Specific Histone Deacetylase 6 Inhibitors Regulate The Proliferation And Migration Of Human Airway Smooth Muscle Cells Through The PI3K/AKT Pathway

Background: Bronchial asthma(asthma)is a common chronic respiratory disease,which is characterized by airway inflammation,airway remodeling and airway hyperresponsiveness.The proliferation,hypertrophy and migration of airway smooth muscle cells play an important role in airway remodeling.Histone deacetylases(HDACs)regulate the deacetylation of histone and non-histone proteins,and also participate in the regulation of smooth muscle cell proliferation,migration,contraction and phenotypic switching and other biological processes.HDAC6 is a member of the histone deacetylase family.Our previous study found that selective inhibition of HDAC6 can effectively improve airway inflammation in asthmatic mice,but the role of HDAC6 in airway remodeling in asthma is still unclear.Objective: To investigate the effect of HDAC6 specific inhibitor Tubastatin A HCl on the proliferation and migration of human airway smooth muscle cells induced by platelet-derived growth factor BB and its possible molecular mechanism.Methods: Human airway smooth muscle cells were routinely cultured in a smooth muscle cell culture system.The airway smooth muscle cells were pretreated with serumfree airway smooth muscle cell medium containing 1,2,4,8,16 and 32 u M final concentrations of TSA for 1 h,and then PDGF-BB solution was added to the medium to make the final concentration of PDGF-BB in the medium 20 ng/ml,and the cells were cultured for 48 h.The Cell proliferation of each group was detected by cell Counting Kit-8(CCK-8),and the appropriate concentration of TSA was selected.In the following experiment,we divided human airway smooth muscle cells into 4 groups: group A: Control group,group B: PDGF-BB(20 ng/ml)group,group C: PDGF-BB + TSA(32u M)group,group D: TSA group.The migration of the four groups of cells was detected by cell scratch test.Western blot was used to detect the expression of HDAC6 protein expression levels,Phosphoinositide 3-kinase(PI3K)protein expression levels,Protein kinase B(AKT)protein expression levels,p-PI3 K protein expression levels and p-AKT protein expression levels in each group.Results: 1.CCK-8 results showed that compared with the Control group,compared with the Control group,the cell viability of PDGF-BB group and different concentrations of TSA(1,2,4,8,16,32 u M)+PDGF-BB group were 194.30% ± 19.31%,180.42% ±10.08%,174.33% ± 0.77%,166.32% ± 3.54%,161.32% ± 3.71%,142.00% ± 3.60% and98.35% ± 3.15% respectively.Compared with the Control group,PDGF-BB(20 ng/ml)significantly increased cell viability,while pretreatment of cells with 32 u M TSA for 1h reduced PDGF-BB-induced cell viability from 194.30% ± 19.31% to 98.35% ± 3.15%(P<0.001),and there was no significant difference in cell viability between the cells treated with 32 u M TSA combined with PDGF-BB and the control group(P>0.05),so TSA at32 u M concentration was selected for the next experiment.2.The results of scratch test showed that PDGF-BB could significantly promote the migration of human airway smooth muscle cells.The migration distances of PDGF-BB group,PDGF-BB+TSA group and TSA group were 2.98 ± 0.10 times,1.62 ± 0.09 times and 0.66 ± 0.05 times of Control group,respectively.Wound healing assay showed that PDGF-BB significantly promoted the migration of human airway smooth muscle cells(P<0.001).TSA pretreatment partially inhibited PDGF-BB-induced cell migration(P<0.001),and TSA alone mildly inhibited cell migration(P<0.05).3.Western Blot results showed that compared with the control group,PDGF-BB stimulation significantly increased the protein expression levels of HDAC6,p-PI3 K and p-AKT in human airway smooth muscle cells,and the ratio of p-PI3K/PI3 K and pAKT/AKT were increased.Pretreatment of the cells with 32 u M TSA for 1h effectively reduced the protein expression levels of HDAC6,p-PI3 K and p-AKT induced by PDGFBB,and the ratio of p-PI3K/PI3 K and p-AKT/AKT decreased.TSA treatment alone decreased the expression of HDAC6 protein and the ratio of p-PI3K/PI3 K and pAKT/AKT.Conclusions: Tubastatin A HCl,a specific HDAC6 inhibitor,can inhibit the proliferation and migration of human airway smooth muscle cells induced by PDGF-BB,and its mechanism may be related to PI3K/AKT signaling pathway.