goblet cell proliferation,mucus secretion and fibrosis in OVA-induced asthmatic airway remodeling mice.5.TGF-?1 inhibitor group could down-regulate the expression of autophagy-related proteins LC3I/II,Beclin-1,Atg5 and Atg7 in the lung tissue of OVA-induced asthmatic airway remodeling mice,while autophagy inhibitor CQ group was able to significantly enhance the expression of TGF-?1 induced autophagy-related proteins LC3I/II,Beclin-1,Atg5 and Atg7.Also TGF-?1 induced p62 degradation was significantly inhibited by CQ,resulting in an increase in the degraded substrate p62.6.TGF-?1 inhibitor CQ can inhibit the expression of smooth muscle proliferative protein in the lung tissue of asthmatic airway remodeling mouse model.7.TGF-?1 inhibitor CQ can reduce airway hyperreactivity.Conclusion:1.TGF-?1 mediates mitophagy by activating TAK1 and AMPK? to activate PINK1-Parkin pathway,and TGF-?1 mediated autophagy is involved in regulating RASMC cell proliferation and cell function.2.TGF-?1 promotes airway remodeling in asthma by activating the PINK1-Parkin pathway through the TAK1-AMPK? signaling axis to mediate autophagy.3.Targeted regulation of TGF-?1 mediated mitophagy may be a new strategy for the treatment of airway remodeling in asthma... |
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