Inhibitory Effect of Bergenin on TLR-4/NF-κB Signal Pathway in Reducing Allergic

Weiming WU, Pengfei GE, Jianqiao LI, Yuefeng WANG

1.Guangxi Wuzhou Red Cross Hospital, Wuzhou 543002, China; 2.The Second Affiliated Hospital of Guilin Medical University, Guilin 541004, China; 3.Guilin Medical University, Guilin 541199, China

Abstract [Objectives]To explore the effect and possible mechanism of bergenin in relieving allergic rhinitis(AR)in mice.[Methods]50 C57/BL6 mice were randomly divided into blank group(n=10), model group(n=10)and high(100 mg/kg), medium(50 mg/kg)and low(25 mg/kg)dose bergenin groups with 10 mice in each group.Except for the blank group, the other mice were sensitized by basic ways combined with attack to replicate the AR model.From the 15th d of modeling(from the second d after the end of the basic modeling), the drug group was given bergenin orally for 15 d, and the blank group and model group were given the same volume of normal saline once a day.24 h after the last establishment of the model, the content of interleukin 4(IL-4), IL-6, TNF-α and IL-1β in nasal lavage fluid and serum of mice in each group was detected by ELISA.The expression of TLR-4, NF-κB and p-NF-κB in nasal mucosa of mice was detected by Western blot.[Results]Compared with the blank group, the content of inflammatory factors IL-4, IL-6, TNF-α and IL-1β in nasal lavage fluid and serum of model group was significantly increased, and the protein expression of TLR-4 and p-NF-κB was significantly increased.After the intervention of bergenin, the content of IL-4, IL-6, TNF-α and IL-1β in nasal lavage fluid and serum and TLR-4 and p-NF-κB protein in tissue was significantly inhibited in bergenin group.[Conclusions]Bergenin can effectively reduce allergic inflammation in AR model mice, and its mechanism may be related to inhibition of inflammation and down-regulation of TLR-4/NF-κB signal pathway.

Key words Allergic rhinitis, Bergenin, Inflammatory response, TLR-4/NF-κB signal pathway

1 Introduction

Allergic rhinitis(AR), as a common chronic allergic respiratory disease, is mainly caused by the interaction of genes and environment.40% of the patients in the world are plagued by varying degrees of AR, and the incidence of the disease has shown an upward trend in recent years[2-3].The main drugs for clinical treatment of AR are corticosteroids and antihistamines, which often lead to collective hormone resistance, somnolence and other adverse reactions[4-5].Therefore, it is particularly urgent to seek drugs for the prevention and treatment of AR.

The main clinical symptoms of allergic rhinitis are sneezing, nasal congestion, nasal pruritus and watery nose.The main cause of allergic rhinitis is inflammation caused by the interaction of IgE and other immunoglobulins under the action of allergen[6].Inflammatory cells in allergic rhinitis include eosinophils and mast cells, and the inflammatory process involves many different allergic mediators, such as histamine, interleukin-1β(IL-1β), IL-4, IL-6 and tumor necrosis factor-α(TNF-α)[7].IL-1β, IL-6 and TNF-α play a role in inflammatory response, T cell activation and B cell survival.The increased expression of these cytokines indicates excessive inflammatory response[8].It has been found that NF-κB(nuclear factor kappa-B)and TLR-4 protein are highly expressed in the nasal mucosa of AR mice, and inhibition of NF-κB/TLR-4 signal pathway can improve the symptoms of AR in mice.Other studies have confirmed that bergenin has a significant inhibitory effect on RAW264.7 cell inflammatory factors induced by LPS[10].To sum up, it is suggested that NF-κB/TLR-4 inflammatory signal pathway is closely related to AR, and bergenin has good anti-inflammatory effects, suggesting that bergenin can interfere with AR by inhibiting NF-κB/TLR-4 signal pathway.Therefore, the main purpose of this study was to explore the alleviating effect and possible mechanism of bergenin on more obvious rhinitis caused by ovalbumin by establishing a mouse model of allergic rhinitis, in order to provide a reference basis for its clinical development and prevention and treatment of AR drugs.

2 Materials and methods

2.1 Materials

2.1.1Animals.50 SPF grade C57BL/6 mice, 6 weeks old and weighing 18-21 g, were purchased from Hunan SJA Laboratory Animal Co., Ltd., license number: SCXK(Xiang)2019-0004.Feeding environment: the temperature was(22±2)℃, the relative humidity was 55%-65%, and the light cycle was 12 h alternating between light and dark.

2.1.2Drugs.Bergenin(Bidepharm, purity≥98.65%, batch: RMC804)(Shanghai Aladdin Biochemical Technology Co., Ltd.); OVA-aluminum hydroxide[Al(OH)3](Sigma Company); IL-6, TNF-α and IL-1β Elisa kits(Jiangsu Meimian Industry Co., Ltd.); PVDF membrane and chemiluminescence reagents(Millipore Co., Ltd.); rabbit anti-NF-κBp 65 antibody, rabbit anti-TLR-4 antibody and rabbit anti-GAPHD antibody(Nanjing Bioworld Biotechnology Co., Ltd.).

2.1.3Instruments.FRESCO 21 high speed frozen centrifuge(Thermo Fisher Scientific Company); Epoch 2HS microplate reader(Bio-Tek Co., Ltd.); electrophoresis apparatus and transmembrane instrument(Bio-Tek Co., Ltd.); automatic chemiluminescence image analysis and detection system(Shanghai Tianneng Technology Co., Ltd.); Eppendorf pipette(Eppendorf Co., Ltd.).

2.2 Methods

2.2.1Animal grouping, modeling and drug administration.50 C57/BL6 mice were randomly divided into blank group, model group and high(100 mg/kg), medium(50 mg/kg)and low(25 mg/kg)dose bergenin groups with 10 mice in each group.The sensitization caused by basic ways combined with attack was used to replicate the AR model[11].The three times of basic sensitization were carried out on the 0th, 7thand 14thd of modeling: the mice in the model group and bergenin group were intraperitoneally injected with OVA-Al(OH)3suspension(50 μg OVA+2 mg Al(OH)3+600 μL saline)in the above 3 d; the mice in the blank group were given the same volume of normal saline in the same way.Sensitization caused by attack was carried out once a day on the 21stto 27thd of basic modeling.The mice in the model group and bergenin group were infused with 10 μL OVA suspension on each side of the nasal cavity(1 mg OVA was added to 40 μL normal saline), once a day; the mice in the blank group were infused with the same volume of normal saline in the same way.When mice frequently scratch their nose, have a runny nose and sneeze, it means that the modeling is successful[12].From the 15thd of modeling(from the second d after the end of the basic modeling), the drug group was orally given bergenin according to different doses for 15 d, and the blank group and model group were given the same volume of normal saline once a day.

2.2.2Collection and preparation of samples.24 h after the last sensitization, the mice were anesthetized with pentobarbital sodium.The blood was taken from the carotid artery of the mice and placed on ice for 2 h.Then the supernatant was obtained by 3 000 r/min centrifugation for 10 min.Then the neck was cut open quickly, the trachea was separated, and 1 mL of normal saline was injected into the trachea.The mouse nasal effluent was collected with 2 mL centrifuge tube, which was centrifuged like the serum treatment method, and the supernatant was pipetted.The nasal lavage fluid and serum stored separately were frozen in a refrigerator at-80 ℃ for Elisa test.Then the nasal mucosa was quickly stripped from the nasal cavity and immediately frozen in liquid nitrogen and stored in a refrigerator at-80 ℃ for Western blotting protein detection.

2.2.3Detection of inflammatory cytokines by ELISA.The whole experiment was performed according to the instructions of ELISA kit, and the content of IL-4, IL-6, TNF-α and IL-1β in serum and nasal lavage fluid of mice was determined.

2.2.4Detection of protein by Western blotting.First of all, the nasal mucosa tissue was cut up and 20 mg tissue was precisely weighed, then 200 μL lysate was added to ensure the complete lysis of the homogenate.The lysate containing tissue was mixed and centrifuged at 4 ℃ in a 12 000 r/min centrifuge for 2 min.The supernatant after centrifugation was obtained.The protein concentration in nasal tissue was determined by BCA method, and then 20 μg samples were taken for polyacrylamide gel electrophoresis(SDS-PAGE).The electrophoresis conditions were as follows: constant voltage 80 V, 30 min, then constant voltage 120 V, 70 min.After the membrane transfer was completed, the target protein was then transferred to the PVDF membrane; fresh sealant containing 5% skim milk powder was used to seal at 24 ℃ for 2 h, and then washed with TBST for 3 times for 8 min.The diluted first antibody(1∶800)was added and incubated at 4 ℃ for 8 h, washed twice for 10 min each time.Then the second antibody(1∶3 000)was incubated at room temperature for 80 min.After washing with TBST again, electrochemiluminescence reagent was added to develop.With β-actin as the internal reference, the grayscale of protein bands was analyzed(using Image J software), and the relative expression of TLR-4, NF-κB and NF-κB p65 protein was calculated.

3 Results and analysis

3.1 Effects on IL-6, IL-1β, TNF-α and IL-4 in serum of AR miceCompared with the normal group, the content of IL-6, IL-1β, TNF-α and IL-4 in the serum of the model group was significantly higher than that of the normal group(P<0.01).The serum level of IL-6, IL-1β, TNF-α and IL-4 in the medium and high dose bergenin group was significantly lower than that in the model group(Table 1).

Table 1 Effects of bergenin on IL-6, IL-1β, TNF-α and IL-4 in serum of AR mice n=10)

3.2 Effects on IL-6, IL-1β, TNF-α and IL-4 in nasal lavage fluid of AR miceCompared with the normal group, the content of IL-6, IL-1β, TNF-α and IL-4 in the nasal lavage fluid of the model group was significantly higher than that of the normal group; the level of IL-6, IL-1β, TNF-α and IL-4 in nasal lavage fluid of mice in medium and high dose bergenin groups was significantly lower than that in model group(P<0.01 orP<0.05)(Table 2).

Table 2 Effects of bergenin on IL-6, IL-1β, TNF-α and IL-4 in nasal lavage fluid of AR mice n=10)

3.3 Effects on TLR-4, NF-κB and NF-κB p65 protein in nasal tissues of AR miceCompared with the normal group, the expression of TLR-4, NF-κB and protein in the nasal lavage fluid of the model group was significantly higher than that of the normal group(P<0.01); the level of TLR-4, NF-κB and NF-κB p65 protein in nasal cavity of mice in medium and high dose bergenin groups was significantly lower than that in model group(P<0.01 orP<0.05, respectively)(Fig.1).

Note: A.Normal group; B.Model group; C.Low dose group; D.Medium dose group; E.High dose group.

4 Discussion

At present, it is widely recognized that Th1/Th2 differentiation imbalance is the pathogenesis of AR, in which Th2 immune response theory occupies a dominant position[13].Th2 cells mainly participate in the humoral immune process of allergy and synthesize Th2 cytokines IL-4, IL-5 and IL-13, thereby playing a role in inflammatory response and participating in the development of atopic diseases[14-15].IL-4 and IL-13 can induce B cells to differentiate into plasma cells, and produce specific IgE that binds to mast cells and basophils to further activate and release inflammatory mediators[16].IL-5 has the ability to recruit and activate other immune cells, and can recruit a large number of eosinophils in inflammatory sites[17].On the other hand, Th1 cells release IFN-γ and IL-2, which inhibit the development of Th2[18].Therefore, overactivity of Th2 and deletion of Th1 have become key factors in the pathogenesis of allergic inflammation and AR.

In recent years, the relationship between TLR-related signal transduction pathway and Th1/Th2 balance has become a hot topic in the pathogenesis of specific diseases.Toll-like receptors are germline-coded pattern recognition receptors that recognize different molecules, thus promoting inflammation[19].TLR4-mediated immune response plays a key role in nasal mucosal defense response[20].NF-κB is a multicellular transcription factor that regulates the cascade between a variety of immune and inflammatory cytokines and inflammatory transmitters and plays an important role in TLR4-mediated inflammation regulation[21].TLR4/NF-κB signal pathway is the main regulator of inflammatory and immune processes, which is widely involved in the pathophysiology of AR.It is reported that TLR4/NF-κB signal pathway may be involved in Th1/Th2 imbalance.In the nasal mucosa of AR mice, TLR4/NF-κB signal pathway was activated, the expression of NF-κB in the nucleus of nasal mucosa was increased, and Th2 cytokines were overreleased, which promoted the increase of IgE, inflammation of mucosal epithelial cells and mucus secretion.Other studies have shown that silencing TLR4 can significantly inhibit the expression of Th2 cytokines IL-4, IL-6 and IgE, thus reducing airway eosinophil production and mucus production, while increasing the expression of Th1 cytokines IL-2, IFN-γ and TNF-α[22].NF-κB inhibitor can enhance the expression of IL-2 and IFN-γ, decrease the expression of TNF-α and inhibit anaphylaxis[23].In this study, allergen ovalbumin induced the overexpression of TLR4 and p-NF-κB, and increased the content of TNF-α and IL-6 in nasal lavage fluid.After intervention with different doses of bergenin, the protein expression level of TLR4 and p-NF-κB was decreased compared with the model group, while the expression level of IL-4, TNF-α, IL-1 and IL-6 was inhibited, and the imbalance of Th1/Th2 response was restored, thus maintaining the normal immune defense function of nasal mucosa and relieving the nasal symptoms of AR model mice.The effect of high dose bergenin group was better than that of other treatment groups.This suggests that the inhibition of TLR4/NF-κB signal pathway may be the basis of bergenin’s anti-inflammatory effect.

To sum up, bergenin has a certain protective effect on ovalbumin-induced AR mice, and its mechanism may be related to the regulation of TLR4/NF-κB signal pathway to inhibit inflammation.The results of this study provide experimental basis for AR and drug development and application, but the specific mechanism needs to be further studied.