Red ginseng aqueous extract improves mucociliary transport dysfunction and histopathology in CF rat airways

Abstract

Background: We previously discovered that Korean red ginseng aqueous extract (RGAE) potentiates the TMEM16A channel, improved mucociliary transport (MCT) parameters in CF nasal epithelia in vitro, and thus could serve as a therapeutic strategy to rescue the MCT defect in cystic fibrosis (CF) airways. The hypothesis of this study is that RGAE can improve epithelial Cl^- secretion, MCT, and histopathology in an in-vivo CF rat model.

Methods: Seventeen 4-month old CFTR^-/- rats were randomly assigned to receive daily oral control (saline, n = 9) or RGAE (Ginsenosides 0.4mg/kg/daily, n = 8) for 4 weeks. Outcomes included nasal Cl^- secretion measured with the nasal potential difference (NPD), functional microanatomy of the trachea using micro-optical coherence tomography, histopathology, and immunohistochemical staining for TMEM16a.

Results: RGAE-treated CF rats had greater mean NPD polarization with UTP (control = -5.48 +/- 2.87 mV, RGAE = -9.49 +/- 2.99 mV, p < 0.05), indicating, at least in part, potentiation of UTP-mediated Cl^- secretion through TMEM16A. All measured tracheal MCT parameters (airway surface liquid, periciliary liquid, ciliary beat frequency, MCT) were significantly increased in RGAE-treated CF rats with MCT exhibiting a 3-fold increase (control, 0.45+/-0.31 vs. RGAE, 1.45+/-0.66 mm/min, p < 0.01). Maxillary mucosa histopathology was markedly improved in RGAE-treated cohort (reduced intracellular mucus, goblet cells with no distention, and shorter epithelial height). TMEM16A expression was similar between groups.

Conclusion: RGAE improves TMEM16A-mediated transepithelial Cl^- secretion, functional microanatomy, and histopathology in CF rats. Therapeutic strategies utilizing TMEM16A potentiators to treat CF airway disease are appropriate and provide a new avenue for mutation-independent therapies.

Keywords: CFTR; Calcium-activated chloride channel; Cystic fibrosis; Cystic fibrosis rat; Functional microanatomy; Ginsenoside; Micro-optical coherence tomography; Mucociliary clearance; Mucociliary transport; Nasal potential difference; Red ginseng; TMEM16A; TMEM16A potentiator.

Figure 1.

Representative NPD tracings showing PD after serial administration of 1) amiloride (100 μM), 2)Cl⁻ free forskolin (20 μM), 3)INH-172 (10 μM) and GlyH-101 (10 μM), and 4) UTP (150 μM) (A), followed by summary data (B) in RGAE-treated and control CF rats. RGAE treatment caused NPD hyper-polarization with UTP (control = −5.96 +/− 2.3 mV, RGAE = −12.51 +/− 1.04 mV, p <0.05; unpaired t test), indicating potentiation of UTP-mediated Cl⁻ secretion through TMEM16A.

Figure 2.

Representative μOCT images (A) and summary data for ASL (B), PCL (C), CBF (D), and MCT (E) recorded in tracheas excised from control and RGAE-treated CF rats (*P<0.05, **P<0.01; unpaired t test). RGAE significantly improved all markers of functional anatomy (ASL – airway surface liquid, PCL – periciliary liquid, CBF – ciliary beat frequency, MCT – mucociliary transport, RGAE – red ginseng aqueous extract). White line = ASL depth. Representative μOCT videos are provided as supplementary data.

Figure 3.

Representative hematoxylin/eosin-stained maxillary sinus mucosa (A) of 10× H&E stained mucosa from control and ginseng-treated CF rats. Summary data regarding number of goblet cells (B) and epithelial height (C). RGAE-treated CF rats demonstrate marked improvement in histopathology (*P<0.05, **P<0.01, unpaired t test).

Figure 4.

Representative Alcian Blue – Periodic Acid Schiff staining of nasal septum epithelium (A) from control and ginseng-treated CF rats. Summary data demonstrates significant reduction in intracellular mucus in RGAE-treated CF rats (*P<0.05, unpaired t test).

Figure 5.

Representative TMEM16A immunohistochemical staining between control and RGAE-treated CF rats (A). Summary data demonstrates no increase in TMEM16a staining intensity in RGAE treated CF rats (B).