Carbocisteine Ameliorates Glucocorticoids Resistance In COPD Via HDAC2 Mediated Regulation

Chronic obstructive pulmonary disease(COPD)is a chronic inflammatory disease which mainly affects the small airways and lung parenchyma,leading to progressive airway obstruction and not fully reversible airflow limitation.Inhibition of airway inflammation becomes a key factor to control the progressive development of COPD,but the anti-inflammatory agent,glucocorticoids(GCs)produces the poor response to COPD patients.Such GCs resistance or insensitivity is found to be closely related to the down-regulation of HDAC2 expression/activity partly caused by oxidative stress.Carbocisteine(S-CMC)is commonly used as clinical expectorant and thiol antioxidant,which is also reported to reduce COPD exacerbations.However,whether S-CMC improves the GCs resistance by regulation of HDAC2 needs to be defined to provide novel theoretical evidence for S-CMC in clinical COPD treatment.Firstly,the animal model of COPD with GCs resistance was established by 12 weeks’ cigarette smoking to explore the effects of S-CMC on improving GCs resistance.The effects of dexamethasone(DEX,i.p.)and S-CMC(i.g.)during the last 6 weeks’ cigarette smoking were examined by the pathologic features of COPD rats by ELISA,Western Blot,histochemical&immunohistochemical staining,differential blood count,muscle tension experiment in vitro and lung function test,etc.In the COPD rats with GCs resistance,DEX did not significantly improve the chronic airway inflammation,emphysema,airway remodeling and lung function impair.The co-treatment of S-CMC could reverse the above-mentioned features and effectively inhibited the process of COPD.Simautaneously,down-regulation of HDAC2 expression/activity was ameliorated by S-CMC treatment.Secondly,the cellular model with GCs resistance was established though cigarette smoke extracts(CSE)-induced cellular inflammation to further explore the effect of S-CMC for improving DEX sensitivity by regulating HDAC2.The levels of IL-8 and TNF-α in A549 cells supernatant were determined by ELSA.S-CMC could significantly improve the anti-inflammatory effects of DEX in terms of both efficacy(Imax)and potency(IC50).The decrease of HDAC2 expression/activity induced by CSE was counteracted by S-CMC alone and more remarkedly by S-CMC plus DEX.The lentivirus transfection of shRNA to silence the expression of HDAC2 could deminish the aboved effects of S-CMC.Furthermore,the effects of S-CMC on regulating HDAC2 and improving DEX sensitivity could be potentiated by the thiol reducing agent DTT,and inhibited by thiol oxidant diamide.S-CMC up-regulated HDAC2 leading to enhancing histone H4 deacetylation and inhibiting the IL-8 gene transcription,which could also be blocked by glutathione synthetase inhibitors.The direct interaction of HDAC2 with GSH but not with S-CMC was found by enzyme function experiment and SPR,implying the effect of S-CMC on regulating HDAC2 may be GSH-dependent.Thirdly,the molecular mechanism of S-CMC was explored on HDAC2 SUMOylation.Both SUMO1 and SUMO2/3 modifications in HDAC2 were found by immunofluorescence and Co-IP in 16 HBE cells under the physiological condition.Through three SUMO softwares,site-specific mutation and Co-IP,K462 site and K51 site were revealed to be the modification sites of SUMO1 on HDAC2.The mutation of K51 site could depress the effects of HDAC2 on histone H4 deacetylation and IL-8 transcription.CSE could increase SUMO1 but not SUMO2/3 modification of HDAC2,which could be counteracted by S-CMC in a GSH dependent manner.Taken together,S-CMC could improve the GCs resistance by up-regulation of HDAC2 and the finding may provide new theoretical basis for S-CMC in clinical COPD treatment.