Effects of Moringa Extract on Aminoglycoside-Induced Hair Cell Death and Organ of Corti Damage.

Abstract

Hypothesis:

Moringa extract, a naturally occurring anti-oxidant, protects against aminoglycoside-induced hair cell death and hearing loss within the organ of Corti.

Background:

Reactive oxygen species (ROS) arise primarily in the mitochondria and have been implicated in aminoglycoside-induced ototoxicity. Mitochondrial dysfunction results in loss of membrane potential, release of caspases, and cell apoptosis. Moringa extract has not previously been examined as a protective agent for aminoglycoside-induced ototoxicity.

Methods:

Putative otoprotective effects of moringa extract were investigated in an organotypic model using murine organ of Corti explants subjected to gentamicin-induced ototoxicity. Assays evaluated hair cell loss, cytochrome oxidase expression, mitochondrial membrane potential integrity, and caspase activity.

Results:

In vitro application of moringa conferred significant protection from gentamicin-induced hair cell loss at dosages from 25-300 μg/ml, with dosages above 100 μg/ml conferring near complete protection. Assays demonstrated moringa extract suppression of ROS, preservation of cytochrome oxidase activity, and reduction in caspase production.

Conclusion:

Moringa extract demonstrated potent antioxidant properties with significant protection against gentamicin ototoxicity in cochlear explants.

Introduction

Aminoglycosides remain among the most widely used antibiotics worldwide, owing to their widespread availability, low expense, high treatment efficacy, and low incidence of allergic reactions. They continue to be first line therapy for conditions ranging from neonatal sepsis to multi-drug resistant tuberculosis.¹ In addition, aminoglycosides have found application as “read through therapy,” a novel form of stop codon suppression treatment for genetic diseases caused by nonsense mutations.^2–4 Side effects of aminoglycosides include risk for ototoxicity, vestibulotoxicity, and nephrotoxicity.⁵ Studies of aminoglycoside-induced hearing loss indicate an incidence anywhere from a few percent to 33%.⁵ In higher risk populations, such as dialysis patients, hearing loss occurs in approximately 60% of patients.^{5, 6} Few treatment options are available to mitigate these risks, but a growing body of literature supports anti-oxidant based strategies to attenuate acquired hearing loss.^{1, 7–11} Identification of an inexpensive, readily accessible, and well-tolerated otoprotective agent could potentially decrease the global burden of aminoglycoside-induced hearing loss.

Drug-induced hearing loss is linked to the generation of reactive oxygen species (ROS) within the cochlea, leading to loss of hair cells in the organ of Corti.⁵ Aminoglycosides form ternary chelation products with transition metals and polyunsaturated lipids. These complexes catalyze the formation of free radicals, which cause mitochondrial dysfunction. Ongoing mitochondrial damage causes loss of mitochondrial membrane potential, release of caspases, and apoptosis.^12–14 Moringa Oleifera, a plant rich in the flavonoid quercetin, vitamin C, and beta-carotene, is an important source of phenolic antioxidants, which can scavenge ROS and attenuate cellular damage.¹⁵ Moringa has been researched in translational and clinical studies, demonstrating its anticancer activity and antioxidant effects, including several studies demonstrating nephroprotective effects against aminoglycosides.^16–20 Moringa has recently also shown efficacy in animals models of several clinical conditions and pathogens associated with oxidative stress.^21–24 Whether moringa extract may protect against drug-induced hearing loss remains unknown.

We sought to investigate moringa extract as a potential otoprotectant for aminoglycoside-induced injury. Using murine organ of Corti explants (organotypic culture), we undertook an in vitro investigation of ROS, bioenergetics, and cell death pathway in the presence of gentamicin with and without moringa extract.

Materials and Methods

Animals:

Male and female CBA/J mice obtained from Harlan Sprague-Dawley Co. (Indianapolis, IN) were used for breeding. All mice were maintained on a 12-hour light/12-hour dark schedule with a regular diet and free access to water throughout the experiment, with a one-week acclimation period for those in the same habitats. Procedures were performed in accordance with the animal protocol #PRO00005637 under the primary investigator (MJB). All experimental protocols were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA) and University of Michigan’s Unit for Laboratory Animal Medicine (ULAM).

Explant Preparation & Treatment:

Neonatal mice were humanely euthanized on post-natal day 2-3. Temporal bones were dissected with cochleae extraction and immersion in cold Hank’s Balanced Salt Solution. Lateral wall tissues and the auditory nerve bundle were removed leaving only the organ of Corti and spiral ganglion cells. These were placed on a culture dish prepared by polymerizing 15 μL of rat tail collagen solution for 15 minutes on a 35-mm dish. One mL of culture medium consisting of Basal Medium Eagle, 1% serum-free supplement (Gibco, Grand Island, New York), 1% bovine serum albumin, 5 mg/mL glucose, and 10 U/mL penicillin G, was added. Each explant was then incubated for 4 hours (at 37°C, 5% CO₂) after which an additional 1.5 mL of the same culture medium was added to submerge explants. In all cases after dissection, explants were incubated for 2 days at 37°C, with 5% CO₂ to allow recovery from stress. After this time, the initial medium was exchanged with new media prior to addition of any treatment conditions.

Moringa Extraction and Dosing:

Organic Moringa Leaf Powder (Zen Principle, Incline Village, NV) was extracted twice with distilled water at temperature 60–70 °C for 48 hours, using previously described methods.²⁵ Extract was filtered using Whatman no. 1 filter paper and concentrated in a rotary evaporator under reduced pressure, resulting in viscous residue, which was lyophilized for use in extract and resuspended for gavage. For in vitro studies, moringa extract was resuspended in 100% dimethyl sulfoxide (DMSO) and then diluted using the culture medium, resulting in a final DMSO content of 0.5% or lower. A matched concentration of DMSO was added to media for control studies.

Experimental Design:

In vitro studies were performed comparing organ of Corti explants in an aminoglycoside injury treatment group (4μM gentamicin; Sigma-Aldrich, Saint Louis, MO) with and without addition of moringa extract. Studies included explants treated with vehicle or moringa to control for the potential effects of each substance on organ of Corti explants. Multiple moringa extract concentrations were used to evaluate gentamicin-induced outer hair cell loss. All subsequent assays included experimental groups that were administered 4μM gentamicin, 200 μg/mL moringa extract, or both. Gentamicin solution was prepared daily from a frozen stock to ensure standard potency. Care was taken to avoid exposure to humidity due to the hygroscopic properties of gentamicin. The solution was diluted with culture medium to achieve experimental concentration. 4μM gentamicin was selected as prior studies suggest low doses are more characteristic of aminoglycoside therapies administered to human subject.^{1, 26, 27} For all assays, pre-treatment with moringa was performed 1 hour prior to gentamicin. All experiments were conducted for 72 hours post Gentamicin treatment.²⁸

Evaluation of Mitochondrial Membrane Potential:

The 4μM gentamicin was administered to organ of Corti explants and compared to untreated, negative controls. At 72 hours after treatment, explants were exposed to 5,506,60-tetraethylbenzimidazolyl-carbocyanine iodide (DePsipher, Trevigen) at 37°C for 30 min. This potential-sensitive dye fluoresces Green (510/527 nm, corresponding to monomeric form) when the mitochondrial transition pore is open. The dye fluoresces red (585/590 nm, corresponding to aggregated form) when inside an intact mitochondrion. Imaging was performed immediately after 30 min dye-loading time to detect the ratio of aggregate/monomer form outer hair cells in each explant. Captured images confirmed that gentamicin administration disrupted the electrochemical gradient across the mitochondrial membrane.

Evaluation of Hair Cell Loss:

Explants were incubated with 4 μM gentamicin with and without moringa extract. Various concentrations of 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 300 μg/mL moringa extract were used. At 72 hours, explants were fixed with 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO) overnight at 4°C and permeabilized with 3% Triton X-100 (Sigma-Aldrich, St Louis, MO) for 30 minutes in phosphate-buffered saline (PBS) (Sigma-Aldrich, St Louis, MO) at room temperature (22-24 °C). Explants were then washed with PBS three times, incubated with rhodamine-phalloidin (1:100) (Sigma-Aldrich, St Louis, MO) or Alexa Fluoro 488 phalloidin (Thermo Fisher Scientific, Ann Arbor, MI) at room temperature for 30 minutes, and again washed three times with PBS. The explants were then mounted on a slide with FluoroGel (Fisher Scientific, Waltham, MA) and examined under Leitz Orthoplan upright light microscope under 50x oil immersion lens.

The right eye objective had a 0.19-mm calibrated scale superimposed on the visual field for reference. Specimens were oriented to show a single row of inner hair cells and all three rows of outer hair cells in the visual scale. Each 0.19 mm frame was evaluated for the presence or absence of inner and outer hair cells, starting from apex and moving down the base of the organ of Corti. The proportion of hair cells lost in each 0.19 mm frame was recorded in a computer program (KHRI cytocochleogram, version v.3.0.6, Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI, USA), allowing comparison with previously established normative data. The percentage of missing hair cells was plotted as a function of distance from the apical turn.

Evaluation of ROS generation:

Gentamicin-induced ROS production was examined via DHE assays in explants treated with 4μM gentamicin, 200 μg/mL moringa extract, both, or neither (negative control) (N=6 for each group). After 72 hours, explants were stained with 20μM dihydroethidium (DHE; Sigma-Aldrich, Saint Louis, MO) at 37 degrees Celsius in a humidified CO₂ (5%) incubator for 20 min. DHE solution preparation: 1 mg DHE was dissolved in 317 μl DMSO for 10 mM stock solution; then 25 μl DHE stock solution was diluted in 12.5 mL Milli-Q pure H₂O for a 20μM staining solution. They were next fixed for 1 hour in freshly prepared 4% paraformaldehyde at room temperature, stained with 2 μg/ml Hoechst stain (Thermo Fisher Scientific, Ann Arbor, MI) for one hour, mounted on slides, and examined immediately under confocal microscopy. Red (ethidium) staining indicated product resulting from reaction between DHE and superoxide using an emission wavelength of 580nm and an excitation wavelength of 480 nm. Blue color indicated nuclear staining secondary to Hoechst stain.²⁹ Mean fluorescence intensity was measured using ImageJ software (U. S. National Institutes of Health, Bethesda, MD) at every 25um of length along the organ of Corti image. Measurements were taken along the OHC regions of each sample.

Evaluation of Cytochrome Oxidase Function:

Explants were treated with 4μM gentamicin, 200 μg/mL moringa extract, both, or neither (negative control). After 72 hours, histochemical staining was carried out using 3,3’ diaminobenzidine (Sigma-Adrich, Saint Louis, MO). Differential Intensity Contrast microscopy images of outer hair cells were utilized to determine extent of cytochrome oxidase activity, a correlate for overall mitochondrial function. Brown staining indicates regions of cytochrome oxidase activity due to creation of insoluble product after addition of 3,3’ diaminobenzidine.

Evaluation of Caspase Activation:

Explants were exposed to 4 μM gentamicin with and without 200 μg/mL moringa extract. Moringa extract and negative control were also performed. At the end of the experimental period of 72 hours, but before fixation, explants were treated with FAM-VAD-FMK (Biomol Research Laboratories, Plymouth Meeting, Pennsylvania), a fluorescein-labeled peptide inhibitor of caspase enzymes. They were next fixed for 1 hour in 4% paraformaldehyde at room temperature, stained with 2 μg/ml Hoechst stain for one hour, Phalloidin (Sigma-Adrich, Saint Louis, MO) stain for 20 minutes, mounted on slides, and examined immediately under confocal microscopy (with appropriate interval PBS washings). FAM-VAD-FMK results in green staining indicating the amount of active caspases. Hoeschst stain in blue and Phalloidin stain in red label the DNA and actin respectively. Mean fluorescence intensity of FAM-VAD-FMK stained cochlear samples was measured using ImageJ software at every 25um of length along the OHC region of the organ of Corti.

Statistical Analysis:

Quantitative data were evaluated by one-way Analysis of Variance (ANOVA) and Student’s t-test with a Newman-Keuls significance set at (p<0.05) using SPSS Biostatistics software. For hair cell death, comparisons included cytocochleogram data after treatment conditions.

Results

We first evaluated the effect of gentamicin on mitochondrial membrane potential. 4μM gentamicin was administered to organ of corti explants and compared to untreated, negative controls (each group N=6). At 72 hours post-treatment, explants were stained with DePsipher. As seen in the representative image in Figure 1, captured images of the hair cells of the basal turn demonstrated a decrease in the orange, aggregated form of DePsipher in the gentamicin treated group. The presence of this orange signal suggests an intact electrochemical gradient across the mitochondrial transmembrane. The monomeric form of DePsipher (green) indicates open mitochondrial channels and is present in both groups. These results suggest that gentamicin administration disrupts the electrochemical gradient across the mitochondrial transmembrane of organ of corti hair cells possibly leading to mitochondrial dysfunction.

Figure 1:

Representative image (N=6) of the basal turn hair cells after mitochondrial membrane potential (MMP) assay. 4μM gentamicin (GM) exposure results in a decrease in the aggregate form of the DePsipher reagent within mitochondria. Orange: aggregated form of DePsipher inside mitochondria indicating normal mitochondria and MMP; Green: monomeric form of DePsipher outside mitochondria.

We next evaluated the effect of moringa extract at various doses on gentamicin-induced hair cell loss in vitro. Organ of corti explants exposed to 4 μg/mL gentamicin were treated with moringa extract concentrations of 25, 50, 100, 150, 200, and 300 μg/mL respectively, as well as a gentamicin alone, moringa alone, and untreated control (each group N=6). Cytocochleogram analysis of outer hair cell (OHC) death with gray fluorescence microscope was performed. Figure 2A demonstrates that exposure to gentamicin alone resulted in an average loss of 70% of OHCs. When combined with moringa extract, the percent loss of OHCs decreased significantly (p<0.05) at all concentrations. Notably at a range of 100-300 μg/mL, moringa protected against damage almost completely. Moringa or vehicle alone did not result in any OHC cell loss. In Figure 2B, representative confocal microscopy depicts protection of gentamicin-induced hair cell loss in the presence of 200 μg /mL moringa. These results indicate a does dependent protective effect of moringa extract on hair cell loss from gentamicin induced damage.

Figure 2:

(2A) Cytocochleogram data demonstrate protective effects against gentamicin (GM) induced outer hair cell (OHC) loss in the presence of moringa extract at 25 ug/mL and greater concentrations. Moringa alone and vehicle alone result in no OHC loss. (2B) Representative confocal images of explant OHCs stained with Rhodamine-phalloidin or Alexa Fluor-488-phalloidin demonstrate protection from GM-induced hair cell loss in the presence of moringa extract (*** = p<0.05 for all comparisons; N=6 for all groups; OHC: Outer Hair Cells; IHC: Inner Hair Cells)

To evaluate the protective effect of moringa extract, we studied the effect of moringa on ROS generation. Organ of corti explants exposed to 4 μM gentamicin with or without 200 μg/mL moringa were stained with DHE to evaluate the potential for moringa to mitigate gentamicin-induced ROS production (both N=6). In hair cells, DHE is oxidized to ethidium in the presence of superoxide radicals leading to a red, fluorescent product, while Hoechst staining labels the DNA blue. The mid-basilar turn was chosen as apex to base analysis of ROS induced cochlear injury indicates increased injury in the base compared to the apex. Figure 3A confocal images from the mid-basilar turn of the cochlea demonstrate that 200 μg/mL moringa prevented gentamicin-induced ROS generation in outer hair cells (OHC). DHE alone as well as merged DHE and Hoechst images are included. The bar chart in Figure 3B shows the percent average mean fluorescence intensity +/− SEM of DHE along each 25um of the OHC compared to the control. The gentamicin group demonstrated a statistically significant increase from all other groups (p ≤0.05, one way ANOVA, N=6). This result suggests that moringa is capable of reducing the production of ROS seen in the presence of gentamicin.

Figure 3:

(3A) Representative confocal image of murine organ of Corti explant demonstrates suppression of gentamicin (GM) induced reactive oxygen species (ROS) by 200 μg/mL moringa extract (ME) via dihydroethidium (DHE) reaction with superoxide. DHE alone is shown alongside merged image of DHE and blue Hoechst nuclear staining. (N=6) (3B) Bar graft of average mean fluorescence +/− SEM of DHE along each 25um of the OHC region illustrates that explants treated with GM alone demonstrated significantly more ROS generation than any other group when compared to control. Red: ethidium, a product of DHE and superoxide; Blue: Hoechst nuclear staining. (* = significant difference from all other groups, p ≤0.05, via one way ANOVA, N=6).

We additionally evaluated the effect of moringa on cytochrome oxidase activity. Explants exposed to 4 μM gentamicin with and without 200 μg/mL moringa extract were evaluated using 3,3’ diaminobenzidine, which results in a brown insoluble compound at the site of cytochrome oxidase activity. Differential Intensity Contrast microscopy images were taken of the basal turn. As seen in representative image in Figure 4, gentamicin alone reduces cytochrome oxidase activity in outer hair cells, leading to a decrease in dark brown aggregate within outer hair cells. This effect is not seen in the presence of moringa extract alone or alongside gentamicin. This suggests moringa protects against gentamicin induced reduction in cytochrome oxidase activity.

Figure 4:

Representative confocal image of cytochrome oxidase assay using 3,3’ diaminobenzidine demonstrates decreased explant cytochrome oxidase activity after gentamicin (GM) alone, illustrated by loss of dark brown outer hair cell morphology, whereas normal morphology and staining is preserved when moringa extract (ME) is administered with GM indicating continued cytochrome oxidase activity. Brown color corresponds to 3,3’ diaminobenzidine staining, arising from deposition of insoluble compound at sites of cytochrome oxidase activity. (all groups N=6; OHC: outer hair cells)

The effect of moringa extract on caspase activity was also evaluated. Explants exposed to 4 μM gentamicin with and without 200 μg/mL moringa extract were treated with FAM-VAD-FMK, a green fluorescein-labeled peptide inhibitor of caspase enzymes, which allows for direct quantification of the amount of active caspases through quantification of the green fluorescence intensity. Hoeschst stain in blue and Phalloidin stain in red label the DNA and actin respectively in both the control and the experimental groups (both N=6). In the confocal images of the mid-basilar turn seen in Figure 5A, the green staining in the gentamicin-alone group corresponds to caspase activity, which is diminished when gentamicin is administered in the presence of moringa extract. The bar charts in Figure 5B show percent mean fluorescence intensity +/− SEM of FAM-VAD-FMK along each 25um of the OHC region compared to control. This result indicates that moringa prevents against gentamicin induced activation of caspases associated with programmed cell death.

Figure 5:

(5A) Representative confocal images of caspase assay demonstrates significant caspase activity in those explants treated with gentamicin (GM) alone. This activity is absent in explants treated with gentamicin plus moringa extract (ME). Green: FAM-VAD-FMK, covalently binds to active caspases; Red: Phalloidin actin staining; Blue: Hoechst nuclear staining. The FAM-VAD-FMK alone image used for the analysis in 5B is included. (5B) Bar graft depicts percent mean fluorescence intensity of FAM-VAD-FMK along every 25um of the OHC region (+/− SEM) compared to control. (* = significant difference from all other groups, p ≤0.05, via one way ANOVA, both groups N=6).

Discussion

Aminoglycoside ototoxicity is responsible for outer hair cell damage within the inner ear. While antioxidant therapies have been shown to alleviate this damage without decreasing antibacterial efficacy, there is still no FDA approved treatment to prevent such hearing loss.^{1, 30} We demonstrated that moringa, a potent antioxidant, confers protection against gentamicin-induced hair loss in vitro. Our assays demonstrated moringa extract suppression of ROS production, preservation of mitochondrial bioenergetics, and reduction in cell death pathway activation.

Aminoglycoside ROS generation occurs primarily in the mitochondria and results in respiratory chain inhibition from ROS insult.^{12, 31} A disruption of the respiratory chain leads to disrupted signaling pathways that mediate cellular responses to stress, cellular growth, and differentiation. Gentamicin, a frequently used aminoglycoside, stimulates the consumption of oxygen, reduction of cytochrome c, and conversion of hydroethidine to ethidium, all independent indicators of superoxide radicals.³² Utilizing murine organ of Corti explants, a well-established tool for modeling cochlear physiology, we were able to examine the powerful mitigating effect of moringa extract on the multitude of aminoglycoside injury cellular pathways. These findings are consistent with demonstration of superoxide suppression by moringa in other model systems.³³

The moringa plant has been a medicinal option for centuries, across the world, for its anti-inflammatory, anti-spasmodic, antihypertensive, and anti-oxidant properties.³⁴ No adverse effects have been seen in human studies.³⁵ Its medicinal properties arise principally from vitamin C, carotenoids, and the flavonoid quercetin.³⁶ Flavonoids have potent anti-inflammatory, antiviral, antioxidant, and free radical scavenging activity.^{31, 37} Other antioxidants, such as aspirin, glutathione, d-methionine, n-acetyl cysteine, pomegranate extract, misoprostol, and garlic – have putative otoprotective effects against aminoglycosides; Moringa extract has been primarily studied for nephroprotection against aminoglycosides.^{5, 16–18, 38–40}

The analysis demonstrated potent effects of moringa in attenuating aminoglycoside-induced injury. Gentamicin ototoxicity was reduced in a dose-dependent fashion, with wide therapeutic window and with no ototoxicity or discernible adverse effects at high concentrations. Experiments probing the underlying mechanism of protection revealed suppressed ROS production on DHE assay and intact cytochrome oxidase activity on 3,3’ diaminobenzidine (DAB) staining, with attendant reduction of active caspases, evidenced through a lack of FAM-VAD-FMK covalent binding. This study is the first demonstration of the otoprotectant effects of moringa in cochlear organotypic culture. Moringa contains a combination of antioxidants, and we hypothesize that their combined action accounts for the salutary effects observed.

This study has several limitations. For histologic analysis, some labelling could be improved upon. For example, while phalloidin-labeled stereocilia bundles provide some visual representation of OHC loss, there may be better combinations of dye labels that could better illustrate both OHC loss and capture any aminoglycoside induced damage to surrounding supporting cells. Furthermore, many other antioxidants are available, and we did not directly compare the extent of otoprotection from moringa to that achieved with chelating agents, such as deferoxamine and 2,3-dihydroxybenzoate, which exploit the critical role of transition metal complexes in oxidative stress related injury to the inner ear.^{14, 32} However, free radical formation by some chelators, such as succimer and trientine, may outweigh protective effects of these treatments.³² Increasing glutathione production via N-acetylcysteine, or inhibiting the signaling pathways directly involved with ROS-induced mitochondrial dysfunction such as STAT 1, also offer additional therapeutic avenues.^{1, 6, 41}

Several areas are in need of future investigation. Refinement of pharmacokinetics are necessary to optimize treatment regimen for any translational application, and there is a need to establish whether administration of the protectant hampers therapeutic efforts, as when protection results from inactivating the therapeutic agent or competes with the therapeutic agent at site of action.^{42, 43} Assay analysis of the bioactive sub-components of moringa is also needed to elucidate which molecules may be refined into more potent otoprotective treatments through indirect and direct antioxidant activity.⁴⁴ Isolation and independent testing of these components would also address potential limitations in batch variation between lots of moringa. Last, further mechanistic studies are needed to dissect the molecular events that mediate protection; evaluating specific loci of mitochondrial dysfunction and protection may afford greater insights in the future.

Conclusion:

Moringa extract demonstrates potent protective effects from gentamicin-induced ototoxicity in murine cochlear explants, mediated by reduced oxidative stress and stabilizing mitochondrial bioenergetics. While further work is needed to evaluate the translational potential along a wider range of aminoglycosides, combining moringa or its most active components with other therapeutic options may provide therapeutic avenues for hearing protection in humans.