Sambucus Ebulus Fruits Extract Ameliorates Eosinophilic Nasal Polyposis: An in vitro Study

Background: The Sambucus Ebulus (ES) L. has an anti-inammatory role by reducing the expression of pro-inammatory genes and free radicals. The aim of this study was to evaluate the anti-inammatory effect of hydroalcoholic extract of SE fruits on eosinophilic nasal polyposis (NP) in Vitro. Method: The chemical composition of SE fruits extract was determined by gas chromatography-mass spectrometry. Polyp Samples were collected from 40 patients and were cut into four pieces (control and case groups). Tissue fragments were treated with 0 (control), 50, 315 and 1000µg/ml of SE FRUIT extract in a culture medium for 24 hours. Apoptosis was detected by TUNEL reaction reagent. Real time-PCR was employed to evaluate the expression of Bax and Bad genes. ELISA was used for assay of IL-5 and GM-CSF. Results: Our ndings showed SE fruits extract increased apoptosis in nasal polyp tissues compared to the control group (P<0.001). Also, GM-CSF level in the experimental groups was signicantly lower than that in the control group (P<0.001). In contrast, the level of IL-5 was not signicantly different in the treatment and control groups (P>0.05). Bax and Bad expression showed a signicant increase in the experimental groups compared to the control group (P<0.001). Conclusion: Our ndings showed that SE fruits hydroalcoholic extract ameliorates NP trough elevation of apoptosis and reduction of inammatory markers. Our experimental data revealed that SE fruits extract plays an important role in the treatment of nasal polyp through inducing apoptosis and reducing the survival of inammatory cells.

plays an important role in the treatment of nasal polyp through inducing apoptosis and reducing the survival of in ammatory cells.

Background
Nasal polyposis (NP) is one of the most common forms of chronic rhinosinusitis with a high recurrence rate [1]. It is characterized by progression of an in ammatory mass into the nasal cavity, leading to complications such as nasal obstruction, reduce sense of smell, headache, and consequently reduce quality of life [2,3,4]. NP as a multifactorial disease is usually associated with diseases such as asthma and other respiratory diseases, including cystic brosis and aspirin sensitivity [3,5]. It is reported that, the prevalence of NP in various populations is 1-4% [6].
Eosinophilic NP is the most common type of NP. Approximately 65-90% of all NP occurs by in ltration of eosinophils into polyp tissues during in ammatory processes [7]. Eosinophils and other cells are located in NP produce cytokines that sustaining the in ammatory process and invoking new eosinophils, which in turn result in tissue damage. Therefore, eosinophils are should be removed from polyp tissues by apoptosis to inhibit the in ammation process [8]. It is noteworthy that, the apoptosis rate of eosinophils in polyp tissues is less than the nasal mucus of healthy people.
The β common cytokine family such as interleukin-5 (IL)-5, and granulocyte-macrophage colonystimulating factor (GM-CSF) result in the survival of these cells via reducing the eosinophilic apoptotic index [8,9]. Besides, GM-CSF plays critical roles as a resolution of in ammatory responses and in the development of chronic in ammation [10]. Therefore, it seems that therapeutic strategies that decrease and inhibit in ammation would be an appropriate approach. Corticosteroids are prescribed as the most common medicine in clinics to reduce the in ammatory process in sinonasal polyposis [11,12]. Steroids, like many other pharmacological agents, are not completely safe and have complications on cardiac, neurological, musculoskeletal, as well as digestive system [13]. Therefore, it is necessary to nd an appropriate drug with minimal side effects.
Herbal medicines have been traditionally used in different communities. Of noted, the long-standing position of herbal medicines in public health has attracted scienti c and research centers in the last century. Sambucus Ebulus (SE), belonging to the Caprifoliaceae family, is a perennial herb with green leaves that appears in the late spring with white or creamy owers [14]. In traditional Iranian medicine, it is used topically to treat in ammations caused by insect bites and also some herbs such as nettle [15,16]. It has been revealed that leaf extract of SE had an inhibitory effect on in ammatory cytokines IL-1 (IL-1α & IL-1 ) and TNF-α [17]. Moreover, other studies revealed that SE contains several compounds, including phenolic acids, avonoids, steroid, vitamin C, caffeic acid, anthocyanins and cyanogen lycosides [18,19]. In another study, it has been shown that SE hexanic extract from fruits and leaves in the northern part of Iran has the most anti-in ammatory effect [16]. In another study, it has been shown that SE hexanic extract from fruits and leaves in the northern part of Iran has the most anti-in ammatory effect [18,20].
Therefore, due to the presence of various bene cial components in this plant and its possible effect on reducing the in ammatory process, the purpose of the present study is to determine the therapeutic effects of the hydroalcoholic SE fruit extract on preventing and treating of NP and its possible role in apoptosis and in ammation.

Materials & Methods
SE fruits extraction SE fruits were collected in Guilan (a province in the north of Iran: with herbarium number, 301HGUM) dried at room temperature and were kept away from direct sunlight. SEF (500g) were soaked in 6 liters of ethanol 70% to prepare a hydroalcoholic extract. Concentration and drying of the extracts were performed after centrifugation using a rotary vacuum evaporator. Then, the extract was transferred to an oven and completely dried at 40°C. The dried extract was weighted and dissolved in Phosphate-buffered saline (PBS) under laminar hood and sterilized using a 0.22µm lter. Our working solution was prepared by dilute DMEM-F12 for the required doses (X1 = 50, X2 = 315, and X3 = 1000 μg / ml).
Separation and identi cation of SE fruits by gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS conditions
All analysis was carried out on a GC-MS system from Agilent Technologies (Wilmington, DE, U.S.A.) GC-MS system has been used in this study consist of a model 7890A gas chromatograph, a model 5970B mass selective detector and electron ionization (EI) mode (70 eV). A fused-silica capillary column (30 m × 250 m i.d., 0.25 m lm thickness) from Scitech Scienti c was also used. The temperature program of GC-MS was as follows: initial temperature was 120 °C, held for 2 min, increased to 140 °C at a rate of 2°C /min, then to 220 °C at a rate of 3 °C/min, and nally to 320 °C at a rate of 7 °C/min and held for 15 NP tissues with ≥40% eosinophils which were con rmed by Hematoxylin and Eosin (H&E) (Fig.1) were considered in the study. The samples were washed 3 times with 10% PBS solution supplemented with 1% penicillin-streptomycin antibiotics and antimycotic (antifungal). Then, the specimens were carefully cut with a sterile blade under a microscope in sterile condition. 4 pieces (approximately 3 mm 3 ) were selected for TUNEL tests from the NP tissue samples of each patient. Other remaining specimens were cut into smaller pieces (approximately 1mm 3 ) and prepared for culture for genes and proteins analyses. The NP tissue was cultured in DMEM-F12 supplemented with 1% penicillin-stereptomycin antibiotics, 10%FBS and various doses of SE fruits extract in 6well plate in 37º C with 5% Co2. The cells were treated with 0 (as a control), 50, 315 and 1000μg/ml [21] of SE fruits extract for 24 hours.
Apoptosis detection with Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) Apoptosis was detected using in situ cell death detection kit (Roche, Germany) in NP tissues according to company manual. Brie y, the 5µm sections were dewaxed and rehydrated according to the standard protocols (by heating at 60˚C, rising in xylene for 10 minutes and then rehydration through a graded series ethanol and ddH2O). Afterward, the slides were washed with PBS and incubated with Proteinase K for 20 minutes at 37 ° C. The specimens were incubated for 10 minutes with a permeablization solution and rinsed again with PBS. In the next step, 50µl of TUNEL reaction solution was poured onto each tissue sample and incubated for one hour at 37 ° C, and after the nal washing, they were observed with a Zeiss LSM 5 uorescent microscope. Apoptotic positive cells were counted at least in 5 elds in each group. A surface of 1-2 mm in each group was considered for counting. In this protocol, TUNEL-Enzyme solution and TUNEL-Label solution were used to color the positive control samples from 1 to 9, while for negative control samples; only TUNEL-label solution was used. Apoptotic cells in this tissue were bright spots indicating apoptotic cells marked during TUNEL. The nucleus of the cells was identi ed as blue with 4 ', 6diamidino-2-phenylindole (DAPI). 4 ', 6-diamidino-2-phenylindole (DAPI) was applied as a counterstaining and the nucleus represent a blue color. The percentage of apoptotic cells was determined by dividing the number of apoptotic cells on the total number of cells.

Real-Time PCR for expression of Bax and Bad
Total RNA was extracted from cells using the InnetiPREP RNA mini kit (Analytikiena, Germany) according to company protocol. The contaminating DNA from RNA preparation was removed using Sinacolon (Iran) kit according to the manufacturer's protocol. cDNA was synthesized using BioFACT(BioFACT, Korea) kit according to the manufacturer's instructions. Real-Time PCR was performed using SYBR-Green methods through the Step One Plus™ real-time PCR instrument to measure the quantitative expression of Bax and Bad genes in different groups. The Ct value was calculated by one step software (Applied Biosystems) using the automatic threshold setting. The relative expression of Bax and Bad relative to the reference gene of GAPDH was performed in triplicates using the ΔΔCT method. The intended genes were ampli ed with primers shown in Table 1.

Statistical analysis
All data were presented as mean ± Standard deviation (SD). The data were analyzed by Statistical Package for the Social Sciences (SPSS) software Version 16. First, the data were normalized through the One-Sample Kolmogorov-Smirnov test, and after con rming the normality of the data, they were analyzed by one-way Analysis of variance (ANOVA). The signi cance level was considered as P<0.05.

Results
Gas chromatography-mass spectrometry analysis Major chemical composition of SE fruits extracts is shown in Table 2. The main constituents with medical and therapeutic properties of this plant were phenolic compounds that are found in great numbers in the fruits of this plant, which are associated with organoleptic, nutritional, and antioxidant properties. Immuno uorescence TUNEL assay was investigated to detect apoptosis following treatment of SE fruits extracts on NP tissues. Figure 2 shows the presence of the fragmented genomic DNA indicates the formation of apoptotic bodies and the apoptotic nucleus is seen in light green (Fig. 2). Our data showed that apoptosis of eosinophils signi cantly increased in the groups that received different doses of the SE fruits extract compare to control group (P<0.0001). Furthermore, the induction of apoptosis pattern was dose-dependent and signi cantly was higher at 1000 μg/ml of SE extract (P=0.004) (Fig. 3A).

SE fruits extract increased expression of anti-in ammatory factors
To con rm the anti-in ammatory effect of SE FRUIT extract, real-time PCR was performed for gene expression analysis. High expression of Bax indicated anti-in ammatory effect after treatment with 50, 315, and 1000 µg/ml of the SE FRUIT extract for 24 hours. The Bax expression in different doses of SE FRUIT extract was signi cantly higher than that in the control group (P<0.0001) (Fig. 3B). In addition, expression of Bad increased in NP after treatment with different doses of the SE FRUIT extract (P<0.0001). A comparison of Bad gene expression between different treatments demonstrated a signi cant relationship between low and high doses (P=0.033) (Fig. 3C).

SE fruits extract decreased in ammatory cytokine in NP
Evaluation of NP culture medium after treatment with SE fruits extract with ELISA showed that the level of IL-5 was not signi cantly different in the treatment and control groups (P>0.05) (Fig. 3D). However, the level of GM-CSF was signi cantly decreased in groups that received different doses of the SE FRUIT extract compare to control groups (P<0.0001). Although GM-CSF levels increased synchronically with the groups receiving the higher dose, this increase was not statistically signi cant (Fig. 3E).

Discussion
This study showed that the hydroalcoholic extract of SS fruit has anti-in ammatory properties on nasal polyp tissue. Increasing GM-CSF improves the in ammatory conditions caused by nasal polyps. On the other hand, our ndings showed that the percentage of apoptotic cells compared to control groups in nasal polyp tissues treated with different doses of SE FRUIT extract increased signi cantly.
NP disease is recognized as one of the most common cases of chronic rhinosinusitis [1,2]. Currently, functional endoscopic sinus surgery is used for treatment of advanced stages of disease and steroid anti-in ammatory drugs are used for control and treatment at early stages. Surgical treatment is an invasive and high-risk process, and steroids, like many chemical drugs, have various side effects [12,14]. Although medicinal plants have been introduced as a promising strategy in treatment of some diseases, its therapeutic properties are not fully understood. The current study, we analyzed the effects of SEL. (that is a popular and available herbaceous plant in folk medicine in Iran) on Bad and Bax gene expression, level of IL-5 and GM-CSF cytokines after 24 h exposure to SE FRUIT. Our study speci ed that using SE FRUIT increase apoptosis in polyp tissue and raise levels of anti-in ammatory cytokines.
It is suggested that multiple mechanisms appear to be involved in increasing in ammation. First, GM-CSF participates in the resolution of in ammation, and IL-3 and IL-5 may also have such properties [10]. In addition, It has been reported that GM-CSF and IL-5 stimulation increase eosinophil gene expression [22]. Similar to IL-5, another cytokines including IL-3 and GM-CSF cytokines are produced as part of the Th2 in ammatory response and are crucial to eosinophil development and function. Second, the physiological effects of each of these cytokines are mainly controlled by the expression of the cytokinespeci c receptor, which are highly impact on target cells. Third, lymphoid cells produce GM-CSF during in ammation process and amount of systemic GM-CSF increase dramatically in circulation during in ammation process [23]. We found that SE FRUIT extract reduced the amount of GM-CSF signi cantly compare to control group (P < 0.0001) and exerts its anti-in ammation property through GM-CSF inhibition.
Our results indicated that SE FRUIT extract increased the level of IL-5 in 50 and 315 µg in vitro, however, the SEs fruits extract decreased the level of IL-5 at 1000 µg/mg SE FRUIT extract in the NP tissues, but it was not signi cant (P > 0.05). Recently, some studies focused to decrease IL-5 in NP, which is only βc cytokine that have an essential role in differentiation, and proliferation of eosinophils [24]. Van Zele et al.
showed that methylprednisolone as a chemical drug exhibited therapeutic effects on NP by decreasing IL-5, eosinophil cationic protein and immunoglobulin E [25], being consistent with our ndings in reducing IL-5 in high doses of the extract. Some studies revealed that low doses of chemical drugs could not induce their therapeutic potency on NP tissues. Besides, allergic diseases that are diagnosed by tissue in ltration of eosinophils can promote long-term tissue injury and brosis [26]. Therefore, researchers used the therapeutic strategy such as inhibition of IL-5, and they showed that this can improve esophageal in ammation in children [27].
Our ndings decipher that level of GM-CSF, another key cytokine, signi cantly decreased after treatment of NP with SE fruits extract. Many studies have demonstrated the importance of GM-CSF and its signi cant role in the development of NP. Therefore, one of the main goals of researchers is decreasing GM-CSF level and in turn decreases the complications of NP. Taken together, our results indicated that hydroalcoholic extract of SE FRUIT has a signi cant anti-in ammatory effect on NP through suppression of IL-5 and GM-CSF cytokines. Of noted, SE fruits could suppress GM-CSF factor more effectively rather than IL-5, however, at the high dose the results are similar. Since, the cytokines are directly association with in ammatory cells; their decline can be justi ed with anti-in ammatory property of SE FRUIT extracts on in ammatory cells, particularly eosinophil.
On the other hands, several studies have indicated that induction of apoptosis in in ammatory cells of the NP, particularly eosinophils, can play a signi cant role in improving the disease conditions [21,28].
The results of the GC-MS technique in this study showed that 20.9% of the metanolic extract of SEs contained a natural pentacyclic trisperpenic acid (ursolic acid). Ursolic acid is commonly found in various parts of medicinal plants including leaves, owers, and fruits and has proven properties such as antiin ammatory, antioxidant and anti-carcinogenic properties [29]. Ursolic acid has been reported to inhibit the in ammatory factors TNF-α and IL6, LOX-1, VCAM-1, ICAM, IL5, IL13 and IL17 [30]. Another study shows that ursolic acid increases apoptosis in lung cancer cells [31]. In addition, it is also reported that UA via decreasing Bcl-2, Mcl-1, TCTP expression and increasing apoptotic proteins such as TNF-α, Fas, FADD and BAX results in activation of caspase-3 and PARP which induces apoptosis of the liver cancer cells. Therefore, the increase of apoptosis and the decrease of in ammatory factors in this study are probably due to the effect of this substance. Our results of apoptosis detection showed that the percentage of apoptotic cells signi cantly increased in comparison to the control group in the NP tissues treated with different doses of the SE FRUIT extract in dose dependently. It is suggested that the formation of apoptotic bodies probably inhibit the release of intracellular contents and inactivated in ammatory mediators, therefore exerts its anti-in ammatory property [32].
Furthermore, high expression of pro-apoptotic genes Bax and Bad con rmed the results obtained from in situ cell death detection with TUNEL. It has been revealed that, the imbalance between Bcl2 and Bax gene expression causes caspase9 activation and leads to apoptosis, indeed, direct activation of caspase3 is another way for causing apoptosis [33]. Our ndings decipher that SE fruits extract increased signi cantly two pro-apoptotic key genes Bax and Bad in NP tissues (P < 0.0001). On the other hand, Beta-sitosterol, one of major component of SEs extract (Table2) has a crucial role in inducing apoptosis in NP tissue [34].
Researchers have been reported Beta-sitosterol as an inducer of apoptosis through the activation of P53 gene and induction of cell cycle arrests may have contributed to increased apoptosis in NP tissue after receiving SEs fruit extracts. Several investigations on drug treatment of NP have shown that inducing transduction of pro-apoptotic genes increases the expression of these genes in NP, leading to apoptosis induction in in ammatory cells, and as a result, the treatment of NP [35].

Conclusion
Our results demonstrated that hydroalcoholic extract of SE fruits reduced the process of in ammation of the NP by inducing apoptosis of in ammatory cells such as eosinophil as well as by reducing in ammatory cytokines in dose-dependently. Thus, hydroalcoholic extract of SE fruits may play bene cial and signi cant roles in the treatment of patients with eosinophilic NP at the early stages of the disease and could be possibly used in the clinical applications after comprehensive studies. We suggest, the presence of diverse and effective compounds in SEs fruits could provide the opportunity for further comprehensive researches in the eld of anti-in ammatory mechanisms and induction of apoptosis in vitro and in vivo studies. Availability of data and materials

Abbreviations
The data is available after the reasonable request from the responsible author.

Competing interests
The authors declare that there was no con ict of Interest during performing this study.