Background: Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist. Methods: Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses. Results: Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-β1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-β1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-β1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP. Conclusions: Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs.

The role of histone deacetylases 6 (HDAC6) has been elucidated in various neurodegenerative diseases. However, the effect of HDAC6 on retinal degenerative processes remains unknown. The aim of this study was to elucidate the potential role of HDAC6 in the retinal ischaemia and reperfusion (I/R) injury model. The retinal pathological lesion was evaluated by haematoxylin and eosin (H&E) staining. HDAC expression or activity was detected by immunohistochemistry, Western blotting assays or colorimetric assays. The expression of apoptotic- and autophagic- related proteins were quantified by Western blotting and RT-PCR. The expression of peroxiredoxin 2 (Prx2) was determined by RT-PCR and ELISA. The levels of acetylated α-tubulin and acetylated histone 3 in the retina were assayed by Western blotting. We found that I/R-induced reduction of the retinal thickness was ameliorated, and the survival of RGCs was increased by the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) as well as by tubacin (an HDAC6 selective inhibitor). The decreased expression of THY (thymus cell antigen) in the I/R-induced retinas was also reversed by TSA and tubacin. Elevated HDAC6 expression and activity in the retina from I/R injury were significantly inhibited by tubacin, which also attenuated I/R-mediated apoptosis by decreasing TUNEL-positive RGCs and Bax expression and increasing Bcl-2 expression. Additionally, tubacin increased the expression of autophagy-related gene Beclin 1 and microtubule-associated protein 1 light chain 3B (LC3B) and the levels of Prx2. Furthermore, the protective effect of tubacin was associated with acetylated α-tubulin and was independent of acetylated histone 3. Our findings suggest that tubacin exhibits neuroprotective effects after I/R retinal injury, and HDAC6 may be a potential therapeutic target for the retinal neurodegenerative disease of glaucoma.

Background: Glaucoma is a neurodegenerative disease that shares similar pathological mechanisms with Alzheimer’s disease (AD). Drug treatments for glaucoma increasingly rely upon both lowering of intraocular pressure (IOP) and optic nerve protection, as lowering of IOP alone has been unsatisfactory. Huperzine A (HupA) is an acetylcholinesterase inhibitor (AChEI) used for AD. This study investigated the potential of HupA as a treatment for glaucoma. Methods: The ability of HupA to lower IOP via causing pupil constriction was assessed using New Zealand rabbits. The retinal neuroprotective effects of HupA were assessed in vivo using rat retinas subjected to ischemia-reperfusion (I/R) and in vitro using primary retinal neurons (PRNs) suffering from oxygen-glucose deprivation (OGD). Results: HupA caused pupil constriction in a dose-time dependent manner which was reversed by the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine and the selective M3 mAChR antagonist 4-DAMP. However, HupA had no effect on isolated iris muscle tension and calcium flow indicating an indirect M3 mAChR mediated effect. HupA exerted a neuroprotective effect against I/R and OGD to attenuate the retinal pathological lesion, improve retinal neuronal cell viability, reverse oxidative stress injury by increasing GSH levels and SOD activity, and decreasing MDA content and reduce the retinal neuronal apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 expression with no effect on the calcium flow tests. The effects were abolished by atropine and the selective M1 mAChR antagonist pirenzepine in OGD-induced PRNs suggesting an indirect M1 mAChR-mediated effect via inhibiting AChE activity to increase endogenous ACh level. Furthermore, HupA increased phosphorylated AKT level and decreased the levels of phosphorylated JNK, P38 MAPK and ERK via M1 mAChR antagonists indicating an involvement of activating the M1 mAChR and the downstream AKT/MAPK signaling pathway in the protective effects of HupA. Conclusions: HupA could significantly decrease IOP via activating M3 mAChR indirectly and produce retinal neuroprotective effect through M1 mAChR/AKT/MAPK by increasing endogenous ACh level. These investigations demonstrated that HupA was an effective drug in glaucoma treatment and the clinical application of HupA and other AChEIs for glaucoma patients should be further investigated.

Histone deacetylase (HDAC) 2 plays a vital role in modifying histones to mediate inflammatory responses, while HDAC2 itself is commonly regulated by post-translational modifications. Small ubiquitin-related modifier (SUMO), as an important PTM factor, is involved in the regulation of multiple protein functions. Our previous studies have shown that carbocisteine (S-CMC) reversed cigarette smoke extract (CSE)-induced down-regulation of HDAC2 expression/activity in a thiol/GSH-dependent manner and enhanced sensitivity of steroid therapy. However, the mechanism by which S-CMC regulates HDAC2 is worth further exploring. Our study aimed to investigate the relationships between HDAC2 sumoylation and its deacetylase activity under oxidative stress and the molecular mechanism of S-CMC to regulate HDAC2 activity that mediates inflammatory responses in human bronchial epithelial cells. We found that modification of HDAC2 by SUMO1 and SUMO2/3 occurred in 16HBE cells under physiological conditions, and CSE induced SUMO1 modification of HDAC2 in a dose and time-dependent manner. K462 and K51 of HDAC2 were the two major modification sites of SUMO1, and the K51 site mediated deacetylation activity and function of HDAC2 on histone H4 that regulates IL-8 secretion. S-CMC inhibited CSE-induced SUMO1 modification of HDAC2 in the presence of thiol/GSH, increased HDAC activity, and decreased IL-8 expression. Our study may provide novel mechanistic explanation of S-CMC to ameliorate steroid sensitivity treatment in chronic obstructive pulmonary disease.

Muscarinic acetylcholine receptors (mAChRs) have been identified in airway epithelium, and epithelium-derived chemokines can initiate the migration of airway smooth muscle (ASM) cells. However, the mAChRs that are expressed in airway epithelium and the mechanism underlying the regulation of ASM cell migration are not clear. The aim of this study was to test whether the effects of the epithelium-derived chemokines on ASM cell migration could be modulated by mAChRs. Human epithelial cells (A549 cells) were stimulated with cigarette smoke extract (CSE) or the mAChRs agonist carbachol. IL-8 and TGF-β1 production were measured by ELISA, and human ASM cell migration was measured using the transwell migration assay and scratch assay. The mRNA levels of the mAChRs subtypes and the acetylcholine concentrations were measured using RT-PCR and LC–MS/MS, respectively. ASM cell migration toward CSE-stimulated A549 cells was markedly reduced by Ac-RRWWCR-NH2 (IL-8 inhibitor) and SB431542 (TGF-β1 inhibitor). CSE-induced ASM cell migration was also suppressed by the mAChRs antagonist tiotropium. Interestingly, carbachol-stimulated A549 cells also induced ASM cell migration; this migration event was suppressed by tiotropium, Ac-RRWWCR-NH2 and SB431542. In addition, the effects of CSE on ASM cell migration were significantly and cooperatively enhanced by carbachol compared to CSE alone. Carbachol-induced ASM cell migration was reduced by selective inhibitors of PI3K/Akt (LY294002) and p38 (SB203580), suggesting that it occurred through p38 and Akt phosphorylation, which was inhibited by the M3 mAChR antagonist 4-DAMP. These findings indicate that M3 mAChR may be important therapeutic target for obstructive airway diseases, as it regulates the effects of the epithelial-derived chemokines on ASM cell migration, which results in lung remodeling.