Glycyrrhiza Glabra and Platycodon Grandiorus are Alternative Treatment Options for Vocal Fold Nodules

Background: Vocal fold nodules (VFN) are one of the main causes of hoarseness. In this study, we try to gure out an alternative treatment from our clinical experience summary. Methods: We retrospectively reviewed VFN patients who received traditional Chinese medicine (TCM) treatments from July 2018 to August 2020 and traced their Chinese Voice Handicap Index-10 (VHI-C10) and multidimensional voice program (MDVP) analysis results. Also, we induce inammatory response of porcine vocal fold epithelial (PVFE) cells with 50 ng/mL TNF-alpha. The inamed PVFE cells were separately cultured in the aqueous extract of Glycyrrhiza glabra and Platycodon grandiorus. Results: For these patients (n = 22), the average VHI-C10 score decreased from 17.6 to 6.6 (p < 0.001). MDVP analysis revealed improvements in jitter, shimmer, noise–harmonic ratio, and GRBAS scoring system. Analysis of prescription patterns revealed that the most commonly used herbs were Licorice and Platycodon grandiorus. In the MTT assay of PVFE cells, no adverse effects of our extracts were observed at doses of 1 –200 ug/mL. Western blot analysis revealed downregulation of p65 and mitogen-activated protein kinase pathway proteins. Conclusions: The results from both the clinical and in vitro aspects of this study revealed that the herbs G. glabra and P. grandiorus may offer benecial outcomes as alternative treatments for VFNs.


Introduction
Vocal fold nodules (VFNs) as benign vocal fold lesions, is quite a challenge for otolaryngologists. Hoarse and raspy voices are distinguished symptoms for VFNs. The de nition of VFNs are mucosal lesions occurring on both sides of the vocal fold on the border of the anterior and middle third of the vocal fold (1,2). Such lesions are often found on the super cial layer of the lamina propia (3). Long term exposure to irritants may cause chronic laryngeal in ammation, which may further lead to the formation of VFNs (4).
Medical treatments, voice therapy and excision through microlaryngeal surgery are considered as VFN treatment. Physiological, medical, and psychological factors should be taken into account in the course of diagnosis and treatment. (5,6).
Voice therapy, which can alter voice production patterns to minimize phonotrauma, is the rst-line treatment for VFNs because it typically resolves voice problems and prevents recurrence in most patients. Voice therapy typically involves education on vocal fold mechanics and etiological factors, as well as speci c modi cations to behaviors that exacerbate inappropriate voice production (5). However, because compliance varies among patients, surgery is often necessary for VFN treatment. However, serious complications associated with surgery may occur, including damage to the vocal folds (7). Intracordal steroid injection can be bene cial for managing benign VFNs. However, the technique requires a high level of skill and therefore a well-trained laryngologist. Nevertheless, VFNs may persist after injection. Therefore, there are increasing patients turning to complementary medicine other than western medicine.
(8) Because some of them may fear surgery or be concerned about using steroids, patients have recently shown increased preference for treatments outside the mainstream medical practice (9).
As for physiotherapy, such as massage, spinal manipulative therapy and acupuncture, there are several trials revealing that these treatments might be effective for voice disorders. (10) In an small-scaled acupuncture study, anti-in ammatory signi cantly increased was found in the experiment group. This may be the mechanism why complementary medicine is effective to the voice disorders. (11) When it comes to herbs, though there are scanty reports showing that herbs formula have signi cant effect on vocal nodule and vocal polyp patients. (12,13) However, the methodological quality remained unsatisfactory. Also, the exact contents and the dosage were unclear in the above researches. Therefore, it is hard to ascertain the exact effect of each herb, not to mention the in vitro evidence of the herbs effect.
For in vitro part, some herbal aqueous and ethanol extracts such as clove and ginger exhibit antiin ammatory ability in human tonsil epithelial cells. (14) However, cell types in the upper airway vary. The vocal fold epithelial cells are strati ed squamous. (15) Since it is di cult to obtain healthy human vocal fold epithelial cells without risking vocal function, the fresh porcine vocal fold tissue is readily and easily available. (16) Moreover, compared to other animals, the morphology of porcine vocal fold epithelium is the most similar to that of humans. (17) So far, few researches study the effects of herbs on both vocal fold nodule patients and in ammatory vocal fold cells. In our study, we retrospectively evaluated the clinical effects according to the clinical results of our TCM herbal treatment. Also, we further investigated the effects of these herbs in an in vitro study.

Subjects
The medical records of 22 patients who had received diagnoses of VFNs are based on videostroboscopy or nal pathology from July 2018 to August 2020. Other benign vocal fold lesions were excluded from the present study, such as vocal polyps, vocal cysts, and Reinke's edema. The patients were examined with laryngeal videostroboscopy using a Kay Elemetrics Stroboscopy Unit (Model, Lincoln Park, NJ, USA) at their initial visit and were followed up 3 months after TCM treatment.
Speech pathologist skilled in voice training and an otolaryngologist evaluated the recorded data. Both of them were blinded to the TCM treatment protocols. The present study was approved by the Institutional Review Board of Chiayi Chang Gung Memorial Hospital (No.201901034B0). All participants gave written informed consent prior to the beginning of the study.

Objective and Subjective Voice Analysis
The acoustic parameters recorded automatically were as follows: average fundamental frequency (F0, in Hertz), jitter, shimmer, and noise-to-harmonic ratio (NHR). Jitter is de ned as the cycle-to-cycle variation in fundamental frequency (18,19), and shimmer is expressed in decibels, representing the variability in the peak-to-peak amplitude. NHR is used for evaluation of dysphonic voice by measuring the amount of additive noise in the voice signal (20). The acoustic parameters were measured by the Computerized Speech Laboratory (core model CSL 4500, KayPentax, Lincoln Park, NJ, USA). The duration parameter measured in this study was the maximum phonation time (MPT). A speaker's MPT is measured by a stopwatch, as their best attempt to sustain the vowel /a/ sound at a comfortable intensity. (21). Twentytwo patients completed a pre-and post-test Chinese Voice Handicap Index-10 questionnaire (VHI-C10) (22,23). Pre-and post-TCM perceptual evaluation was performed by using the GRBAS scoring system (G = grade, R = roughness, B = breathiness, A = asthenia, and S = strain; 0 = normal, 1 = mild, 2 = moderate, and 3 = severe). It the difference between the two GRBAS scores exceeded two points, a reassessment is required. The voice parameters mentioned above were measured by a speech pathologist and an otolaryngologist in a double-blinded manner.

Aqueous Extracts of TCM Herb Glycyrrhiza glabra and Platycodon grandi orus
The raw herbs used for the aqueous extracts (AE) were obtained from Chiayi Chang Gung Memorial Hospital (Taiwan), 20 g G. glabra from Batch No. 42050309 and 20 g P. grandi orus from Batch No. N15529. The raw herb materials were separately macerated in 1000 mL reverse osmosis water and then boiled for 10 minutes. These ltrates were combined and concentrated under reduced pressure at 40°C by using a vacuum rotary evaporator to obtain crude AEs. Approximately 4 g of the resulting G. glabra AE (20% yield) and 11 g of the resulting P. grandi oras AE (55% yield) were stored at − 20°C before use.

Primary Culture PVFE Cells
The porcine vocal fold epithelial (PVFE) cells for the primary culture were purchased from GeneDirex, Inc. The PVFE cells were isolated and cultured as reported. (16) Epithelial cells were derived from porcine vocal folds, and then further expanded in culture. Epithelial cells identi cation and characterization were done by immunostaining with pan-Cytokeratin antibodies.
MTT Assay PVFE cells were treated with various concentrations of G. glabra and P. grandi orus; subsequently, the percentages of metabolically active cells were determined based on the mitochondrial conversion of MTT into formazine. In brief, after cells were treated with G. glabra and P. grandi orus for various incubation times, culture media were replaced with DMEM/F-12 (1:3 ratio) containing 0.02% MTT (Sigma-Aldrich) and incubated for 4 h; subsequently, the medium was replaced with 200 µL of dimethyl sulfoxide per well. The results were assessed in a 96-well format plate reader by measuring the absorbance at a wavelength of 595 nm on a Victor Nivo Multimode microplate reader (PerkinElmer, Akron, OH, USA).

In ammatory Response Induction and Culture in TCM Aqueous Extract
The PVFE cells were seeded at a density of 35 ×10 3 cells/well in PLL-coated 24-well plates and incubated overnight at 35°C for 20 h. PVFE cells were then exposed to the in ammatory cytokine tumor necrosis factor alpha (TNF-α) 50 ng/mL for 24 h incubation at 35°C to trigger in ammation. In addition, the corticosteroid dexamethasone was included as a positive control anti-in ammatory drug.

Western Blot Analysis
Cells were collected in lysis buffer containing protease and phosphatase inhibitors for protein isolation. We prepared cellular extracts by sonication. And total protein concentrations were determined for the Western blot analyses. Proteins were separated on 4-20% Tris-Glycine gels (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes. After the membranes were blocked in TBST (10 mM Tris-HCl buffer, pH 8.0; 150 mM NaCl; and 0.1% Tween 20) and 5% (w/v) BSA at room temperature for 60 min, they were incubated overnight at 4°C with antigen-speci c primary antibodies. The primary antibodies used were against phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, Danvers, MA, USA). The blots were under incubation with species-speci c HRP-conjugated secondary antibodies for 2 hours at room temperature. And then, the proteins were visualized through incubation with a chemiluminescent substrate kit (Thermo Fisher Scienti c, Waltham, MA, USA).

Statistics
Experiments were performed at least three times. The clinical and in vitro data were analyzed using SPSS 13.0 (Chicago, IL, USA). The results are expressed as means ± standard deviations. P value less than 0.05 was considered statistically signi cant.

Laryngeal videostroboscopy image outcomes
The patients in this study had husky voices for more than 6 months. VFNs with mild to moderate phonatory gaps were seen under videostroboscopy. Figure 1 presents the bilateral vocal nodules at rest (Fig. 1A) and the glottal gap during phonation (Fig. 1B).
Patients were followed up 3 months after TCM treatment. Videostroboscopy, acoustic analysis, and perceptual assessment were conducted. Figure 1D and 1E presents the post-TCM glottal gap at rest and during phonation, respectively. We found that after TCM treatment, most patients achieved marked improvements in glottal closure, mucosal wave, and the amplitude of mucosal wave under videostroboscopy. No additional morbidities or complications were observed during treatment.

Objective and Subjective Voice Parameters
The acoustic analyses before and after TCM herb treatments are presented in Table 1. No signi cant differences were noted between MPT and NHR among the two groups (P > 0.05). Subjective VHI-C10 questionnaire scores, assessed by a blinded voice therapist, decreased signi cantly after TCM treatment.
Jitter, shimmer, and GRBAS scores also improved signi cantly. A signi cant decrease was also observed in pre-and post-TCM perceptual assessments, which were conducted according to total GRBAS scores ( Table 2).

TCM Medication Review
We pooled the prescribed medications of the study participants and calculated the total doses. The following herbs were prescribed with total dosages of > 1500 g (Fig. 2): P. grandi orus, licorice, Ophiopogon japonicus, Fritillaria thunbergii, and Sepiae Endoconcha (cuttlebone). The total prescribed amount of P. grandi oras and licorice are more than 2000 grams. The average duration and doses of P. grandi oras and licorice for the patients are summarized in Table 2. P65 and mitogen-activated protein kinase Pathway Inhibition by Glycyrrhiza glabra and Platycodon grandi orus in vitro G. glabra and P. grandi orus did not hinder the growth of PVFE cells even at high concentrations (Fig. 3A). In VFE cells treated with 50 ng/mL TNFα, NF-kβ p65, a downstream target of the mitogenactivated protein kinase (MAPK) pathways, was stimulated by phosphorylation. G. glabra, P. grandi orus and the corticosteroid dexamethasone could reduce the expression of phosphorylated p65 in TNFαtreated cells (Fig. 3B). To further study the mechanism of the anti-in ammatory effect of TCM, the change in p-AMPK, p38, extracellular signal-regulated kinases (ERK), and Jun N-terminal kinases (JNK) in response to TCM treatment in VFE cells was investigated (Fig. 3C). G. glabra and P. grandi orus signi cantly inhibited the p38, ERK, and JNK pathways, but their effect on the MAPK pathway was unclear.

Discussion
We collected the retrospective medical records of 22 vocal fold nodule patients treating with TCM from July 2018 to August 2020. We found that the most used herbs were P. grandi orus and licorice. Further, we used the two herbs for porcine VFE culture. And the two herbs signi cantly inhibited the P38, ERK, and JNK pathways in TNF-alpha induced in ammation.
The nished herbal products (FHP) we prescribed are granulated compounds concentrated from Chinese herbal remedies, including single herbs and herbal formulae. (24) They are o cially approved in the national healthcare systems not only in Taiwan, but also in Japan, Korea and mainland China. They are widely used in the East Asia. (25) In our retrospective analysis, patients regularly return to clinic in a two-week to one-month manner. Since our clinic is comparatively remote, most of our patients found that returning to TCM clinic is more convenient than attending voice therapy. From Fig. 2, we can see the most used herbs were Licorice and P. grandi oras. The total amount were more than 2000 grams.
Glycyrrhiza glabra, the licorice species we used here, is brought from Chiayi Chang Gung Memorial Hospital (Taiwan). G. glabra is grown in Eurasia, northern Africa and western Asia.(26) Its rhizomes and roots are the most important medicinal parts, which have been reported to be used alone or in combination with other herbs for treating multiple diseases such as digestive disorders, respiratory tract disorders, epilepsy, fever, sexual disability, and etc. (27) P. grandi orus contains numerous amino acids, vitamins, and trace elements. Also, it has the ability to treat the following disorders: cough and asthma relief, antitumor activity, anti-in ammatory and antibacterial effects, antioxidation, hypoglycemic effects, liver protection, and immunity enhancement (28). Both Glycyrrhiza glabra and P. grandi orus can be used as food and avoring agents.
The combined use of these two herbs can be traced to ancient China. In the Chinese classic Shang Han Lun (English title: Treatise on Cold Damage Diseases, written by ZHANG Zhong-jing, 150-219 A.D.), the two herbs were combined in a 2:1 ratio and were used mainly to treat sore throat and Fei Yon (pulmonary abscess) (29,30). Pharmacologically, they can affect the metabolic process of each other, improving the bioavailability of their compounds (31,32).
Clinically, in ammation is commonly observed in patients with chronic voice disorders, such as vocal fold nodules. For example, an immediate increase in in ammatory cytokines, including TNF-α, is observed in response to vocal fold injury. The effect of TNF-α on the vocal fold epithelium upregulates mucin expression (16). TNF-α may activate the NF-κB p65 pathway in PVFE cells, but this in ammatory pathway is inhibited by the corticosteroid dexamethasone(Dex) and high doses of G. glabra and P. grandi orus. This means that the anti-in ammatory effect of TCM herbs on the vocal folds depends on their accumulation in vocal epithelium cells.
MAPK families are critical in complex cellular programs like proliferation, differentiation, development, transformation, and apoptosis. The MAPK pathways involve a series of protein kinase cascades, which play a crucial role in the regulation of cell proliferation. The three major MAPK families, namely mammalian ERKs, c-JNK, and p38 kinases, are activated by many stimuli. JNK and p38 are activated by cytokines and stressors, and ERK is mainly activated by growth factors (33). Figure 3C shows that G. glabra and P. grandi orus deregulated the activation of p38, p-ERK, and p-JNK. This result may be explained by the bene cial effects of G. glabra and P. grandi orus on vocal nodules due to downregulation of the MAPK pathway (34).
In our prescription pattern, a 1:1 ratio of G. glabra to P. grandi orus may be more bene cial for VFN patients. Our sample size is relatively small. Also, the dosage and duration varied, which may be due to individual difference. For in vitro study, though the above in ammatory pathway down-regulation was seen, whether there exists synergistic effect or other interaction between the herbs are still unknown. Further prospective and functional studies are needed to con rm the results of this study and clarify the mechanism of action.
In conclusion, by reviewing clinical patient data and conducting an in vitro study, we con rmed that TCM herbal treatment is an alternative treatment option for VFNs. Patients' voices exhibited improvement after daily TCM herb use, and VFNs were reduced. From the above results, we may imply that our most prescribed herbs G. glabra and P. grandi orus as potential VFN alternative treatment.    pathways were inhibited by G. glabra and P. grandi orus, but their effect on the MAPK pathway was unclear.