| Allergic rhinitis is a chronic inflammatory disease that affects approximately 10?20%of the global population.In recent years,the incidence of allergic rhinitis is increasing year by year worldwide.Allergic rhinitis is part of a systemic inflammatory disease and is associated with other inflammatory disorders,including asthma,sinusitis,and allergic conjunctivitis.Allergic rhinitis reduces the quality of life of a patient and can affect sleep,school work,work productivity,and social life.Allergic rhinitis has been classified as a chronic respiratory disease due to its high morbidity and its impact on life quality.Allergic rhinitis is not only imposes a heavy economic burden on patients,but also increases the financial burden of national medical insurance.Compared with common diseases such as coronary heart disease,diabetes,coronary heart disease and asthma,it requires more capital investment.There are guidelines for allergic rhinitis for doctors and patients.However,many patients with allergic rhinitis do not seek the help of a primary care doctor or expert,but choose to treat themselves with allergic rhinitis or even ignore it directly.Therefore,when identifying and diagnosing allergic rhinitis symptoms,doctors should carefully ask the patient's history of onset and previous medications to facilitate reference for follow-up treatment.Currently,conventional therapies for allergic rhinitis include avoidance of contact with allergens,saline cleaning of the nasal cavity,oral antihistamines,intranasal corticosteroids,combined with intranasal corticosteroids and antihistamine sprays,leukotriene receptor antagonists.And allergen immunotherapy.Other therapies may be useful in individual patients,including decongestants and oral corticosteroids.However,these drugs only relieve the symptoms of allergic rhinitis in a short period of time,and the inevitable side effects limit their long-term clinical application.Therefore,there is an urgent need to develop novel therapeutic agents that target the pathogenesis of allergic rhinitis,which have low side effects or compliance with allergen avoidance.It is well known that miRNAs are involved in many biological processes such as cell differentiation,immune defense,and apoptosis.In recent years,MiR-146a has become one of the hotspots in the field of inflammatory diseases research,with a strong and extensive anti-inflammatory effect.It has been found that MiR-146a is highly expressed in children with allergic rhinitis treated with allergen-specific immunotherapy.Moreover,MiR-146a is highly expressed on the surface of regulatory T cells,and when effector T cells and myeloid cells are activated,it can induce the expression of MiR-146a.MiR-146a is involved in allergen-specific immunotherapy mainly by regulating the inflammatory response mediated by TRAF6 and IRAK 1/2 through negative feedback.Strikingly,it has been reported in the literature that MiR-146a inhibits the inflammatory response by inhibiting TLR4/TRAF6/NF-?B signaling in rheumatoid arthritis.They also found that MiR-146a may have the potential to treat allergic rhinitis,but the specific mechanism of its anti-inflammatory effects in allergic rhinitis has not yet been elucidated.Objective1.MiR-146a has been shown to have a very effective anti-inflammatory effect among the inflammation-related diseases.Therefore,the aim of this study was to investigate the expression of MiR-146a in patients with allergic rhinitis and its effects on inflammation in patients.2.Investigate whether MiR-146a affects allergic rhinitis by affecting the TRAF6/NF-?B signaling pathway.3.Investigate whether MiR-146a exerts therapeutic potential and its underlying mechanisms for allergic rhinitis in the mouse model of allergic rhinitis.Methods1.The RNA,mRNA and protein levels of MiR-146a,TRAF6 and other related molecules in the nasal mucosa of three groups of mice and patients were detected by reverse transcription quantitative polymerase chain reaction and Western blotting.2.Immunohistochemistry was used to detect the expression of TRAF6 in patients with simple nasal septum deviation and allergic rhinitis.3.In vitro culture of nasal mucosal tissues of patients with allergic rhinitis,treat with different concentrations of MiR-146a analogues,then detect the protein expression levels of key molecules in the TRAF6/NF-?B signaling pathway by protein western blotting.4.Female mice were randomly divided into three groups:the allergic rhinitis group(AR),the control group(Ctrl)and the MiR-146a group(MiR-146a).Mice were stimulated with ovalbumin to establish a mouse model of allergic rhinitis.For mice in the allergic rhinitis group,mice were sensitized on day 0,7,and 14 by intraperitoneal injection of 100 ?l of physiological saline containing 100 ?g of ovalbumin and 2 mg of aluminum hydroxide.The mouse nasal cavity was then stimulated daily with ovalbumin solution(soluted in physiological saline at 40 mg/ml,20?l/mouse)for 21-28 days.For the control group,an equal amount of physiological saline solution containing aluminum hydroxide and no ovalbumin was normally injected,and then the mouse nasal cavity was stimulated with physiological saline instead of the ovalbumin solution on days 21-28.For the MiR-146a group,on days 0,7,and 14,mice were sensitized by intraperitoneal injection of 100 ?l of physiological saline containing 100 ?g of ovalbumin and 2 mg of aluminum hydroxide,and then used daily for 21-28 days.Before the ovalbumin solution administered intranasally 30 minutes,the MiR-146a analog was administered intranasally(dissolved in physiological saline at a concentration of 0.1 mg/ml,20?l/mouse).The sneezing frequency and the number of nasal friction events were recorded in all mice at 28 days,and the recording process lasted 10 minutes.Two hours after the last stimulation of the mouse's nasal cavity,blood and nasal lavage fluid of each mouse were collected for subsequent analysis in the case of mouse anesthesia.5.Enzyme-linked immunosorbent assay is used to detect the concentration of related proteins in nasal lavage fluid and serum,including ovalbumin-specific IgE,leukotriene C4,eosinophil cationic protein,and interleukin-2,IL-4,IL-5 and IL-13 and interferon-y levels.6.We Count the total number of white blood cells in the nasal lavage fluid of the three groups of mice using a blood cell counter.Eosinophils,neutrophils and lymphocytes were stained by Wright's-Giemsa assay and cell counts were performed later.Results1.(1)The RNA level of MiR-146a was significantly down-regulated in patients with allergic rhinitis compared with patients with simple nasal septum deviation.Moreover,the mRNA and protein levels of the downstream target gene TRAF6 of MiR-146a were significantly up-regulated in patients with allergic rhinitis.(2)HE staining results showed that the nasal mucosa tissue structure of patients with simple nasal septum deviation was relatively normal,but staining of patients with allergic rhinitis showed a large amount of inflammatory cell infiltration.And immunohistochemistry results showed that TRAF6 was significantly elevated in patients with allergic rhinitis.(3)Compared with patients with simple nasal septum deviation,the levels of proinflammatory cytokines IL-4,IL-5 and IL-13 in serum of patients with allergic rhinitis were significantly increased.2.Compared with untreated,the protein expression level of TRAF6 in the nasal mucosa of patients with allergic rhinitis decreased after treatment with MiR-146a analogue;the protein level of p-I?B? was significantly down-regulated;the protein level of I?B? was significantly increased;p65 protein levels decreased.Moreover,the level of change in these proteins has a concentration dependence of the MiR-146a analog.3.(1)The results of HE staining showed that there were glandular epithelial edema and submucosal gland hyperplasia in the nasal mucosa of mice with allergic rhinitis compared with the normal control group.After the intervention of MiR-146a mimic,nasal mucosal glandular epithelial edema and glandular hyperplasia were reduced in mice with allergic rhinitis.(2)Compared with the control group,the RNA expression level of MiR-146a in the nasal mucosa of the mice with allergic rhinitis was significantly down-regulated(P=0.002).And as the downstream target gene of MiR-146a,the mRNA level(P=0.004)and protein level(P<0.01)of the TRAF6 in the nasal mucosa of the allergic rhinitis mice group were apparently higher than those of the control group.Increase.(3)Compared with the control group,the untreated allergic rhinitis mice had severe sneezing and runny nose symptoms(P<0.01),and the average number of sneezing in the control group within 10 minutes was 11.50 times,but the average number of sneezing in 10 minutes of untreated rhinitis mice was 56.75 times.After receiving MiR-146a analog treatment,the number of sneezing in allergic rhinitis mice was significantly reduced(P=0.007),and the average number of sneezing in 10 minutes was reduced to 35.5 times.(4)The average number of nasal frictions in the control group was 14 times in 10 minutes,and the average number of nasal friction in the untreated allergic rhinitis mice was 62.25 times in 10 minutes.The number of nasal frictions in the untreated allergic rhinitis mice was distinctly higher than that of the control mice(P<0.01).After receiving MiR-146a analog treatment,the number of nasal frictions in allergic rhinitis mice was obviously reduced(P=0.0013),and the average number of nasal frictions in 10 minutes was 41.25 times.(5)The average content of ovalbumin-specific IgE in the serum of the control mice was 0.1833 pg/ml,while the content of the untreated allergic rhinitis mice was 0.5133 pg/ml,which was conspicuously higher than that of the control mice(P=0.0381).After treatment with MiR-146a analog,the average level of ovalbumin-specific IgE in the allergic mice group was 0.3233 pg/ml,which was conspicuously lower than that of untreated allergic rhinitis mice(P<0.01).For serum leukotriene C4,the average content of the control mice was 129.8 pg/ml,and the average content of untreated allergic rhinitis mice was 239.8 pg/ml,which was conspicuously much higher than the control mice's level(P=0.0094).After receiving the MiR-146a analog treatment,the average level of leukotriene C4 in the group of allergic rhinitis mice was reduced to 159.7 pg/ml,which was conspicuously lower than that of untreated allergic rhinitis mice(P<0.01).For the Eosinophil cation protein in serum,the average content of the control mice was 156.4 pg/ml,and the average content of the untreated allergic rhinitis mice was 589.8 pg/ml,which was conspicuously higher than that of the control mice(P<0.01).After treatment with the MiR-146a analog,the average level of Eosinophil cation protein in the group of allergic rhinitis mice was 276.3 pg/ml,which was conspicuously lower than that of untreated allergic rhinitis mice(P<0.01).(6)The average number of eosinophils in the nasal lavage fluid of the control group was 5.43×106 cells/ml,and the average number of allergic rhinitis mice was 26.16.10×106 cells/ml,which was conspicuously higher than that of the control group(P=0.0009).After receiving the MiR-146a analog treatment,the average number of eosinophils was 16.83×106 cells/ml,which was conspicuously lower than that of untreated allergic rhinitis mice(P<0.01).For leukocytes in nasal lavage fluid,the average number of control mice was 10.6×106 cells/ml,and the average number of allergic rhinitis mice was 54.49×106 cells/ml,which was conspicuously higher than that of the control group(P<0.01).After receiving MiR-146a analog treatment,the average number of white blood cells in the nasal lavage of allergic rhinitis mice was 27.66×106 cells/ml,which was conspicuously lower than that of untreated allergic rhinitis mice(P=0.0006).For lymphocytes in nasal lavage fluid,the average number of control mice was 3.273×106 cells/ml,and the average number of allergic rhinitis mice was 15.83×106 cells/ml,which was conspicuously higher than that of the control group(P=0.0002).After receiving MiR-146a analog treatment,the average number of lymphocytes in the nasal lavage of allergic rhinitis mice was 9.618×106 cells/ml,which was conspicuously lower than that of untreated allergic rhinitis mice(P<0.01).For neutrophils in nasal lavage fluid,the average number of control mice was 1.773×106 cells/ml,and the average number of allergic rhinitis mice was 12.50×106 cells/ml,which was conspicuously higher than that of the control group(P<0.01).After receiving the MiR-146a analog,the average number of lymphocytes in the nasal lavage of allergic rhinitis mice was 8.118×106 cells/ml,which was conspicuously lower than that of untreated allergic rhinitis mice(P<0.01),(7)The average level of serum IL-4 in the control group was 23.82 pg/ml,and the average level of serum IL-4 in allergic rhinitis mice was 116.8 pg/ml,which was conspicuously higher than that in the control group(P=0.0063).After receiving MiR-146a analog treatment,the average level of serum IL-4 in mice with allergic rhinitis was 55.52 pg/ml,which was conspicuously lower than that of the untreated group of allergic rhinitis mice.For IL-5 in serum,the average level of control mice was 31.15 pg/ml,and the average level of allergic rhinitis mice was 250.1 pg/ml,which was conspicuously higher than that of the control group(P=0.0038).After receiving the MiR-146a analog treatment,the average level of serum IL-5 in allergic rhinitis mice was 122.2 pg/ml,which was conspicuously lower than that of the untreated group of allergic rhinitis mice.For serum IL-13,the average level of control mice was 37.82 pg/ml,and the average level of allergic rhinitis mice was 183.5 pg/ml,which was conspicuously higher than that of the control group(P=0.0228).After receiving MiR-146a analog treatment,the level of serum IL-5 in allergic rhinitis mice was 88.85 pg/ml,which was conspicuously lower than that of the untreated group of allergic rhinitis mice.(8)The average level of serum IL-2 in the control group was 411.6 pg/ml,and the average level of serum IL-2 in the mice with allergic rhinitis was 203.6 pg/ml,which was conspicuously lower than that in the control group(P<0.01).After receiving the MiR-146a analog treatment,the average level of serum IL-2 in allergic rhinitis mice was 289.8 pg/ml,which was conspicuously higher than that of the untreated group of allergic rhinitis mice.For serum IFN-y,the average level of control mice was 53.27 pg/ml,and the average level of allergic rhinitis mice was 20.30 pg/ml,which was conspicuously lower than that of the control group(P=0.0002).After receiving MiR-146a analog treatment,the average level of serum IFN-y in mice with allergic rhinitis was 35.11 pg/ml,which was conspicuously higher than that of the untreated group of allergic rhinitis mice.(9)Compared with the control group,the protein expression level of TLR4 and its downstream molecule TRAF6 in the nasal mucosa of allergic rhinitis mice was significantly increased.After treatment with the MiR-146a analog,the expression levels of both proteins were conspicuously down-regulated in allergic rhinitis mice.In addition,NF-?B was conspicuously activated in the group of allergic rhinitis mice,and the ratio of p-I?B? to I?B? and the expression level of p65 protein in the nucleus were conspicuously increased in the cytoplasm compared with the control group.Similarly,the ratio of p-I?B? to I?B? in the cytoplasm and the protein expression level of p65 in the nucleus were conspicuously down-regulated in allergic rhinitis mice after treatment with MiR-146a analog.Conclusions1.This study found that the RN A level of MiR-146a in patients with allergic rhinitis is significantly down-regulated,and the mRNA and protein levels of its downstream target gene TRAF6 are conspicuously up-regulated.Moreover,the levels of pro-inflammatory factors IL-4,IL-5 and IL-13 were conspicuously increased in patients with allergic rhinitis.2.The TRAF6/NF-?B signaling pathway was affected after treatment of nasal mucosa which was cultured in vitro with MiR-146a analog.Therefore,this study suggests that in patients with allergic rhinitis,the expression of MiR-146a may be down-regulated to affect the TRAF6/NF-?B signaling pathway,thereby promoting inflammation.3.This study found that the RNA level of MiR-146a was significantly down-regulated in mice with allergic rhinitis,and the mRNA and protein levels of downstream target gene TRAF6 were significantly up-regulated.MiR-146a analogues can alleviate allergic symptoms in mice with allergic rhinitis.Moreover,the MiR-146a analog can reduce the production of ovalbumin-specific IgE in serum,reducing the number of inflammatory cells.In addition,MiR-146a analogues can regulate Th1/Th2 cell imbalance in mice with allergic rhinitis to some extent,thereby affecting the expression of pro-inflammatory and anti-inflammatory cytokines.In terms of mechanism,this paper found that the anti-inflammatory effect of MiR-146a on allergic rhinitis may play a role in inhibiting the TLR4/TRAF6/NF-?B signaling pathway to some extent.Therefore,MiR-146a can be considered as a potential therapeutic target for allergic rhinitis in the future. |
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