Efficacy of lianhuaqingwen granules in the management of chronic rhinosinusitis without nasal polyps

Journal Pre-proof Efficacy of lianhuaqingwen granules in the management of chronic rhinosinusitis without nasal polyps

Lin Lin, Fei Dai, Guoqiang Ren, Jinjin Wei, Zheng Chen, Xinyue Tang PII:

S0196-0709(19)30861-0

DOI:

https://doi.org/10.1016/j.amjoto.2019.102311

Reference:

YAJOT 102311

To appear in:

American Journal of Otolaryngology--Head and Neck Medicine and Surgery

Received date:

11 September 2019

Please cite this article as: L. Lin, F. Dai, G. Ren, et al., Efficacy of lianhuaqingwen granules in the management of chronic rhinosinusitis without nasal polyps, American Journal of Otolaryngology--Head and Neck Medicine and Surgery(2019), https://doi.org/ 10.1016/j.amjoto.2019.102311

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© 2019 Published by Elsevier.

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Efficacy of lianhuaqingwen granules in the management of chronic rhinosinusitis without nasal polyps Lin Lin1*#, MD, Fei Dai1#, MM, Guoqiang Ren2#, MM, Jinjin Wei1, MM, Zheng Chen1, MM, Xinyue Tang1, MM 1

Department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital

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North of Fudan University, Shanghai, China Department of Pathology, Huashan Hospital North of Fudan University, Shanghai,

*Corresponding author: Lin Lin

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Lin Lin, Fei Dai and Guoqiang Ren contributed equally to this work.

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#

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China

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Department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital

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North of Fudan University, No. 12 Wulumuqi Middle Road, Shanghai, 200040, China E-mail: [email protected]

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Tel: 86 21 52889999

Fax: 86 21 62489191

The running head: Lianhuaqingwen granules improve CRSsNP Disclosures: The authors have no financial conflicts of interest.

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Efficacy of lianhuaqingwen granules in the management of chronic rhinosinusitis without nasal polyps ABSTRCT Objectives: Chronic rhinosinusitis (CRS) is a complicated disease with clinical symptoms that are impacted by the absence or presence of nasal polyps (CRSsNP or

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CRSwNP). Understanding of the different treatments of CRS is very significant in selecting appropriate therapies and preventing exacerbation relevant to this chronic

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medicine lianhuaqingwen granules on CRSsNP.

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inflammation. This study was aimed to evaluate the effect of Chinese traditional

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Materials and methods: CRSsNP patients were enrolled and randomized into placebo

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or lianhuaqingwen (LHQW) granules treatment group (placebo or LHQW group).

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Their clinical symptoms were scored using Visual Analog Scale (VAS) and Sino-Nasal Outcome Test (SNOT)-22. Nitric oxide (NO) from nasal cavity and sinus

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and nasal resistance were also examined. Then, nasal biopsy samples and nasal lavage fluid (NLF) were obtained from these patients, and histologic characteristics of nasal mucosa and T cell subpopulations patterns in the NLF were evaluated. Finally, inflammatory mediators in the NLF were assessed in both groups. Results: One hundred and forty patients with CRSsNP finished this one-month study. VAS and SNOT-22 scores and nasal resistance were all decreased distinctly after the treatment of LHQW, but not after placebo. However, the nasal NO concentration was increased in LHQW administration group in comparison with placebo group. There were significant differences in above parameters between these two treatments. 2

Journal Pre-proof Histologic changes in nasal mucosa were improved only in LHQW group. CD4+ and CD8+ T cells were all downregulated in the LHQW treatment group, but not in placebo group. Inflammatory mediators from the NLF were decreased in LHQW treatment group compared to placebo group. Furthermore, there were significant changes between these two groups in CD4+ and CD8+ T cell subpopulations and

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concentrations of inflammatory substances. Conclusion: These findings demonstrate that LHQW granules treatment may control

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the inflammation in nasal mucosa and result in the improvement of CRSsNP. This

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Chinese medicine might become a promising therapy in the management of this

rhinosinusitis;

Lianhuaqingwen;

Placebo;

Treatment;

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Chronic

Inflammation

1. Introduction

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Keywords:

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disease.

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The incidence of chronic rhinosinusitis (CRS) accounts for 1% to 9% of the total population around the world [1]. It has shown that CRS affects 10.9% of the population in Europe and 12.5% in USA. The prevalence of this chronic disease runs up to 8% of the whole population in mainland China [2]. CRS is generally defined as chronic inflammation in the mucosa of nasal cavity and the paranasal sinuses, and characterized clinically by two or more symptoms: one is nasal obstruction, and the other is nasal discharge. Additional symptoms can comprise facial pressure or pain, smell reduction or loss, or both [1]. CRS is a heterogeneous condition consisting of some disease variants with 3

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corresponding pathological mechanisms. Generally, there are two relevant CRS phenotypes which are characterized by their clinical characteristics: CRS without NP (CRSsNP) and with nasal polyps (CRSwNP) [1]. Treatments of CRS include medication and surgery. Generally, medication therapy requires combining relevant medicines including topical nasal glucocorticoids or oral

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glucocorticoids, antibiotics, mucolytic agents, nasal decongestants, and saline lavage, etc. The above regimen is aimed to reduce nasal mucosal edema, alleviate the local

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infection, and facilitate sinus drainage. CRS patients who do not benefit from full

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medicinal treatments should refer to functional endoscopic sinus surgery (FESS) [1].

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FESS is an effective therapy modality for CRS patients who fail medications, which

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can improve the severe symptoms and quality of life. Nonetheless, there are a small

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number of patients who cannot benefit from FESS, and 7–10% of these patients may have somewhat exacerbation of olfactory dysfunction [3]. In addition, patients with

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refractory CRS, have sustained, uncontrolled symptoms and objective inflammatory findings despite full medical and surgical therapies [4]. Therefore, novel therapeutic strategies for CRS should be exploited and developed. Lianhuaqingwen (LHQW) is a Chinese prescription, composed of 13 herbs, that is derived from appropriate and necessary addition and reduction of combined Yinqiao San and Maxing Shigan decoction. The former, deriving from the Chinese traditional medicine monograph Wenbing Tiaobian of Qing Dynasty, was primarily administered in the management of “Warm disease” characterized by clinical symptoms like thirst, fever and headache. The latter, stemming from the Chinese works Shanghan Lun in 4

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Han Dynasty, was often used to treat disease like early stage of measles, pneumonia and bronchitis [5]. LHQW has become a useful method in the treatment of seasonal influenza for many years, and this prescription performs a broad-spectrum curative effect on a number of influenza viruses, even the recently identified H7N9. The pathogenesis may be associated with its regulation on the immune responses to viral

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infection [6,7]. It has been shown that LHQW can disrupt bacterial biofilms, reduce the number of viable cells located at the intramembrane in vitro, and can also regulate

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cellular immunity in mice. Therefore, this Chinese herb medicine was used to control

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the airway inflammatory state in those patients with acute exacerbation of chronic

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obstructive pulmonary disease [8]. Based on above researches, we assume that

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LHQW may control the inflammation in upper airway mucosa and improve CRSsNP.

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In this present study, we scored clinical symptoms of CRSsNP patients using Visual Analog Scale (VAS) and Sino-Nasal Outcome Test (SNOT)-22, and examined nasal

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nitric oxide (NO) and nasal resistance, and assessed T cell subpopulations patterns and inflammatory mediators in the nasal lavage fluid (NLF) of these subjects after LHQW granules administration.

2. Materials and methods 2.1. Study design A prospective, randomized, single-center, double-blinded, controlled pilot study was designed in this current investigation. Relevant recruitment was held in the department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital North of Fudan University, with all patients enrolled between October 2017 and May 2018. 5

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2.2. Study population One hundred and forty four outpatients (aged between 18 and 75 years), who visited the Department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital North of Fudan University, were enrolled in the study. They were given a diagnosis of CRSsNP in accordance with European Position Paper on Rhinosinusitis

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and Nasal Polyps 2012 [1]. The investigators recorded the relevant sino-nasal medical history, along with the rhinological examinations including nasal endoscopy and the

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computed tomography (CT) scans of paranasal sinuses. The exclusion criteria

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included subjects aged < 18 or > 75 years, inability to conform to the study program,

a

history

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current

smoking,

granulomatous

disease,

diabetes,

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study,

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significant psychological problems, treatment with steroids or antibiotics during the

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immunocompromise, allergic rhinitis, cystic fibrosis, asthma, primary ciliary dyskinesia, pregnancy, active fungal infection, head and neck irradiation, or other

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systemic or local diseases inhibiting the participation of subjects in the study procedure such as recurrent oral ulcer and glossitis, or medical and/or surgical therapies impacting the investigation such as the use of long-term acid-suppression therapy on gastroesophageal reflux disease and partial or total gastrectomy for gastric cancer. 2.3. Study protocol Patients with CRSsNP were randomly divided into two groups: placebo and LHQW group, and 72 patients were distributed to each group. Placebo (Chinese traditional medicine

Quasiword,

Z20100040;

production 6

batch,

1712033;

production

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specifications, 6 g/packet) and LHQW granules (Chinese traditional medicine Quasiword, Z20100040; production batch, 1710002; production specifications, 6 g/packet) were all offered by Beijing Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, China). The raw material of placebo only comprises 1 ingredient (lonicera confusa DC.) according to Pharmacopoeia of People's Republic of China (Version 2010). The

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raw material of LHQW comprises 13 ingredients as shown in Table 1. Randomization was carried out using a validated computer program that matched treatments to drug

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numbers in a 1:1 ratio. Blinding was achieved by a double-dummy technique. Two

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groups of patients received the same number of granule packets (placebo or LHQW).

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Placebo and LHQW were indistinguishable in appearance and taste; all packets and

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labels were identical except for the drug numbers. These two groups of patients were

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administered placebo or LHQW granules orally (6 g/time, 3 times/day, for 30 days). There were totally two study visits. At the first, patients received the examinations

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which comprised clinical history, nasal endoscopy, CT scan, evaluations of Visual Analog Scale (VAS) and Sino-Nasal Outcome Test 22 (SNOT-22). At the second (i.e. after 30-day medical intervention), scores of VAS and SNOT-22 were reevaluated. Patients were required not to administer any other medicines to treat CRSsNP during the whole study. The study flowchart is described in Fig. 1. The present study was approved by the Ethical Committee of Huashan Hospital of Fudan University (no. 2017-364), and signed informed consent was acquired from all these outpatients. 2.4. Measurements of nasal NO Nasal NO measurements were made as follows. Patients were required to stop 7

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eating and drinking at least for 1 h before measurements. The detailed procedures were made through the NIOX MINO® Airway Inflammation Monitor (Aerocrine AB, Solna, Sweden). The air from nasal cavity was gained directly from one nostril through the inherent flow of electroluminescence analyzer. The target airflow rate was settled as 0.25 L/min, and aspiration/insufflation flows were ~5 mL/s. The probe was

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linked to a polystyrene nasal olive, and then inserted into the vestibulum nasi. The contralateral nostril was left open. Patients were required to exhale orally in order to

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2.5. Rhinomanometric measurements

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final concentration of nasal NO was recorded.

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close the soft palate. A NO plateau of nose manifested steady state after ~20 s. The

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Rhinomanometry (RM) was performed at the first and second visit (30-day

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follow-up). We used a NR6-rhinomanometer (GM Instruments Ltd, Glasgow, UK) to gain the rhinomanometric recordings. And the total inspiratory nasal resistance was

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recorded at a radius of 200 Pascal.

2.6. Samples and NLF preparation Samples of mucosa were taken endoscopically from middle meatus with a cup forceps device under local anesthesia for histochemical analysis. Nasal irrigation was performed in accordance with the published procedure [9], and NLF was collected for evaluation of contents of inflammatory mediators with enzyme-linked immunosorbent assay (ELISA). 2.7. Histochemical analysis The specimens were obtained and embedded in paraffin, and sectioned at a 8

Journal Pre-proof thickness of 4 μm, and finally placed onto glass slides. Each slide was allowed to dry in a 37°C incubator overnight and then baked at 60°C for 30 min. The specimens were deparaffinized through xylenes, rehydrated in graded alcohols, and rinsed in distilled water. Then they were dehydrated in graded alcohols, cleared in xylene, and coverslipped. The specimen sections were visualized with hematoxylin/eosin (H&E)

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staining. They were immersed in Harris hematoxylin (Thermo Scientific, Waltham, MA, USA) for 5 min, and then rinsed in tap water. Next, they were lay in 0.5% acid

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alcohol for 5 s, blued in 1% ammonia, and rinsed in deionized water. Finally, the

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sections were immersed in an alkaline eosin solution (pH = 4.4) for 20 s. The numbers

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of infiltrating inflammatory cells were detected microscopically at a high-power field

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2.8. Flow cytometry analysis

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of ×400 magnification in a blinded manner.

The cell suspension was centrifuged for 10 min at 200 × g at 4 °C, and cells were

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harvested for flow cytometric analysis. The cells were washed using 200 μL flow cytometry medium [FCM, 1% BSA and 0.1% sodium azide in phosphate-buffered saline (PBS)] and incubated using 50 μL rabbit serum for 5 min. Then the cells were washed three times using FCM, and incubated using 50 μL of primary antibody at 4 °C for 30 min. They were washed again, and incubated with 50 μL secondary antibody (biotin-conjugated rabbit anti-human IgG; DakoDenmark A/S, Glostrup, Denmark)

followed

by

50

μL

R-phycoerythrin-conjugated

streptavidin

(RPEstreptavidin; Dako). For double CD3/CD4 or CD3/CD8 staining, the cells were incubated first with anti-CD3 antibody (Abcam, Cambridge, MA, USA), then with 9

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biotinylated goat anti-human IgG followed by RPE-streptavidin. These Cells were incubated with human serum, and then incubated with FITC-conjugated rabbit anti-human CD4 or CD8 antibody (Abcam, Cambridge, MA, USA). Finally, cells were resuspended in 100 μL FCM, and examined through a Becton-Dickinson fluorescence-activated cell scanner (FACScan; Becton-Dickinson, Mountain View,

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CA, USA) equipped with the LYSYS II software analysis program. The obtained data were analyzed with the WinMDI 2.8 software.

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2.9. ELISA analysis

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IL-4 in the NLF was measured using a 96-well microplate (Corning Inc., New York,

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NY, USA) which coated by 1 μg/mL IL-4 antibody (MyBioSource, Inc., San Diego,

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CA, USA). The microplate was incubated overnight at 4°C, and then was incubated in

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3% bovine serum albumin (Calbiochem, La Jolla, Calif., USA) at 37°C for 1 h. Samples were then added at 1:10 dilution, and followed by incubation at 37°C for 1 h.

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After washing, IL-4 antibody which had been biotinylated with a biotinylation kit (American Qualex International Inc., San Clemente, Calif., USA) was then added at a concentration of 1 μg/mL, and then incubated at 25 °C for 1 h. Next, streptavidin peroxidase at 1.5 μg/mL was added, and also followed by incubation at 25°C for 1 h. Finally, tetramethylbenzidine (TMB) substrate [12.5 mL citric phosphate buffer, 200 μL of TMB stock solution (6 mg/mL in dimethyl sulfoxide), 100 μL 1% H2O2] was added in order to produce the color reactions. The reactions were terminated after the addition of 6 N H2SO4. The optical density was detected at 450 nm with a microplate reader (MTP-32; Corona Electric, Ibaraki, Japan). Concentrations of IL-5, ECP, IFN-γ, 10

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MPO and TNF-α in the NLF were evaluated with the corresponding ELISA kits (all purchased from MyBioSource, Inc., San Diego, CA, USA). 2.9.1. Statistical analysis Data were analyzed with the Prism 6.0 (GraphPad Software Inc, San Diego, CA, USA), a commercially available statistical software. Kruskal-Wallis test was used for

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comparisons between these two groups. If the initial Kruskal-Wallis test was significant, a nonparametric Mann-Whitney test was then applied. p < 0.05 was

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3. Results

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considered statistically significant.

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3.1. Patient characteristics

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One hundred and forty patients completed the present study. Four subjects were

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excluded because of their poor adherence to the medical treatments (Fig. 1). Demographic and clinical characteristics appeared similar between these two groups

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(Table 2), and there were no significant differences in age and gender between the placebo and LHQW group (Fig. 2A and B). 3.2. Adverse effects

At each study visit, the patients were required to tell whether they had any discomforts and problems. The incidence of adverse effect was 4.3% for the placebo group and 11.4% for the LHQW group during the whole study period. The overall adverse effect incidence was 7.9%. Adverse effect included stomachache and diarrhea. These events were thought mild, and all resolved over the course of this investigation. Events like vomit, cough, allergy, or other systemic side effects were not found during 11

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this research. 3.3. Comparisons of clinical examinations There were no statistical differences between the placebo and LHQW group in the scores of VAS (placebo: with a median of 6.0, range, 5.0-7.0; LHQW: with a median of 6.0, range, 4.5-7.5) (Fig. 2C) and SNOT-22 (placebo: with a median of 58.0, range,

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48.0-66.0; LHQW: with a median of 60.0, range, 48.0-66.0) (Fig. 2D), and the concentrations of nasal NO (Fig. 2E) and the values of nasal resistance (Fig. 2F)

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before treatments. We also found there were no significant changes in all above

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parameters for the treatment of placebo (Fig. 2G, H, I and J). However, after the

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administration of LHQW, VAS (placebo: with a median of 5.5, range, 5.0-8.0; LHQW:

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with a median of 4.5, range, 3.0-5.5) and SNOT-22 scores (placebo: with a median of

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55.5, range, 48-68; LHQW: with a median of 46.0, range, 35.0-56.0) and nasal resistance were decreased (Fig. 2K, L and N), and nasal NO was enhanced (Fig. 2M).

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Furthermore, there were statistical changes between these two treatment groups (Fig. 2O, P, Q and R). The results clearly demonstrate that LHQW can improve symptoms of CRSsNP patients through relieving nasal blockage and elevating nasal immunity [10]. 3.4. Comparisons of histochemical analysis Defects of epithelial cell cilia can impact mucociliary clearance and lead to chronic sinonasal inflammatory condition [10]. And outside factors such as microbial invaders and pollutant particles can directly and indirectly damage normal cilia function through particular production of inflammatory mediators [11]. Accordingly, we 12

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performed histochemical analysis to evaluate nasal epithelial cell cilia of these CRSsNP patients. Morphologically, defects of nasal mucosal cilia of the subjects, including short cilia, regions of cilia loss and irregular arrangement, were all observed in Fig. 3A and C microscopically. There were no obvious changes after the placebo treatment (Fig. 3B), however, morphological improvements including regeneration

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and regular arrangement of the cilia were shown after the application of LHQW granules (Fig. 3D). Also, inflammatory cells infiltrating in nasal mucosa were counted

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in this research. We found there were no significant differences before treatments (Fig.

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3E). Placebo did not reduce the counts of inflammatory cells (Fig. 3F), but LHQW

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decreased the infiltrating of these cells greatly (Fig. 3G). Statistical differences were

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also found between the two groups (Fig. 3H). The findings verify that LHQW

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granules may restore the function of mucociliary clearance of nasal mucosa and can alleviate the damages from inflammatory cells.

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3.5. Comparisons of T cell subpopulations in the NLF Former studies from other scholars suggest that CRSsNP may be a disease characterized by a T helper (Th)1 signature, however, subsequent research indicates that this chronic inflammation includes cytokine profiles associated with Th1, Th2 and Th17 characteristics alone or in combination modality (also with comparatively lower concentrations of Th2 type cytokines) [12]. Thus we assessed CD4+ and CD8+ T cell subpopulations in the study to further analyze the efficacy of LHQW on CRSsNP. The data clearly indicated that there were no statistical differences in subpopulations of the T cells before these two treatments (Fig. 4A and B). Placebo 13

Journal Pre-proof treatment did not decrease CD4+ and CD8+ T cell populations statistically (Fig. 4C, D, E, F, G and H). However, LHQW granules did reduce their percentages of total NLF cells whether in CD4+ or CD8+ T cell (Fig. 4I, J, K, M, L and N). In addition, there were statistical differences in these two cell populations between the two groups (Fig. 4O and P). These data show that LHQW treatment can regulate the proliferation of T

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cell subtypes that may control the endotypes of CRSsNP. 3.6. Comparisons of inflammatory mediators

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On account of endotypes of CRSsNP, we examined Th1 and Th2 type cytokines

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and mediators in this investigation, such as interleukin (IL)-4, IL-5, eosinophil cation

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protein (ECP), interferon (IFN)-γ, neutrophil myeloperoxidase (MPO) and tumor

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necrosis factor (TNF)-α. The study indicated that there were no significant changes in

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expressions of above inflammatory mediators before initiations of the two treatments (Fig. 5A, B, C, D, E and F). The administration of placebo could not downregulate

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their concentrations (Fig. 5G, H, I, J, K and L). Nonetheless, LHQW granules therapy could lessen their productions in the NLF of these patients (Fig. 5M, N, O, P, Q and R). Furthermore, there were statistical changes in contents of all these substances between these two treatment groups (Fig. 5S, T, U, V, W and X). The results illustrate that the LHQW therapy may ameliorate local inflammatory reactions through the regulation of productions of cytokines and relevant mediators.

4. Discussion The objectives of relevant treatments of CRS comprise elimination of the microbial infection, alleviation of chronic inflammation, and maintaining of sinonasal drainage. 14

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Additionally, CRS may be associated with pathological factors such as cystic fibrosis, sinonasal anatomic obstruction, allergies, gastroesophageal reflux and immunologic diseases [13]. Accordingly, the management of these pathological elements should also be considered. However, even though combinations of optimized medications and ESS were provided to patients with CRSsNP, some of them would appear to be

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caught in a sustained state of this morbidity. Hence, innovative medications should be exploited and utilized for this intractable disease. Perhaps because of the limited

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success of conventional therapy and the nature of the disease, herbal medicines are

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becoming increasingly popular and are frequently applied by adults with CRSsNP

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[14], although some scholars assumed that no appropriate evidences supported any

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[15].

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herbal medical therapies that could be beneficial in the management of this condition

LHQW can provide anti-inflammatory and immune regulatory effects that may be

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derived from the components of Chinese traditional medicine, and can be applied to treat chronic obstructive pulmonary disease with acute exacerbation [8]. On the basis of the above research, we hypothesized that LHQW may control the inflammatory condition of CRSsNP. Thus we conducted this study in our tertiary hospitals. The study was designed as a randomized, blinded, and controlled trial, and there were no significant differences in age, gender, VAS and SNOT-22 scores and other relevant parameters before these two treatments between placebo and LHQW groups. All of the above factors reduced potential bias. In the current study, we tried to utilize this herb medication to treat CRSsNP. The 15

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data demonstrated that LHQW treatment reduced VAS and SNOT-22 scores and nasal resistance compared to placebo. Scores of VAS and SNOT-22 are often subjectively used to evaluate clinical symptoms of CRS like nasal obstruction [1] and nasal resistance is always objectively used to assess the nasal airflow with RM. The decreases of these parameters indicated the improvement of clinical symptom. Some scholars

defined

the

relationship

between

mild/moderate/severe

and

VAS

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patient-classification rated measure of severity for CRS: ‘mild’ as being 0-3 inclusive,

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‘moderate’, as >3-7 inclusive and ‘severe’ as >7-10 inclusive [16]. Other investigators

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proposed a validated definition for stratification of the SNOT-22 score system, with

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‘Mild’ being defined on the SNOT-22 score as 8-20 inclusive, ‘moderate’ as >20-50

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and ‘severe’ as >50 [17]. In this present study, the median VAS score was decreased

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from 6.0 to 5.5 in the placebo group, and from 6.0 to 4.5 in LHQW group. All the median values were within the range of moderate. However, the median SNOT-22

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score was reduced from 58.0 to 55.5 in the placebo group, and from 60.0 to 46.0 in LHQW group. The median value of LHQW group was improved from severe to moderate degree. Some studies demonstrated that VAS scores for total nasal symptom score and individual symptoms correlated significantly with SNOT-22 [18]. The incoordinate phenomenon in VAS and SNOT-22 scores might be attributed to the size of the population in this current study. It is reported that NO present a first-line defense through its antibacterial and antiviral action and its enhancement of ciliary motility [19]. High levels of NO are found in normal paranasal sinuses, and the lack of this mediator may induce the sinus 16

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inflammation [20]. Epithelial cells located at paranasal sinuses were identified as the primary production of NO, and the inducible NO synthase (iNOS) would produce most of the NO in sinuses [20]. Nasal NO measurement may become a useful measure in the diagnosis, assessment and management of CRS patients [20]. The data of our study showed that LHQW upregulated the content of the nasal NO compared to

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placebo treatment, which resulted in the promotion of nasal immunity of these patients. The mRNA of iNOS was not examined in this experiment on account of

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ethical issues.

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Based on the histological analysis, we found LHQW could repair nasal epithelial

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cell cilia after 30-day treatment, which was very important for the function of

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mucociliary clearance of nasal mucosa. We observed morphological changes

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including the restoration, regeneration and ordered arrangement of the nasal mucosa epithelial cell cilia located at the middle meatus. Furthermore, the active treatment of

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LHQW also limited numbers of inflammatory cells invading the nasal mucosa in comparison with placebo, accompanied by decreased productions of ECP produced from eosinophils and MPO released by neutrophils according to the data. It has been shown that eosinophils and neutrophils can coexist in nasal mucosa of CRS, and the numbers of these cells depend on the endotypes of the disease [5]. As a result, we examined these two inflammatory substances degranulated from the corresponding cells in the investigation. Ultimately, all of above outcomes could contribute to the ameliorations of damages originated from chronic inflammation of CRSsNP to the nasal mucosa tissue. 17

Journal Pre-proof It should be emphasized that this herb could limit the proliferation of CD4+ and CD8+ T cells derived from nasal mucosa. T cells is a major constituent which can induce the adaptive immunity [10]. Defining the inflammatory endotypes of CRS is contingent on detailed analyses of cytokine signatures associated with Th1, Th2, and Th17 inflammation [12]. Type-2 cytokines like IL-4 and IL-5 are mainly secreted

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from CD4+ T cells, and CD8+ T cells can secrete type-1 cytokine like IFN-γ and TNF-α [21]. Exclusive characteristics which can distinguish CRSsNP from other CRS

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subtypes remain under violent controversy [22]. Many studies show that CRSsNP

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may not display more “type-1” features than CRSwNP [23]. To further investigate the

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efficacy of this traditional medicine on CRSsNP, we evaluated inflammatory

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mediators in the NLF. The acquired data indicated that LHQW markedly

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downregulated expressions of type-2 cytokines such as IL-4 and IL-5 and type-1 cytokine like IFN-γ and TNF-α. The result clearly demonstrated that LHQW

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performed its immunomodulatory effects not only on influenza virus infections but also on chronic inflammation in nasal cavity and parasinus. The mechanisms of actions of anti-inflammation and regulations of cellular immunity from LHQW are not very clear. However, we might find some reasonable interpretations from its ingredients according to the theory system of Chinese traditional medicine. Among these components, Forsythia suspense (Thunb.) Vahl, Mentha haplocalyx Briq., and Ephedra sinica Stapf can dispel the cold; Gypsum Fibrosum, Lonicera japonica Thunb., and Isatis indigotica Fortune can eliminate internal qi and endogenous heat; Armeniaca sibirica (L.) Lam. and Houttuynia cordata 18

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Thunb. can detoxify and remove carbuncles; Rheum palmatum L. can eliminate hollow viscera and lung heat; and Rhodiola rosea L. can tonifying qi and yin and remove evil. Generally, these thirteen herbs can modulate the human’s immunity and boost the body’s resistance, and this Chinese prescription is appropriate for the treatment of inflammatory diseases.

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This study has several limitations, such as a small number of subjects and a short duration. In addition, it is a single-center study, so the level of evidence is somewhat

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low. Also, we did not differentiate the endotypes of CRSsNP in accordance with

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histological characteristics because of the heavy workload and the ethical problems.

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As a consequence, more studies on the underlying mechanisms of the efficacy of

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5. Conclusion

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LHQW on CRSsNP are required urgently.

In conclusion, the study demonstrate that LHQW granules treatment may control

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the inflammation in nasal mucosa through reducing inflammatory cell infiltration, T cell subpopulation proliferation and inflammatory mediators. Correspondingly, the treatment can clinically result in the improvement of CRSsNP. This Chinese traditional medicine might be a promising therapy for treating this chronic inflammatory disease. Disclosures The authors have no financial conflicts of interest. References [1] Fokkens WJ, Lund VJ, Mullol J, et al. European Position Paper on Rhinosinusitis 19

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and Nasal Polyps 2012. Rhinol Suppl 2012;23:3 p preceding table of contents:1–298. [2] Shi JB, Fu QL, Zhang H, et al. Epidemiology of chronic rhinosinusitis: results from a cross-sectional survey in seven Chinese cities. Allergy 2015;70:533–9. [3] Lind H, Joergensen G, Lange B, Svendstrup F, Kjeldsen AD. Efficacy of ESS in

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chronic rhinosinusitis with and without nasal polyposis: a Danish cohort study. Eur Arch Otorhinolaryngol 2016;273:911–9.

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[4] McCoul ED, Tabaee A. A Practical Approach to Refractory Chronic Rhinosinusitis.

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Otolaryngol Clin North Am 2017;50:183–98.

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[5] Hsieh CF, Lo CW, Liu CH, et al. Mechanism by which ma-xing-shi-gan-tang

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inhibits the entry of influenza virus. J Ethnopharmacol 2012;143:57–67.

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[6] Chen W, Lim CE, Kang HJ, Liu J. Chinese herbal medicines for the treatment of type A H1N1 influenza: a systematic review of randomized controlled trials. PLoS

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[7] Ding Y, Zeng L, Li R, et al. The Chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of viral propagation and impacts immune function. BMC Complement Altern Med 2017;17:130. [8] Dong L, Xia JW, Gong Y, et al. Effect of lianhuaqingwen capsules on airway inflammation in patients with acute exacerbation of chronic obstructive pulmonary disease. Evid Based Complement Alternat Med 2014;2014:637969. [9] Galvão CE, Saldiva PH, Kalil Filho JE, Castro FF. Inflammatory mediators in nasal lavage among school-age children from urban and rural areas in São Paulo, 20

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Brazil. Sao Paulo Med J 2004;122:204–7. [10] Stevens WW, Lee RJ, Schleimer RP, Cohen NA. Chronic rhinosinusitis pathogenesis. J Allergy Clin Immunol 2015;136:1442–53. [11] Gudis DA, Cohen NA. Cilia dysfunction. Otolaryngol Clin North Am 2010; 43:461–72.

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[12] Gurrola J 2nd, Borish L. Chronic rhinosinusitis: Endotypes, biomarkers, and treatment response. J Allergy Clin Immunol 2017;140:1499–508.

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[13] Piromchai P, Kasemsiri P, Laohasiriwong S, Thanaviratananich S. Chronic

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rhinosinusitis and emerging treatment options. Int J Gen Med 2013;6:453–64.

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[14] Blanc PD, Trupin L, Earnest G, et al. Alternative therapies among adults with a

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reported diagnosis of asthma or rhinosinusitis: data from a population-based

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survey. Chest 2001;120:1461–7.

[15] Guo R, Canter PH, Ernst E. Herbal medicines for the treatment of rhinosinusitis:

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a systematic review. Otolaryngol Head Neck Surg 2006;135:496–506. [16] Lim M, Lew-Gor S, Darby Y, Brookes N, Scadding G, Lund VJ. The relationship between subjective assessment instruments in chronic rhinosinusitis. Rhinology 2007;45:144–7. [17] Toma S, Hopkins C. Stratification of SNOT-22 scores into mild, moderate or severe

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instruments.

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2016;54:129–33. [18] Doulaptsi M, Prokopakis E, Seys S, Pugin B, Steelant B, Hellings P. Visual analogue scale for sino-nasal symptoms severity correlates with sino-nasal 21

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outcome test 22: paving the way for a simple outcome tool of CRS burden. Clin Transl Allergy 2018;8:32. [19] Djupesland PG, Chatkin JM, QianW, Haight JS. Nitric oxide in the nasal airway: a new dimension in otorhinolaryngology. Am J Otolaryngol 2001;22:19–32. [20] Lindberg S, Cervin A, Runer T. Nitric oxide (NO) production in the upper

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airways is decreased in chronic sinusitis. Acta Otolaryngol 1997;117:113–7. [21] Stevens WW, Ocampo CJ, Berdnikovs S, et al. Cytokines in Chronic

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Rhinosinusitis: Role in Eosinophilia and Aspirin Exacerbated Respiratory Disease.

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Am J Respir Crit Care Med 2015;192:682–94.

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[22] Derycke L, Eyerich S, Van Crombruggen K, et al. Mixed T helper cell signatures

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in chronic rhinosinusitis with and without polyps. PLoS One 2014;9:e97581.

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[23] Mahdavinia M, Suh LA, Carter RG, et al. Increased noneosinophilic nasal polyps in chronic rhinosinusitis in US second-generation Asians suggest genetic

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regulation of eosinophilia. J Allergy Clin Immunol 2015;135:576–9.

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Figure legends Fig. 1. The study flowchart. Fig. 2. Differences of demographic characteristics of the patients and comparisons of clinical examinations. A, age. B, gender. C–F: comparisons of pre-treatment. C, VAS score. D, SNOT-22 score. E, NO. F, RM. G–J: placebo treatment. G, VAS score. H,

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SNOT-22 score. I, NO. J, RM. K–N: LHQW treatment. K, VAS score. L, SNOT-22 score. M, NO. N, RM. O–R: comparisons of post-treatment. O, VAS score. P,

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SNOT-22 score. Q, NO. R, RM. Placebo, placebo treatment. LHQW, lianhuaqingwen

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granules treatment. VAS, Visual Analog Scale. SNOT-22, Sino-Nasal Outcome Test

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22. NO, nasal nitric oxide. RM, Rhinomanometry. Pre, pre-treatment. Post,

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****p < 0.0001.

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post-treatment. The values shown are expressed as mean ± SD. n.s., not significant.

Fig. 3. Comparisons of histochemical analysis. A, pre-treatment of placebo. B,

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placebo treatment. C, pre-treatment of LHQW. D, LHQW treatment. E, comparisons of pre-treatment. F, placebo treatment. G, LHQW treatment. H, comparisons of post-treatment. Placebo, placebo treatment. LHQW, lianhuaqingwen granules treatment. Pre, pre-treatment. Post, post-treatment. Open arrow, epithelial cell cilia. Filled arrow, inflammatory cells. Original magnification × 400, Scale bar = 10 μm. The values shown are expressed as mean ± SD. n.s., not significant. **p < 0.01. ****p < 0.0001. Fig. 4. Comparisons of T cell subpopulations. A, comparisons of pre-treatment of CD4+ T cells. B, comparisons of pre-treatment of CD8+ T cells. C, pre-treatment of 23

Journal Pre-proof placebo of CD4+ T cells. D, post-treatment of placebo of CD4+ T cells. E, placebo treatment of CD4+ T cells. F, pre-treatment of placebo of CD8+ T cells. G, post-treatment of placebo of CD8+ T cells. H, placebo treatment of CD8+ T cells. I, pre-treatment of LHQW of CD4+ T cells. J, post-treatment of LHQW of CD4+ T cells. K, LHQW treatment of CD4+ T cells. L, pre-treatment of LHQW of CD8+ T cells. M, post-treatment of LHQW of CD8+ T cells. N, LHQW treatment of CD8+ T cells. O,

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comparisons of post-treatment of CD4+ T cells. P, comparisons of post-treatment of

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CD8+ T cells. Placebo, placebo treatment. LHQW, lianhuaqingwen granules treatment.

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Pre, pre-treatment. Post, post-treatment. Q1 quadrant, CD3+ staining; Q2 quadrant,

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CD3+CD4+ (CD8+) staining; Q3 quadrant, CD4+ (CD8+) staining and Q4 quadrant,

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CD3-CD4- (CD8-) staining. The values shown are expressed as mean ± SD. n.s., not

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significant. *p < 0.05. **p < 0.01. ***p < 0.001. Fig. 5. Comparisons of inflammatory mediators. A–F: comparisons of pre-treatment.

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A, IL-4. B, IL-5. C, ECP. D, IFN-γ. E, MPO. F, TNF-α. G–L: placebo treatment. G, IL-4. H, IL-5. I, ECP. J, IFN-γ. K, MPO. L, TNF-α. M–R: LHQW treatment. M, IL-4. N, IL-5. O, ECP. P, IFN-γ. Q, MPO. R, TNF-α. S–X: comparisons of post-treatment. S, IL-4. T, IL-5. U, ECP. V, IFN-γ. W, MPO. X, TNF-α. IL, interleukin. ECP, eosinophil cation protein. IFN, interferon, MPO, myeloperoxidase. TNF, tumor necrosis factor. Placebo, placebo treatment. LHQW, lianhuaqingwen granules treatment. Pre, pre-treatment. Post, post-treatment. The values shown are expressed as mean ± SD. n.s., not significant. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

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Family

Used part

Weight

Dryopteris crassirhizoma Nakai Houttuynia cordata Thunb. Lonicera japonica Thunb. Forsythia suspensa (Thunb.) Vahl Gypsum Fibrosum Isatis indigotica Fortune Ephedra sinica Stapf Rhodiola rosea L.

Dryopteridaceae Saururaceae Caprifoliaceae Oleaceae – Brassicaceae Ephedraceae Crassulaceae

Rhizoma Whole plant Flower bud Fructus – Root Stem Rhizoma

255 g 255 g 255 g 255 g 255 g 255 g 85 g 85 g

Armeniaca sibirica (L.) Lam.

Rosaceae

Seed

85 g

Glycyrrhiza uralensis Fisch. Pogostemon cablin (Blanco) Benth. Rheum palmatum L. Mentha haplocalyx Briq.

Leguminosae Labiatae Polygonaceae Mentha

Rhizoma Whole plant Rhizoma Menthol

85 g 85 g 51 g 7.5 g

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Table 1 Components of LHQW. Plant

25

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Table 2 Demographic and clinical characteristics of the patients (n = 140). Placebo group

LHQW group

Total no. of patients

70

70

Age (median, range)

54 (18~75)

51 (25~71)

Sex (male/female)

33/37

36/34

No. adverse effect

3

8

No. patients withdrawn

2

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lP

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LHQW = lianhuaqingwen.

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Patient characteristics

26

2

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5