Clin Exp Otorhinolaryngol.  2014 Mar;7(1):13-18.

Protective Effect of Hexane and Ethanol Extract of Piper Longum L. on Gentamicin-Induced Hair Cell Loss in Neonatal Cultures

• Yadav MK
• Choi J
• Song JJ

Abstract

OBJECTIVES
Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Piper longum L. (PL) is a well-known spice and traditional medicine in Asia and Pacific islands, which has been reported to exhibit a wide spectrum of activity, including antioxidant activity. In this study, we evaluated the effect of hexane:ethanol (2:8) PL extract (subfraction of PL [SPL] extract) on GM-induced hair cell loss in basal, middle and apical regions in a neonatal cochlea cultures.
METHODS
The protective effects of SPL extract were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. The anti-apoptosis activity of SPL extract was measured using double labeling by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and myosin-7a staining. The radical-scavenging activity of SPL extract was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.
RESULTS
SPL extract at a concentration of 1 microg/mL significantly inhibited GM-induced hair cell loss at basal and middle region of cochlea, while 5 microg/mL was effective against apical region hair cell loss. The protective effect of SPL extract was concentration dependent and hair cells retained their stereocilia in explants treated with SPL extract prior to treatment with 0.3 mM GM. SPL extract decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. The outer hair and inner hair counts were not decreased in SPL extract treated groups in compare to GM treated explants. Additionally, SPL extract showed concentration dependent radical scavenging activity in a DPPH assay.
CONCLUSION
An anti-apoptosis effect and potent radical scavenger activity of SPL extract protects from GM-induced hair cell loss at basal, middle and apical regions in neonatal cochlea cultures.

Keyword

Gentamicin; Autotoxicity; Inner hair cell; Outer hair cell; Piper longum; Apoptosis; Antioxidant

MeSH Terms

Animals
Apoptosis
Asia
Cochlea
DNA Nucleotidylexotransferase
Ear, Inner
Ethanol*
Gentamicins
Hair*
In Situ Nick-End Labeling
Medicine, Traditional
Mice
Neurons
Pacific Islands
Phalloidine
Piper*
Reactive Oxygen Species
Spices
Stereocilia
DNA Nucleotidylexotransferase
Ethanol
Gentamicins
Phalloidine
Reactive Oxygen Species

Figure

• 

  Fig. 1 Phalloidin labeled outer hair cell and inner hair cell count. (A-C) show hair cell counts per 100 µm length of basal, middle and apical part of cochlea, respectively. Bundles labeled with phalloidin strain were counted under a fluorescence microscope and are presented as the mean±SD. The hair cell count of a single sample was the average of five fields, and 5-11 specimens were assessed for each condition. The results were significant (*P<0.01, **P<0.005) as compared with the gentamicin-alone group. IHC, inner hair cell; OHC, outer hair cells; GM, gentamicin; PL, Piper longum L.

• 

  Fig. 2 Microscopic images of phalloidin labeled inner hair cell and outer hair cell. All examples are from comparable basal, middle and apical regions of explants. (A-C) depict basal, middle and apical region of explants of untreated group. (D-F) depict basal, middle and apical region of gentamicin (GM; 0.3 mM) treated group. (G-I) depict basal, middle and apical region of 1 µg subfraction of SPL extract and GM treated group. (J-L) depict basal, middle and apical region of 5 µg SPL extract and GM treated group. (M-O) depict basal, middle and apical region of 10 µg SPL extract and GM treated group. GM, gentamicin; PL, Piper longum L.; SPL, subfraction of PL.

• 

  Fig. 3 Microscopic images of anti-myosin-7a (red) and TUNEL (green) double-labeled P4 organ of corti explants. All examples are from comparable basal, middle and apical regions of explants. (A-C) depict control group explants. (D-F) show explants treated with gentamicin (0.3 mM). (G-I) show explants treated with 1 µg/mL subfraction of SPL extract and GM. (J-L) show explants treated with 5 µg/mL SPL extract and GM. (M-O) show explants treated with 10 µg/mL SPL extract and GM. GM, gentamicin; PL, Piper longum L.; SPL, subfraction of PL.

• 

  Fig. 4 TUNEL-labeled outer hair cell (OHC) and inner hair cell (IHC) count. The black bar and grey are representing total OHC and IHC counts per 100 µm length of cochlear respectively. TUNEL-labeled bundles were counted under a fluorescence microscope and are presented as the mean±SD of OHCs and IHCs hair cells per 100 µm length of the cochlea. The hair cell count of a single sample was the average of five fields, and 5-11 specimens were assessed for each condition. The results were significant (*P<0.05) as compared with the control group. GM, gentamicin; PL, Piper longum L.

• 

  Fig. 5 Radical scavenging activity of subfraction of Piper longum L. (SPL) extract. The percentage increases in scavenging activity were plotted against the concentration of SPL extract. The results were significant at 1 µg/mL (*P<0.0001). Error bar representing the SD of quadruplicate samples.

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