Investigation of the Immunoprotective Effect of Zinc on Ovalbumin Induced BALB/C Male Mice Based on NF-KB Signaling Pathway.

Allergic rhinitis is one of the common chronic inflammatory diseases of the nasal mucosa. In order to investigate the effect of zinc on ovalbumin induced allergic rhinitis in BALB/C male mice based on NF-KB signaling pathway, thirty BALB/C male mice are randomly divided into three groups: control group, ovalbumin induced allergic rhinitis asthma group and zinc intervention group. The experimental results show that Zinc supplementation in allergic asthma mice with allergic rhinitis correct the immune response of TH2 cells by inhibiting THE NF-KB signaling pathway, reduce the infiltration of inflammatory cells into lung nasal tissue, and reduce airway co-hyperreactivity to improve the clinical symptoms of asthma and play an immune protective role.

1. Introduction

Allergic rhinitis is a chronic inflammatory disease of the nasal mucosa in which atopic individuals are exposed to allergens, resulting in the release of IgE-mediated inflammatory mediators and the participation of a variety of immune active cells, cellular molecules and adhesion molecules. Caused by indoor or outdoor allergens, the disease is clinically characterized by nasal congestion, paroxysmal and repeated sneezing, watery rhinorrhea, nasal itching and post-nasal drip. Current immunopathology has confirmed that chronic allergic inflammation in the upper and lower respiratory tract is similar, and both have similar inflammatory cell exudation, such as the involvement of eosinophils, Th2 cells, mast cells and IgE, and also have similar cytokines, such as IL-4, IL-5, IL-13 and other inflammatory mediators [1, 2]. Therefore, T lymphocytes are closely related to the pathogenesis of allergic rhinitis. Allergen exposure can lead to the transformation of T lymphocytes into Th2 cells, and a large number of studies have confirmed that the Th2 cytokine IL-4 can stimulate B cells to synthesize IgE [3]. Therefore, the level of gE is positively correlated with the severity of variant rhinitis and the reactogenicity of nasal airways. IL-5 can induce IL-4 to generate Ige [4, 5]. In conclusion, the current study confirms that inhibition of TH2 cytokines can reduce the severity of variant rhinitis [6].

The NF-kB signaling pathway is involved in the expression of various genes and regulates various physiological processes such as immune response, inflammatory response, and apoptosis. Some studies have also confirmed that abnormal activation of NF-kB can be observed in variant rhinitis, and some studies can treat variant rhinitis by inhibiting the activation of NF-kB [7, 8]. Zinc (Zn) is a trace element, which recently has been confirmed to participate in the immune response by regulating the NF-kB cell pathway and the TCR cell pathway. Some studies have also shown that the activity of IkBα can be regulated by proteins such as ZIP8 and PKA, and the maintenance of the activities of these protein regulators requires the participation of zinc [9]. Therefore, the level of zinc expression is closely related to the activity of the NF-kB signaling pathway, but the result of whether zinc promotes or inhibits the NF-kB signaling pathway is still inconclusive, and there is no domestic study to explore the effect of zinc supplementation on allergic rhinitis. Therefore, this study selected 30 BALB/C male mice as the research objects to explore the immune protection effect of zinc on ovalbumin-induced allergic rhinitis mice.

The rest of this paper is organized as follows: Section 2 discusses related work, followed by the experimental materials and proposed method in Section 3. The cases analysis and data statistics in Section 4. Section 5 concludes the paper with summary and future research directions.

2. Related work

Allergic rhinitis is a chronic inflammatory disease of the upper respiratory tract whose main clinical features are nasal obstruction, nasal discharge, and sneezing [10]. Immunological features of the disease include TH2-biased eosinophil infiltration [11]. Nowadays, a large number of clinical and animal experiments have found that in mice with OVA-induced variant rhinitis, TH2 lymphocyte levels are ameliorated, inflammatory cells and inflammatory factors are ameliorated, and TH2 cells secrete cytokines IL-4, IL-5, and IL-13 to induce eosinophilia, which can produce a variety of cytotoxic proteins, oxides, neuropeptides and other substances, which can cause mast cells to release histamine and damage respiratory mucosal epithelium. Triggered smooth muscle contraction, resulting in mucosal edema, leading to adverse symptoms [12]. In recent years, a large number of studies have shown that patients with allergic rhinitis have phenomenally reduced serum zinc levels [13]. There have also been studies suggesting that zinc supplementation or maintenance of zinc homeostasis may be a new strategy for the treatment of allergic rhinitis [14]. Therefore, the results of this study are to investigate the mechanism of zinc supplementation in allergic rhinitis mice.

First of all, the results of this study show that compared with NC group, the symptoms of sneezing and nasal scratching in OVA group are phenomenally ameliorated, while zinc can effectively relieve the symptoms after nasal challenge. Compared with NC group, the values of nasal resistance in OVA group and Zn group are phenomenally ameliorated, while compared with OVA group, the values of nasal resistance in Zn group are phenomenally alleviated after nasal challenge. This indicates that zinc supplementation can reduce the nasal resistance value and relieve the symptoms of variant rhinitis. Gomez et al. find that zinc deficiency can lead to phenomenal changes in lung lipid metabolism in male rats, which can lead to an ameliorate in phospholipids and cholesterol and a alleviate in triglyceride content, thus leading to airway hyperresponsiveness in mice [15]. Zhang et al. find that zinc deficiency can aggravate oxidative stress in lung tissue, induce inflammation and lead to pulmonary fibrosis [16]. The results of this study are consistent with the above-mentioned results, indicating that zinc deficiency can lead to airway hyperresponsiveness in mice, and the specific mechanisms combined with the results can be summarized as changes in lung lipid metabolism, oxidative stress, and infiltration of inflammatory cells.

Secondly, the results of this study show that the total number of cells, eosinophils and neutrophils in nasal lavage fluid of OVA group are phenomenally ameliorated, but there is no phenomenal difference between the two groups. Compared with OVA group, the total number of cells, eosinophils and neutrophils in Zn group are phenomenally alleviated, and the lymphocyte count level is phenomenally ameliorated. IgE and IL-4 in OVA group are phenomenally ameliorated, while IFN-γ and IL-10 are phenomenally alleviated. Compared with OVA group, IgE and IL-4 levels in Zn group are phenomenally alleviated, while IFN-γ and IL-10 levels are phenomenally ameliorated, which indicated that zinc supplementation could effectively improve inflammatory indexes, reduce inflammatory cell infiltration and exert immune protection.

Other results indicate that zinc-deficient peripheral blood mononuclear cells that secrete phenomenally less IFN-γ protein show phenomenally higher cytokine mRNA levels compared to cells with high total zinc levels. Wessels find that zinc supplementation attenuates LPS-induced lung injury in a mouse model of ALI [17-20]. The results of Tsai indicate that zinc improves T cell spectrum imbalance and may be a potential adjunct to dust mite-induced allergic hypersensitivity [21-24]. The above results are consistent with the results of this study. It is confirmed that zinc deficiency can ameliorate the expression of TH2 cytokines, promote inflammatory cell infiltration, and lead to the occurrence of allergic rhinitis. Zinc supplementation can improve inflammatory indexes, reduce inflammatory cell infiltration and exert immune protection. The specific reason may be that the present study confirms that zinc participates in the differentiation of T cell subpopulations. Zinc deficiency leads to a alleviate in the function of TH1 cells, but the function of TH2 cells is not affected, which leads to an ameliorate in the level of TH2 cytokines. Among them, IL-4 can promote B cell maturation and convert IgG into IgE, and promote the differentiation of T cells into TH2 cells, thus leading to an ameliorate in IgE level. However, zinc supplementation may alleviate the production of inflammatory cytokines and restore TH1/TH2 cell subpopulations through inhibition of related inflammatory signaling pathways [25, 26].

The results of this study show that compared with NC group, the expression of p-P65 and p-IkBα in OVA group is phenomenally ameliorated, while the expression of p-P65 and p-IkBα in Zn group is phenomenally alleviated, while the expression of IkBα in Zn group is phenomenally ameliorated. Immune protection is achieved through NF-kB signaling pathways. Some paper finds that the mechanism of action in the treatment of allergic rhinitis may inhibit NF-kB signaling pathway on the one hand, and stimulate cAMP-PKA signaling pathway on the other, which may be related to the down-regulation of MUC5AC and MUC5B expression. Other paper finds that regulating the expression of TLR-NF-kB pathway, inhibiting the over-expression of NF-kB, is conducive to regulating the proliferation of eosinophils in the pathological process of allergic rhinitis. These results are consistent with those of the present study, which confirm the important role of NF-kB signaling pathway in the pathogenesis of allergic rhinitis. It has been shown that NF-kB regulates TH2 cell differentiation and TH2 cytokine expression in allergic rhinitis, while activated NF-kB enhances TH2 cell differentiation bias and increases the expression of related cytokines. The core of the activated NF-kB signaling pathway and the phosphorylation and ubiquitination of IkB and IKK components are regulated by a variety of proteases, as in the signaling pathway, TRIM13, TRIM21, and TRIM27 inhibit the activity of the IKK complex, and TRIM9 inhibit ubiquitination of phosphorylated IκB by preventing β-TrCP from binding to IκB. In the nucleus, TRIM19, TRIM20, TRIM39 and TRIM45 inhibit NFkB activity. The IKK complex is also regulated by ZIP8, PKA, and A20. The enzyme activity of the above-mentioned TRIM protein and other related proteins is mostly dependent on the presence of zinc, so the deletion of it is able to lead to the reduction of the above-mentioned enzyme activity, thus leading to the activation of NF-kB [27, 28]. However, the effects of regulatory proteins on NF-kB pathway are different, and there is inhibition and promotion. Therefore, the effects of intracellular zinc supplementation on NF-kB signaling pathway need further investigation.

3. Experimental materials and proposed method

3.1. Experimental materials and main instruments

The experimental animals include the follow aspects: (1) SPF grade male BALB/C male mice aged 6-8 weeks, with an average weight of 18-22g, are raised in SPF grade environment of the Experimental Animal Center of Ningxia Medical University. They are kept stable at about 25 °C, with 70% relative humidity and 12/12h of day and night illumination; (2) all experimental procedures in this study are in accordance with the requirements of the Animal Ethics Committee of Ningxia Medical University.

The experimental instruments and reagents include the follow aspects: (1) portable nebulizer; (2) small animal lung function meter; (3) pressure sensor; (4) precision syringe pump; (5) protein imprinting luminescence system; (6) ovalbumin (Sigma); (7) adjuvant liquid aluminum; (8) enzyme-linked immunoassay kit; (9) mouse anti-human antibody.

3.2. Proposed method

3.2.1. Model preparation and grouping

Thirty male BALB/c mice are randomly divided into three groups by random number table method: non-intervention control group (NC group), allergic rhinitis model group (OVA group) and zinc intervention group (Zn group) on the basis of allergic rhinitis model group. Mice are sensitized by intraperitoneal injection of 0.1ml of 0.01% OVA/AL(OH)3 mixture on day 1 and day 13, and are given nasal drops of 5% OVA saline on day 25 to day 31, with a total of 20ul on each side. For 7 consecutive days. NC mice are given saline intraperitoneally on the 1st and 13th day, and nebulized saline is given for 45min from the 25th day to the 31st day. In the Zn group, 5 mg·kg-1 zinc preparation is administered orally for 7 days, while in the OVA group and NC group, the same dose and physiological saline drops are given. At the same time, the number of nasal scratches and sneezes are counted in the three groups of mice within 15 minutes.

3.2.2. Mouse nasal resistance assay

After the mice are anesthetized with pentobarbital sodium, they are fixed in a supine position and placed on the operating table, then a 15mm incision is made in the middle of the neck, the trachea is separated, and an incision is made with ophthalmic conjunctival scissors at the opening of the thorax. Offering BUXCO company 18G cannula to the nasopharynx, which is connected to the T-tube, and the T-tube is connected to the experimental precision syringe pump and the pressure sensor respectively. The injection/retraction volume is 10ml/10ml, and the post-nasal pressure is measured by the pressure sensor. The parameters are recorded, and then the oral wet cotton ball is given for sealing. The total resistance and intubation resistance are calculated, and the nasal airway resistance of the mice is calculated.

3.2.3. Laboratory index measurement

All mice are anesthetized by intraperitoneal injection of sodium pentobarbital after nasal resistance measurement. Preocular venous blood is removed from the eyeball, centrifuged at 3000 r/min for 15min, and supernatant is taken. Serum IgE, IL-4, IL-10 and IFN-γ levels are measured by enzyme-linked immunosorbent assay. After blood is collected, and the mice are taken into the supine position, with 24G indwelling needle inserted into one nostril of mice. 1ml PBS is used for lavage, recovery amount is recorded, and EP tube is placed under the contralateral nostril to recover all nasal lavage solution. After being shaken well, 100ul of lavage solution is taken for 1000 r/min for 15min. Afterwards, it is centrifuged, and taken cell precipitation, adapting cell count to measure the total number. They are classified into eosinophils, neutrophils, and lymphocytes at high magnification and counted according to their morphological characteristics.

3.2.4. Histopathological preparation of mice HE nasal tissue

The nasal tissues of mice are fixed in formaldehyde and embedded in paraffin for 4um, and then stained with HE. The specific steps are as follows: (1) Dewaxing of conventional sections; (2) dehydration of alcohol gradient and injection of distilled water into the staining solution; (3) put into hematoxylin staining solution for about 5∼15min; (4) remove excess dye solution; (5) put into saturated solution with lithium carbonate for alkalization; (6) dye with 0.1∼0.5% eosin staining solution for 1∼5min; (7) dehydration with gradient alcohol and transparent xylene; (8) drop a proper amount of neutral gum, then seal the film with cover glass, and observe under the microscope. The degree of inflammatory infiltration in nasal mucosa is evaluated. No inflammatory cells are 0 points, a few inflammatory cells are 1 point, more unevenly distributed inflammatory cells are 2 points, a large number of inflammatory cells are evenly distributed are 3 points, and a large number of inflammatory cells are clustered into 4 points.

The SP immunohistochemistry include the follow aspects: (1) Pathological tissues are ished with conventional PBS solution; (2) pathological tissues are fixed with 4% paraformaldehyde, incubated with conventional 3% H2O2 solution for 10min, and then treated with citrate buffer for antigen repair; (3) they are subsequently heated in water bath for 45 min and then cooled, and primary antibodies, i.e. rabbit anti-NF-KB primary antibodies, are incubated for 2h, and biotin-labeled secondary antibodies are added after isling with PBS solution; (4) after rinsing with PBS solution, add DAB chromogenic reagent dropwise and incubate at 37°C for 30 min, rinse with tap water, stain with hematoxylin, dehydrate to clear. Seal with neutral gum, and film under inverted fluorescence microscope.

3.2.5. Measurement of protein expression in nasal tissue by protein imprinting

The expression of proteins related to NF-KB signaling pathway in nasal tissue is determined by Western-bolt method. The sample of nasal mucosa is taken for full grinding, and the cell lysate containing protease inhibitor is added at a ratio of 1:9. Then it is shaken with a low-temperature homogenizer. After being fully lysed for 30 min, it is transferred to a centrifuge tube with a pipette and centrifuged at 12000 r/min at 4°C for 5min, after boiling for 10 min, 50 ug of total protein is taken into sodium dodecyl sulfate polyacrylamide gel. The classified protein is transferred to PVDF membrane with a wet membrane transfer device. Then 5% skim milk powder is applied to seal the membrane at 4°C overnight. Antibodies to p-p65, p65, p-IKBα and IKB α proteins are blocked for 2h at room temperature, three times with TBST solution, then added secondary antibody, incubated in the dark for 1h, and then three times with TBST solution for 10min each time. The ratio of the relative expression of protein to the absorbance value of the target band to the absorbance value of the internal reference protein

3.3. Statistical methods

SPSS 20.0 statistical software is used to process the data. Measurement data are expressed as mean ± standard deviation, T test is used for comparison between two groups, and F test is used for comparison between multiple groups. Enumeration data are expressed as n or percentage, and chi-square test is used. Differences with P<0.05 are considered statistically phenomenal.

4. Cases analysis and data statistics

4.1. Allergic rhinitis modeling behavior score in mice

Compared with NC group, the symptoms of sneezing and nasal scratching in OVA group are phenomenally increased, and the behavioral scores are phenomenally increased, while zinc intervention could effectively relieve the symptoms, and the behavioral scores are alleviated, the difference is statistically phenomenal (P < 0.05), as shown in Table 1.

Table 1

Statistics of behavioral symptom scores in mice.

Group	NC group (n = 10)	OVA group (n = 10)	Zn group (n = 10)	F value	P value
Times of sneezing	1.85 ± 1.62	32.87 ± 5.23	18.52 ± 3.29	10.532	0.001
Times of scratching	12.65 ± 5.34	66.71 ± 15.39	38.42 ± 5.28	9.814	0.002
Total score	4 ± 1.02	5 ± 0.59	4 ± 1.11	9.625	0.023

4.2. Comparison of nasal resistance measurements in mice

There is no phenomenal difference in nasal airway resistance inspiratory and expiratory values among the three groups (P > 0.05). Compared with NC group, the nasal resistance values of OVA group and Zn group are phenomenally ameliorated, and the difference is statistically phenomenal (P < 0.05). The difference is statistically phenomenal (P < 0.05), as shown in Figure 1.

Figure 1

Line chart of three groups of nasal resistance.

4.3. Comparison of parameters of nasal lavage fluid in mice

Compared with the mice in the NC group, the total number of cells, eosinophils, and neutrophils in the nasal lavage fluid of the OVA group are phenomenally ameliorated, and the difference is statistically phenomenal (P < 0.05). There is no phenomenal difference between the two groups (P > 0.05). Compared with the OVA group, the total number of cells, eosinophils and neutrophils in the Zn group are phenomenally alleviated, and the lymphocyte count is phenomenally ameliorated, and the difference is statistically phenomenal (P < 0.05), as shown in Table 2.

Table 2

Comparison of parameters of nasal lavage fluid in mice.

Group	NC group (n = 10)	OVA group (n = 10)	Zn group (n = 10)	F value	P value
Total number of cells	2.14 ± 6.32	12.19 ± 0.52	10.54 ± 00.43	8.416	0.025
Eosinophils	2.15 ± 1.15	40.42 ± 15.13	16.62 ± 2.53	10.534	0.023
Neutrophils	4.65 ± 0.42	16.35±3.85	11.82 ± 5.31	8.415	0.045
Lymphocytes	21.89 ± 8.35	22.65±8.31	42.89 ± 5.67	12.314	0.001

4.4. Comparison of serum parameters in mice

Compared with NC group, IgE and IL-4 in OVA group are phenomenally higher, while IFN-γ and IL-10 are phenomenally lower (P < 0.05). Compared with OVA group, IgE and IL-4 levels in Zn group are phenomenally lower, while IFN-γ and IL-10 levels are phenomenally higher (P < 0.05), as shown in Table 3.

Table 3

Comparison of serum parameters in mice.

Group	NC group (n = 10)	OVA group (n = 10)	Zn group (n = 10)	F value	P value
IgE(ng/ml)	128.07 ± 60.92	625.65 ± 100.59	316.54 ± 82.17	8.541	0.009
IL-4(ng/L)	152.05 ± 5.98	285.34 ± 5.45	172.41 ± 7.72	7.319	0.008
IL-10(pg/ml)	11.45 ± 1.65	8.43 ± 1.85	10.62 ± 2.21	10.542	0.012
IFN-γ(ng/L)	527.61 ± 23.15	272.34 ± 12.75	385.82 ± 30.16	9.413	0.003

4.5. HE staining of pathological sections of mice

The results of HE staining show that there is no inflammatory cell infiltration around the mucosa in NC group, while in OVA group, a large number of inflammatory cell infiltration and exudation of eosinophils and neutrophils are seen under the mucosa. Figure 2 is the comparison of three pathological scores. The pathological grade score of nasal mucosa inflammation show that Zn group is lower than OVA group, and the difference is statistically phenomenal (P < 0.05).

Figure 2

HE staining of pathological sections of mice.

4.6. Changes of nasal-tissue-related proteins in mice

Compared with the NC group, p-P65 and p-IKBα in the OVA group are phenomenally ameliorated, and the expression of IKBα protein is phenomenally alleviated, and the difference is statistically phenomenal (P<0.05). The expression of p-IKBα is phenomenally alleviated, while the expression of IKBα protein is phenomenally ameliorated, and the difference is statistically phenomenal (P<0.05), as shown in Table 4.

Table 4

Changes of nasal-tissue-related proteins in mice.

Group	NC group (n = 10)	OVA group (n = 10)	Zn group (n = 10)	F value	P value
p-P65	0.21 ± 0.05	0.85 ± 0.15	0.45 ± 0.11	8.416	0.005
P65	0.67 ± 0.07	0.65 ± 0.09	0.68 ± 0.08	0.452	0.438
IKBα	0.82 ± 0.09	0.21 ± 0.05	0.65 ± 0.07	6.934	0.001
p-IKBα	0.05 ± 0.02	0.54 ± 0.12	0.32 ± 0.08	7.315	0.003

5. Conclusions

To sum up, mice with allergic rhinitis are supplemented with zinc preparation, which may correct the immune response of TH2 cells by inhibiting NF-KB signaling pathway, down-regulating inflammatory factors, reducing nasal mucosal tissue infiltration of inflammatory cells, and reducing nasal resistance value to improve clinical symptoms and exert immune protection. However, there are also studies showing that zinc can activate P13K-AKT/NFAT signaling pathway, so the mechanism of zinc supplementation to improve allergic rhinitis requires further exploration.