Zinc salicylate reduces airway smooth muscle cells remodelling by blocking mTOR and activating p21(Waf1/Cip1)

 Asthma is characterized by chronic inflammation and tissue remodeling of the airways. Remodeling is resistant to pharmaceutical therapies. This study investigated the effect of zinc salicylate-methylsulfonylmethane (Zn-Sal-MSM) on remodeling parameters of human airway smooth muscle cells (ASMC).

Asthma is the most common chronic inflammatory lung disease worldwide with increasing prevalence

• It is characterized by shortness of breath, airway hyper-responsiveness, chronic airway inflammation, and increased airway wall thickness.
• Two major pathologies described as airway remodeling are:
• increased cell number of fibroblasts and/or smooth muscle and- increased deposition of extracellular matrices.

Cryo-preserved ASMC was used in all experiments

• The cells had been isolated from endo-bronchial tissue biopsies obtained from patients with moderate to severe asthma and non-asthma controls, and characterized by immune-chemistry.
• All experiments were performed in ASMC between passage 3 and 8.

Cell treatment

• Confluent ASMC were deprived of serum for 24 h for cell cycle synchronisation and quiescence.
• Following, the cells were incubated with increasing concentration (0.1-100 µg/mL) of either zinc salicylate-methylsulfonylmethane (Zn-Sal-MSM, 20:80; sourced from Nimni-Cordoba Tissue Engineering and Drug Discovery Laboratory, Division of Plastic and Reconstructive Surgery, Department of Surgery, Keck School of Medicine, University of Southern California, 1333 San Pablo Street, BMT-302, Los Angeles, CA 90033, USA) or zinc chloride for 24 hours, and where then activated by 10 ng/mL human recombinant platelet-derived growth factor-BB.

Cell proliferation, activity, and toxicity

• Cell proliferation was determined by manual cell counts using ASMC and cell numbers determined using an improved Neubauer chamber slide
• MTT assay determined by assay after incubation (4 hours, 37°C) with 500 µg/mL MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide) before being lysed with dimethyl sulfoxide (DMSO) and absorbance measured at 560 nm (micro plate reader, BioTek, Winooski, USA)

Proteome Analysis by Mass Spectrometry

• Secreted proteins were compared in pairs by mass spectrometric analysis which was performed in 200 µL of cell supernatant by SWATH method on a QE-HF platform and analyzed by Spectronaut software against an internal HEK-cell line spectral library (Biocentre, University of Basel).

Western-Blot

• Protein expression was determined by Western-blotting for PCNA, p21(Waf1/Cip1), p27(kip), collagen type I and fibronectin after 24 and 48 h.
• Proteins were extracted in RIPA-buffer, concentration determined by Bradford assay, and equal amount of protein were size fractionated in a gradient PAGE gel (BioRad, Basel, Switzerland) at 100 V, opened current for 1 h.

Phosphorylation of Erk1/2 MAPK by ELISA

• Western blotting and ELISA were used to detect phosphorylation
• ELISA was performed according to the distributor's instructions
• Cells were exposed to either Zn-Sal-MSM alone or 10 min after stimulation with PDGF-BB (10 ng/mL) and lysed after various incubation times

ECM deposition by ELISA

• Cells were seeded into a 96-well plate and grown to confluence, before being serum deprived for 24 h.
• The cells were washed 3x with PBS and fixed for 2 × 5 min in 4% formaldehyde and washed once more with PBS. Unspecific antibody binding was reduced by incubating the fixed cells for 1 h with PBS containing PBS containing 0.01% TWEEN-20 and 2% bovine serum albumin (blocking buffer).
• Collagen type-I, or fibronectin were detected by incubated the cells in blocking buffer containing an antibody specific to either collagen types I and II for overnight at 4°C.

MMP zymography

• Cell supernatant was collected 24 hours after treating the cells with Zn-Sal-MSM at various concentrations.
• Equal amounts of each sample were applied onto a 10% polyacryamide gel containing 0.1% of porcine gelatine and were applied to electrophoresis (125 V, 30 mA, 60 min).
• The gel was then equilibrated in distilled water containing 2.5% Triton X-100 (2 × 30 min) before being incubated overnight in an enzyme activation buffer (50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2, brij-35). MMPs were visualized by negative staining with SimplyBlue Safestain and photographed.

Statistics

• The null hypothesis: Zn-Sal-MSM has no effect on PDGF-BB induced proliferation, ECM deposition, or MMP activity was tested by application of ANOVA, Mann-Whitney U-test, or Student's t-test.

Zn-Sal-MSM inhibited proliferation by up-regulating p21(Waf1/Cip1) expression

• It inhibited PDGF-BB induced proliferation of ASMC in a dose-dependent manner.
• In contrast, ZnCl2, at the same concentrations, was toxic at all concentrations and reduced cell numbers below the seeding density.

Zn-Sal-MSM reduced the deposition of collagen type-I

• PDGF-BB significantly upregulated collagen deposition in ASMC compared to non-asthma tissue donors, but no significant effect on fibronectin deposition (Fig. 4A).
• Preincubation with zinc-salamisam significantly reduced collagen deposition compared to PDGFBB alone at concentrations >10 µg/mL.
• Interestingly, in cells derived from asthma patients, the effect was stronger in cells from people with asthma, but not asthma patients.

Zn-Sal-MSM Modified the Expression of Mitochondria Regulating Proteins

• 42 mitochondria regulating proteins were downregulated, while four were upregulated.
• Interestingly, activating protein 2 (AP2), known as a cell proliferation driving protein, was significantly downregulated by the drug
• Other proliferation controlling proteins including Akt2, PI3K-receptor 1, and TM7SF2 were amongst those proteins that were most significantly affected

Discussion

• Zn-Sal-MSM has the potential to reduce several aspects of ASMC remodeling, an important pathology in asthma, which cannot be reduced or prevented by any available medication.
• The major contributor to airway wall remodeling in asthma are ASMC and it was reported earlier that the mechanism regulating their proliferation is not well controlled in asthma.
• Another pathology included in "remodeling" is the increased deposition of proinflammatory collagen type-I [38]. There is no asthma therapy that reduced collagen Type-I deposition in the asthmatic airway, only short-term application of heat to lastingly reduce the extracellular matrix.

Reference

• 10.1016/j.jnutbio.2020.108563