| Objective: Asthma is one of the most common chronic respiratory diseases.With the change of global climate and environment and the change of people's living standard,the incidence rate of asthma is increasing worldwide,and it has become a serious public health problem.Asthma is a kind of chronic airway inflammation,characterized by persistent airway inflammation,airway hyperresponsiveness and airway remodeling.Airway remodeling may occur in the early stage of the disease and is closely related to the occurrence of severe asthma and refractory asthma.Bronchial epithelial cells are the first barrier for the body to contact allergens and harmful substances in the environment.After exposure to harmful stimuli,bronchial epithelial cells can participate in airway remodeling through epithelial mesenchymal transition(EMT),and play an important role in the defense mechanism of asthma.Thus,how to inhibit EMT process in bronchial epithelial cells and reduce airway remodeling has become the focus of asthma prevention and treatment.Sirtuin is a class III histone deacetylase,which is involved in many physiological processes such as energy metabolism balance,cell anti-stress ability,cell proliferation,genome stability and aging.Recently,studies have shown that sirtuin also plays an important role in inflammation and fibrosis.In asthma research,SIRT1 and its agonists have anti-inflammatory effects,while SIRT2 has pro-inflammatory effects.It has been found that SIRT6 is highly expressed in bronchial epithelial cells isolated from asthmatic patients,thus it might be involved in the pathogenesis of asthma.However,its expression during airway remodeling process and its role in airway remodeling is not clear.To solve these problems,we established a rat model of airway remodeling to explore the expression and location of SIRT6 in airway remodeling of asthma.Furthermore,in vitro experiments were conducted to investigate whether SIRT6 can affect the EMT process and EMT related cell behavior of airway epithelial cells.Finally,the possible mechanism of SIRT6 affecting EMT was clarified.Methods: 1.Rat airway remodeling models,EMT indicators expression and SIRT6 expression(1)The establishment of airway remodeling model in rats with asthma.Forty SD rats were divided into four groups: PBS-4weeks group,OVA-4 weeks group,PBS-8 weeks group and OVA-8 weeks group.The rats in OVA group were sensitized by intraperitoneal injection of OVA and aluminum hydroxide on day 0,7 and 14 of the experiment.The rats were challenged with OVA from day 21 of the experiment,once every other day,and the excitation time was 4 weeks and 8 weeks respectively.Rats in PBS groups were sensitized and challenged with PBS.(2)BALF were collected and the leukocytes in BALF were counted and classified.(3)H&E staining and Masson staining were used to observe the pathological changes and immunohistochemical staining was used to access the expression of Ecadherin,a-SMA and SIRT6 in airway epithelial cells.(4)Western blot was used to access the protein expression of E-cadherin,Ncadherin,vimentin,a-SMA and SIRT6 in the lung tissue of airway remodeling rats.PCR was used to access the m RNA expression of E-cadherin,a-SMA and SIRT6.2.The effects of SIRT6 on TGF-b1 induced EMT indicators,cell migration and proliferation(1)The bronchial epithelial cell line(16HBE)was cultured in vitro.The changes of EMT indicators were identified in epithelial cells induced by TGF-b1.The expression levels of E-cadherin,N-cadherin,vimentin,a-SMA and SIRT6 in 16 HBE cells stimulated by TGF-b1 were detected by Western blot and PCR.(2)SIRT6 was knocked down in 16 HBE cells.The expression levels of E-cadherin,N-cadherin,vimentin,a-SMA and SIRT6 were detected by Western blot and PCR.Whether SIRT6 knockdown affects the changes of EMT index induced by TGF-1 was explored.The migration and proliferation of cells were detected by Transwell and CCK8 assays.Whether SIRT6 downregulation affects TGF-b1 induced cell migration and proliferation was explored.(3)SIRT6 was overexpressed in 16 HBE cells.The expression levels of E-cadherin,N-cadherin,vimentin,a-SMA and SIRT6 were detected by Western blot and PCR.Whether the overexpression of SIRT6 could affect the changes of EMT indicators in TGF-b1 induced cells was explored.SIRT6 was overexpressed in 16 HBE cells.The migration and proliferation of cells were detected by Transwell and CCK8 assays to determine whether the overexpression of SIRT6 affects the TGF-b1 induced cell migration and proliferation.(4)SIRT6 was overexpressed in 16 HBE cells,the m RNA expression levels of IL-6 was detected by PCR.3.The mechanisms of SIRT6 inhibition of EMT indicators,cell cell migration and proliferation.(1)Western blot was used to detect the protein expression levels of Erk1/2,pErk1/2,Smad2,p-Smad2,Smad3 and p-smad3 in airway remodeling rat model and TGF-b1 induced EMT cell model.(2)SIRT6 was overexpressed in 16 HBE cells.The protein expression levels of Erk1/2,p-Erk1/2,Smad2,p-Smad2,Smad3 and p-smad3 were detected by Western blot to determine whether SIRT6 affects the activation of Erk and Smad pathways.(3)SIRT6 was overexpressed in 16 HBE cells,the expression of MMP9 and c-Jun was detected by Western blot and PCR,and the acetylation level of H3K9 was detected by immunofluorescence.(4)The effect of SIRT6 on the transcriptional activity of c-Jun promoter was detected by dual luciferase activity reporter system.Results: 1.In asthmatic airway remodeling model,SIRT6 was localized in bronchial epithelial cells and the expression increased(1)In the animal model of airway remodeling of asthma,the number of inflammatory cells in alveolar lavage fluid increased(P < 0.01),mainly eosinophilic airway inflammation.There were a lot of inflammatory cells infiltration around the trachea.Thickened airway wall(P < 0.01),thickened smooth muscle layer around the bronchus(P < 0.05),and increased collagen deposition around the trachea can be found(P < 0.01).(2)In the OVA-4 weeks and OVA-8 weeks group,the expression of E-cadherin protein in the lung tissue of airway remodeling rats decreased(P < 0.01),while the expression of N-cadherin,vimentin and a-SMA protein in the interstitial cells increased(P < 0.05).In the airway remodeling groups,the expression of E-cadherin m RNA decreased,while the expression of a-SMA m RNA increased(P < 0.01).At the same time,the results of immunohistochemistry showed that E-cadherin decreased in the bronchial epithelial cells of the model group,accompanied by the increase of aSMA(P < 0.05).The results showed that EMT changes were found in the lung and bronchial epithelial cells of the model group.(3)The expression level of SIRT6 protein and m RNA in the lungs of OVA-4 weeks and OVA-8 weeks group were higher than that of the corresponding control group(P < 0.01).Immunohistochemistry showed that SIRT6 was expressed in bronchial epithelial cells,alveolar epithelial cells and inflammatory cells,but SIRT6 was most expressed in airway epithelial cells.The expression level of SIRT6 in airway remodeling group was higher than that in control group(P < 0.05).2.In 16 HBE cells,SIRT6 overexpression inhibited EMT,proliferation and migration induced by TGF-b1(1)After being treated with 10 ng / ml TGF-b1 for 48 hours,the expression of Ecadherin protein decreased(P < 0.001),and the expression of N-cadherin,vimentin and a-SMA protein increased(P < 0.05).The morphology of the cells changed from polygonal and paving stone to long fusiform.The results of immunofluorescence showed that E-cadherin on the cell membrane was lost after TGF-b1 treatment,and the expression of a-SMA in the cytoplasm was increased,and the cells showed EMT changes.After 48 hours of TGF-b1 treatment,SIRT6 expression in 16 HBE cells was significantly up-regulated(P < 0.05).(2)Sh RNA 2 significantly reduced SIRT6 protein expression and m RNA expression in 16 HBE cells(P < 0.001).However,SIRT6 knockdown did not affect the changes of EMT markers induced by TGF-b1.SIRT6 knockdown had no significant effect on TGF-b1 induced cell proliferation and migration(P > 0.05).(3)The SIRT6 expression level in the cells was significantly increased by plasmid transfection and screening(P < 0.001).Western blot showed that SIRT6 could inhibit the down-regulation of E-cadherin protein induced by TGF-b1 and decrease the up-regulation of N-cadherin,vimentin and a-SMA protein induced by TGF-b1(P < 0.05).PCR also showed that overexpression of SIRT6 could attenuate the down-regulation of E-cadherin m RNA induced by TGF-b1 and up-regulation of N-cadherin,vimentin and a-SMA m RNA levels(P < 0.05).The results of immunofluorescence showed that over expression of SIRT6 could reduce the loss of E-cadherin on the cell membrane,inhibit the increase of a-SMA in the cytoplasm,and maintain the epithelioid morphology.(4)Overexpression of SIRT6 attenuated TGF-b1 induced cell migration(P < 0.01).CCK8 showed that over expression of SIRT6 could also reduce the cell proliferation level at 72 hours,96 hours and 120 hours(P < 0.05;P < 0.05;P < 0.01).(5)In 16 HBE cells,overexpression of SIRT6 reduced the m RNA expression of EMT related cytokines IL-6(P < 0.01).3.SIRT6 inhibits EMT by regulating Smad3 activation and c-Jun expression(1)In the lung tissue of airway remodeling model,the expression levels of pERK1 / 2,p-Smad2 and p-smad3 were higher than those of the corresponding control group,suggesting that ERK pathway and Smad were activated(P < 0.05;P < 0.05;P < 0.05).In 16 HBE cells,the expression levels of p-ERK1/2,p-Smad2,and p-smad3 increased 24 hours and 48 hours after TGF-1 treatment(P < 0.05),suggesting that ERK pathway and Smad were activated in the cell model.(2)In 16 HBE cells,overexpression of SIRT6 decreased the expression of psmad3 induced by TGF-b1(P < 0.05),indicating that SIRT6 could inhibit the activation of Smad3.Over expression of SIRT6 did not affect the expression of pERK1/2 and p-Smad2(P > 0.05).(3)MMP9 is an important indicator of cell migration.Western blot showed that overexpression of SIRT6 reduced the expression of MMP9 induced by TGF-b1(P < 0.05).PCR showed that overexpression of SIRT6 also reduced the expression of MMP9 at m RNA level(P < 0.05).(4)The protein c-Jun can promote the transcription of MMP9.In SIRT6 overexpressed cells,the protein and m RNA expression levels of c-Jun decreased(P < 0.05;P < 0.05).At the same time,it was found that the acetylation level of H3K9 in cells decreased significantly.SIRT6 could inhibit the transcription activity of c-Jun promoter(P < 0.05).It is suggested that SIRT6 might inhibit the expression of MMP9 by inhibiting the transcription of c-Jun and down regulating the expression of c-Jun.Conclusions: 1.In the airway remodeling model of asthma,SIRT6 is mainly located in the bronchial epithelial cells.The increased expression of SIRT6 in this model was accompanied by EMT of airway epithelial cells.2.In 16 HBE cells,TGF-? 1 can up regulate SIRT6.Over expression of SIRT6 can inhibit the loss of epithelial markers,inhibit the increase of stromal markers,and help maintain epithelial cell morphology;SIRT6 can inhibit TGF-?1 induced cell proliferation and cell migration.SIRT6 also inhibited the expression of IL-6.3.SIRT6 inhibits EMT by regulating TGF-?1/Smad3 signaling pathway.SIRT6 can reduce the acetylation of histone H3K9 and c-jun promoter activity,which may be one of the mechanisms of SIRT6 inhibiting cell proliferation and migration. |
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