Antipharyngitis Effects of Syringa oblata L. Ethanolic Extract in Acute Pharyngitis Rat Model and Anti-Inflammatory Effect of Ir-Idoids in LPS-Induced RAW 264.7 Cells

Acute pharyngitis is an inflammation of the pharyngeal mucous membrane and submucous lymphoid tissues. Unsatisfactory treatment and repeated occurrences might cause chronic pharyngitis and other complications. Syringa oblata L. (S. oblata) is a traditional Chinese medicine that exhibited a significant therapeutic effect on various inflammatory diseases. Its commercial drug Yan Li Xiao (YLX) capsule is used as a cure for the treatment of inflammatory diseases, such as bacillary dysentery, tonsillitis, bronchitis, and acute gastroenteritis. However, studies about S. oblata relieving acute pharyngitis are rare. In this study, high-performance liquid chromatography (HPLC) was used to analyze the chemical profile of S. oblata, and the bioactive phytoconstituents were isolated and identified by nuclear magnetic resonance (NMR) and mass spectrometry. An ammonia-induced acute pharyngitis rat model was established to estimate the protective effect of S. oblata in vivo for the first time. The severity of pharyngitis was observed by appearance index and HE staining. The cytokines expression was performed by ELISA. Crucial proteins expression associated with TLR4/NF-κB/MAPK and NLRP3 inflammasome signaling pathways were analyzed by western blotting and immunohistochemistry. Furthermore, the anti-inflammatory effect of isolated compounds was evaluated by suppressing lipopolysaccharide- (LPS-) induced NO generation and regulating the cytokines levels in RAW 264.7 cells. The results showed that S. oblata exhibited a protective effect in the ammonia-induced acute pharyngitis rat model, and the compounds exert potential anti-inflammatory properties against LPS-activated RAW 254.7 cells.


Introduction
Acute pharyngitis is the most common seasonal nonspecific inflammation among upper respiratory tract diseases [1][2][3]. Patients with pharyngitis mainly manifest severe itchy throat, cough, pain, congestion, swelling, fever, dryness, and loss of appetite [4]. Additionally, untimely treatment can lead to various complications, including tympanitis, laryngitis, nasosinusitis, bronchitis, and myocarditis [5,6]. To date, several treatment strategies, including antibiotics, antiviral drugs, and glucocorticoid hormones, are used for pharyngitis in clinical to relieve pain. However, these drugs have potential risks and side effects [7][8][9]. Current research mainly focused on infectious pharyngitis induced by virus and bacteria, while both preclinical and clinical epidemiological studies for noninfectious pharyngitis caused by an allergic reaction, smoking, alcohol intake, and so on are lacking [10]. erefore, the development of safer and more effective therapeutic alternatives for noninfectious pharyngitis was limited under this situation.
Accumulative evidence shows that pharyngitis is inflammation in the mucosa and submucosa tissue and related to immune responses [11]. Irregular immune homeostasis and repeated acute inflammatory may result in carcinogenesis of chronic ear-nose-throat disease and nasopharyngeal mucosa [12]. Toll-like receptors (TLRs), a member of the pattern recognition receptors (PRRs) recognizing pathogen-associated molecular patterns (PAMPs) in the pathogen, play a crucial role in the defense against invading pathogens [13,14]. TLR4 is a key pattern recognition receptor in the TLRs family and activates the immune response by recognizing invasive pathogens. Myeloid differentiation primary response gene 88 (MyD88) is one of the main adaptor proteins for TLR4 promoting signal transduction. e nuclear transcription factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) can be activated via interaction of dimerized TLR4 with its downstream effector MyD88 to regulate the release of inflammatory cytokines for controlling inflammation, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and other proinflammatory cytokines [15,16]. As an important part of inherent immunity, nucleotide-binding oligomerization domain-(Nod-) like receptor family pyrin domain-containing 3 (NLRP3) plays an important role in the process of inflammatory response and disease. e activation of NLRP3 could recruit inflammasome-adaptor protein (ASC) and procaspase-1 to form a multimeric protein complex and accelerate procaspase-1 that is converted to mature caspase-1, which promote the maturation and secretion of cytokine precursors pro-IL-1β and further lead to inflammatory reactions [17][18][19]. Accordingly, the pathogenesis and progress of pharyngitis were closely related to TLR4/NF-kB/MAPK and NLRP3 inflammasome pathway [20,21]. RAW 264.7 cells play an important role in the inflammatory processes. Once activated, inflammatory mediators, including nitric oxide (NO) and inflammatory cytokines, might be excess production to promote the inflammatory response [22].
Syringa oblata Lindl. has long been used to treat diseases such as vomiting, nausea, diarrhea, rheumatic pain, and kidney deficiency [23]. Modern pharmacological studies confirmed that the leaves of S. oblata, extensively applied to treat various inflammatory illnesses and the activities mentioned above, might attribute to iridoid glycosides, which, as physiologically active substances, showed remarkable activities [24]. Yan Li Xiao (YLX) capsule is one of the commercial medicines and mainly consists of water extract and powder of S. oblata leaves. It is used as a cure for the treatment of bacillary dysentery, tonsillitis, bronchitis, acute gastroenteritis, and so on. Furthermore, clinical observation demonstrated that YLX displays a higher treatment rate for children's acute infections among clinical cases [25]. However, there were no systematic studies performed to evaluate the potential effects of S. oblata in pharyngitis, and molecular mechanisms are still not clearly elucidated. e advantages and application of S. oblata as remedy in the treatment pharyngitis should be further explored.

Preparation of S. Oblata Ethanol Extract.
e leaves of S. oblata were collected from the Heilongjiang University of Chinese Medicine. Dried leaves (408.0 g) were extracted with 75% ethanol at room temperature for 3 d by the maceration method with intermittent stirring and shaking. After filtration, ethanol was evaporated with a rotary evaporator at reduced pressure and temperature not exceeding 50°C to obtain a solid mass of extract (95.0 g). e production yield of the extract was 23.3%. Finally, the extract was dissolved in H 2 O for subsequent bioassays. e extract (80.0 g) was suspended in H 2 O (3 L) and with petroleum ether (PE) (3 × 3.0 L, 24 h each), EtOAc (5 × 3.0 L, 24 h each), and n-butanol (3 × 3.0 L, 24 h each) to give EtOAc (9.4 g) and n-butanol (3.3 g) fractions.

2
Evidence-Based Complementary and Alternative Medicine e structure was identified by NMR spectra, recorded on Bruker DPX 600 instrument (Bruker, Karlsruhe, Germany). HR-ESI-MS was conducted using a Waters Synapt G2-Si High-Definition Mass Spectrometer (Waters Inc., Milford, MA, USA).

HPLC Analysis.
Liquid chromatography was performed using a Waters 2695 HPLC system equipped with a ELS detector (Waters Inc., Milford, MA, USA). e chemical profiling of S. oblata extract was performed using a Waters SunFire ® C18 column (5 μm, 4.6 mm × 250 mm). e mobile phases were A (acetonitrile) and C (waters), with gradient elution performed as 10%-80% (0-40 min). e flow rate was 1 mL/min and the injection volume was 10 μL. Meanwhile, the contents of compounds isolated from S. oblata leaves were measured. 2020-0001) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China). e animals were feed water and food with free access for a week in a clean environment at a temperature of 25°C ± 1°C and relative humidity of 45% to 55% under 12 h light/dark cycle. All animal experiments were strictly approved by the Animal Ethics Committee of Heilongjiang University of Traditional Chinese Medicine (Heilongjiang, China) on 16 March 2021 (number 2021031604). 70 rats were randomly divided into 7 groups (n � 10 in each group), including the control group, model group, DEX group, YLX group, and three different doses of S. oblata extract groups. According to the previous study, the acute pharyngitis model was induced using a dose of 15% ammonia water sprayed into the pharyngeal mucosal twice per day for 3 consecutive days, and the rats in the control group were treated similarly with distilled water into the pharyngeal. After the model was made, the positive control group was administered with DXM (5 mg/kg). e rats in other treatment groups were administered with YLX (270 mg/kg) and S. oblata extracts (high: 270 mg/kg; middle: 135 mg/kg; low: 68 mg/kg), respectively. Furthermore, the rats in the control and model groups were given the same  Evidence-Based Complementary and Alternative Medicine volume of distilled water. After 24 h of the last administration, all rats were killed to collect the blood and pharyngeal tissue for the following experimental indicators analysis. e experimental design is shown in Figure 2.

Evaluation of Appearance
Index. Based on behavioral and physical marking criteria [26], the changes of appearance indexes, including diet, drink, activities, mouth scratch, mouth hair loss, cough, saliva secretion, and pharyngeal swelling, were observed, and the scores were recorded using assessment criteria in Table 1.

Cytokines Levels
Assays. e blood samples were centrifuged at 3500 rpm for 10 min to obtain serum, and the pharyngeal tissues were ground with PBS (1 : 9, w/v) as a homogenization medium to obtain 10% tissue homogenate. e levels of cytokines in serum (IL-6, IL-1β, TNF-α, IFN-c, IL-4) and inflammation mediators (PGE2, EGF, and COX-2) in pharyngeal were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. e OD value was measured at 450 nm using a microplate reader, and the contents were quantified by standard curves and analyzed using GraphPad 8.0 software.

Histological Examination of Pharyngeal.
For histological evaluation, the rat pharyngeal samples were collected and fixed in 4% paraformaldehyde fixing solution for 24 h at room temperature. en, it was embedded in paraffin and sliced. Samples were cut into 4 μm thick slices and stained sequentially with hematoxylin and eosin (HE). Finally, the section was observed under a light microscope for evaluating pathological changes of pharyngeal. e severity of pathological damage was scored under blinded conditions from 0 to 4 as described in Table 2.

Immunohistochemistry Staining of Pharyngeal.
e expression of NLRP3, MyD88, p-ERK1/2, and p-p38 in pharyngeal tissue was evaluated by immunohistochemistry, and the sections used for immunohistochemistry staining came from paraffin-embedded tissues. Paraffin sections were deparaffinized into the water and repaired with antigen in the repair box of antigen repair buffer, rinsed with PBS (pH 7.4). 3% H 2 O 2 was added to sections and incubated for 25 min at room temperature and away from light. e sections were blocked with 3% BSA for 30 min after being rinsed with PBS. e sealing fluid was gently shaken off, a portion of PBS containing the first antibodies (NLRP3, MyD88, p-ERK1/2, and P-p38) was added to the sections and incubated overnight in a humid box at 4°C, rinsed with PBS, and then incubated with a secondary antibody at room temperature for 50 min. After that, they were rewashed with PBS, and diaminobenzidine solution (DAB) was added at room temperature for 10 min. e sections were observed using a light microscope (E100, Nikon, Japan). e above process of washing with PBS was 3 times and 5 minutes each time.

Anti-Inflammatory Activity In Vitro
2.5.1. Cell Culture. RAW 264.7 cells were purchased from Stem Cell Bank, Chinese Academy of Sciences, and cultured in Dulbecco's modified Eagle's medium (DMEM) solution that contained 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere at 37°C with 5% CO 2 .

Cell Viability.
Cell viabilities were determined using the MTT method [27]. In brief, RAW 264.7 cells (1 × 10 6 cells per well) were seeded into 96-well plates and incubated for 24 h until adhesion. Cells were treated with various concentrations of the samples (5.5, 11.0, 22.0, 44.0, 88.0, 176.0 μM) for 24 h with and without treating with LPS (1.0 μg/mL). After incubation, 10 μL MTT solution (5 mg/ mL) was added and further incubated at 37°C for 4 h. 100 μL DMSO was added to each well and the absorbance was determined at 570 nm. All assays were performed in triplicate.

No Production.
e Griess method was performed to evaluate the compounds' inhibitory effects on the production of NO in RAW 264.7 cells according to the previously reported method [27]. Briefly, RAW 264.7 cells were seeded into 96-well plates, incubated for 24 h, and then induced with LPS (1.0 μg/mL) in a serum-free medium containing various concentrations of the tested compounds. N-Monomethyl-larginine (L-NMMA, 5 μM) was used as a positive control. After 24 h, the 100 μL supernatants and NO detection kit (Beyotime, China) were mixed at 37°C. e 4 Evidence-Based Complementary and Alternative Medicine absorbance was determined at 570 nm using a microplate reader. All assays were performed in triplicate.

Cytokines Levels
Assays. RAW 264.7 cells were plated into 96-well plates (1 × 10 6 cells/well) and cultured for 24 h at 37°C with 5% CO 2 . After pretreating with various compounds for 24 h, the 1.0 μg/mL LPS was used to stimulate RAW 264.7 cells. e supernatants were collected after 24 h and centrifuged at 12000 rpm for 10 min. e release of cytokines was measured by ELISA following the manufacturer's instructions. DXM was used as a positive control. e absorbance was determined at 450 nm using a microplate reader.
2.6. Statistical Analysis. Each experiment was performed at least three times, and data were presented as the mean-± standard error of the mean (SEM). e statistical significance between different groups was calculated by oneway analysis ANOVA using GraphPad 8.0 software. e value of p < 0.05 was considered to be significant. p < 0.01 was considered very significant. e structures of compounds were determined by comparing the NMR data with previous studies [25,28]. Additionally, the contents of four compounds were 10.21, 32.63, 28.43, and 15.83 mg/g, respectively.

Results and Discussion
Syringalactone

Alleviated the Pathological Symptoms of Acute Pharyngitis Rats.
e severity of lesions on the pharyngeal was evaluated according to score criteria. As shown in Figure 4, rats in the control group exhibited normal activity, diet, weight, and other behaviors. However, compared with control group, the pharyngeal swelling, mucous secretions increased, the weight and activity ability of rats decreased seriously, and the mouth scratch behaviors and cough increased during modeling. Additionally, some of them lost their mouth hair gradually. As expected, these symptoms in treatment groups significantly alleviated in different degrees and reduced the pathology score compared to the model group.

Regulating the Expression of Cytokines in Acute Pharyngitis Rats.
e expression of inflammation cytokines in serum (IL-6, IL-1β, TNF-α, IFN-c, IL-4) and inflammation mediators (PGE2, EGF, and COX-2) in tissue were assessed using ELISA kits. e results showed that the levels of IL-6, IL-1β, TNF-α, IFN-c, PGE2, and COX-2 were significantly increased in ammonia-induced acute pharyngitis rats compared to the control group, while the levels of IL-4 and EGF were decreased. In contrast, the balance between cytokines was regulated after treatment with S. oblata and YLX. e production of IL-6, IL-1β, TNF-α, IFN-c, PGE2, and COX-2 was dramatically inhibited in treatment groups, whereas the release of IL-4 and EGF was promoted ( Figure 5).

Relieving Histopathological Damage in Acute Pharyngitis Rats.
Hematoxylin and eosin staining (HE) was used to evaluate the histopathological changes of pharyngeal tissue in ammonia-induced acute pharyngitis. As shown in Figure 6(a), the control group showed no evidence of damage in the pharyngeal mucosa epithelium. In contrast, an abnormal structure was observed in the model group, with massive inflammatory cell infiltration in the submucosa, connective tissue hyperplasia, and glandular atrophy. However, treated with S. oblata extracts, especially at a dose of 270 mg/kg, the pathological damage significantly ameliorated. Additionally, it could be observed that the microscopic scores between the control group and model group were remarkably different. e microscopic scores of S. oblata (270 mg/kg and 135 mg/kg) (Figure 6(b)) were lower than the model group, suggesting that the S. oblata alleviated acute pharyngitis in a dose-dependent manner.

Inhibiting the Expression of TLR4/NF-κB/MAPK Signaling Pathway.
Inflammatory is a physiological immune response and mainly regulated via TLR4/NF-κB/MAPK signal pathway in macrophages [15]. e TLR4/NF-κB/ MAPK signal pathway could promote and amplify the inflammatory response during cell and tissue damage and pathogen invasion. us, the expression of key proteins was examined by western blot and immunohistochemistry, respectively. As shown in Figures 7(a)-7(b), ammonia caused a dramatically increased in the expression of TLR4, MyD88, NF-κB, JNK1/2/3, p-JNK1/2/3, ERK1/2, p-ERK1/2, p38, and p-p38 compared to the control group while the protein expression in treatment groups significantly reduced compared to the model group. Moreover, immunohistochemical staining further confirmed this result (Figure 8). e expression of MyD88, p-ERK1/2, and p-p38 was downregulated significantly by administration of S. oblata, suggesting that S. oblata alleviated acute pharyngitis via inhibiting the activation of the TLR4/NF-κB/MAPK signaling pathway.

Attenuating the Expression of NLRP3 Signaling
Pathway. NLRP3 inflammasome plays a critical role in the development and progression of many diseases via Evidence-Based Complementary and Alternative Medicine regulating the expression of inflammatory mediators. us, the expression of the related protein in the NLRP3 signaling pathway was assayed by Western blot analysis to determine the effect of S. oblata on NLRP3 inflammasome activity. As illustrated in Figures 9(a)-9(b), NLRP3, ASC, and caspase-1 were abnormally elevated in the model group compared with the control group, while the expression was downregulated significantly in the treatment groups. Additionally, the expression of NLRP3 was measured by immunohistochemical staining (Figure 9(c)), and a similar result was also observed, indicating that S. oblata reduced the acute pharyngitis by inhibiting the expression of the NLRP3 inflammasome signaling pathway.

Effect of Compounds on Cell Viability in RAW 264.7 Cells.
e MTT assays were used to evaluate the effect of compounds syringalactone A (1), syringopicroside (2), oleuropein (3), and ligstroside (4) on cell viability in RAW 264.7 cells. e four iridoids at the concentration ranging from 5.5 to 176.0 μM had no cytotoxic in RAW 264.7 cells ( Figure 10). erefore, these concentrations of test compounds were selected for the subsequent experiments' antiinflammatory effects. Figure 11, test compounds significantly decreased NO production with concentrations 44.0, 88.0, 176.0 μM. Furthermore, all compounds showed an evidently dose-dependent manner except for syringopicroside (2). Hence, the 44.0, 88.0, and 176.0 μM were used for the analysis of other inflammatory cytokines.

Effect of Compounds on Cytokines Levels in RAW 264.7
Cells. To further investigate the inflammatory activity, the inhibitory effects of the four compounds were assessed using Elisa. e result showed that treatment of cells with LPS caused a significant elevation in IL-6 and TNF-α levels while decreasing the IL-4 level. However, pretreatment with test 3.4. Discussion. S. oblata is a traditional herbal medicine that has anti-inflammatory property, and its commercial drug YLX capsule is extensively applied in a variety of acute and chronic inflammatory diseases [29]. e ammonia-induced acute pharyngitis rat model has been widely used as an experimental model in pharyngitis pathogenesis studies due to being highly consistent with clinical indicators as well as the characteristics of easy, reliable, and short duration. erefore, an ammoniainduced acute pharyngitis rat model was applied to evaluate the therapeutic effects of S. oblata in vivo in the present study for the first time. e administration dosage was calculated by coefficient commutation of somatotypes according to the human clinical dosage of the YLX capsule. Preexperiment results indicated that the selected dosage had no obvious side effects and was suitable for further experiments. e administration of S. oblata could significantly ameliorate symptoms of pharyngitis in varying degrees. Additionally, HE results provide intuitive morphologically pathological characterizations that the rats treated with S. oblata exhibited complete epithelial structure of the mucosa and few lymphocytes infiltration.
Cytokines, mainly released by activated lymphocytes and monocytes, are acknowledged to be responsible for the initiation and progression of inflammation [30]. Particularly, IL-6, IL-1β, and TNF-α are considered the most important proinflammatory cytokines. Previous studies revealed that cytokines play a synergistic role in triggering the production of anti-inflammatory cytokines and inflammatory mediators, causing more severe inflammation [31]. us, the levels of cytokines were determined in serum and tissue in our study. e data showed that the levels of IL-6, IL-1β, TNF-α, IFN-c, PGE 2 , and COX-2 remarkably increased, while the levels of IL-4 and EGF decreased in the model group. As predicted, the S. oblata displayed an antiinflammatory effect via reversing the abnormal regulation of pro-and anti-inflammatory cytokines. Evidence-Based Complementary and Alternative Medicine e protective mechanisms of S. oblata in ammoniainduced noninfectious acute pharyngitis were also explored by western blot and immunohistochemistry. TLR4/NF-kB/ MAPK signaling pathways, which are responsible for the expression of inflammatory cytokines and chemokines, are the major signaling cascade in initiating and propagating inflammatory response in acute pharyngitis, as reported previously [16]. As the first TLR protein identified, TLR4 is associated with a variety of inflammatory reactions. e myD88-dependent pathway is the main signals transferred method after stimulation and the interaction between TLR4 and MyD88 in the structure domain of TIR triggers a cascade reaction of downstream signals, followed by multiple downstream signaling pathways activated such as the NF-kB and MAPK classical signaling pathways to induce the release of inflammatory cytokines [15,16].
ree major characterized subfamilies of MAPKs, ERK1/2, p38, and JNK1/2/3 were able to activate transcription regulators of inflammation in the nucleus. e phosphorylation of p38, ERK1/2, and JNK1/2/3, related to a signaling cascade involving MAPK kinases responsible for phosphorylation and activation of the MKKK and MKK, is required for MAPK activation [32]. In this present study, the relevant protein expressions involved in TLR4/NF-κB/MAPK signaling pathway, including TLR4, MyD88, NF-kB, p-JNK1/2/3, JNK1/2/3, p-ERK1/2, ERK1/2, p-p38, and p38 were upregulated in the model group compared control group, indicating that TLR4/NF-κB/MAPK signaling pathway was activated by ammonia. In contrast, the levels of proteins were significantly decreased after administration for 5 days. Additionally, the effect of S. oblata in the dosage of 270 mg/ kg and 135 mg/kg was better than that of 68 mg/kg. us, we speculated that S. oblata and Yan Li Xiao may exert a protective effect in ammonia-induced acute pharyngitis via blocking TLR-4/NF-κB/MAPK signaling pathway.
NLRP3 inflammasome is an important multiprotein complex, which mainly consists of a sensor (NLRP3), an adaptor (ASC), and an effector (caspase-1). It is a major intracellular inflammatory pathway, mainly driving autoimmune and inflammatory disease processes by regulating the productions of proinflammatory cytokines [33]. Assembly of NLRP3 triggered a cascade process, including cleavage procaspase-1 into active caspase-1, and then converts the pro-IL-1β and pro-IL-18 into mature and biologically active IL-1β and IL-18 from secrete into extracellular, respectively [34]. Several studies indicated that both MAPK and NF-κB play important roles in cellular responses to inflammation-induced stress and NLRP3 activation [35]. erefore, inhibition of NLRP3 inflammasome activity has been proposed as a strategy for treating inflammatory diseases. In our study, ammonia significantly increased the protein expression of NLRP3, ASC, and caspase-1 in model control.  cytokines levels. Interestingly, test compounds exhibited a significant effect in a dose-dependent manner except for syringopicroside. is phenomenon may be caused by the contradictory effect of syringopicroside on macrophages. e result of syringopicroside in vitro was consistent with the previous study [36]. e phenomenon might be related to the contradictory effect of syringopicroside on macrophages. erefore, the mechanisms of compounds that display an anti-inflammatory effect need to be further investigated.

Conclusions
In this study, the ammonia-induced acute pharyngitis model was used for evaluating the therapeutic effect of S. oblata. S. oblata significantly regulated the balance between proinflammatory and anti-inflammatory cytokines. e results indicated that S. oblata could alleviate ammonia-induced acute pharyngitis by inhibiting the activation of TLR4/NF-κB/MAPK and NLRP3 inflammasome signaling pathways. Furthermore, four iridoids were isolated from the leaves of S. oblata and identified by NMR methods. Results indicated that all of them exerted antiinflammatory activity in LPS-stimulated RAW 264.7 cells in different degrees. is study provided a potential basis for future clinical treatment and research in inflammationrelated diseases.