Catechin Alleviates The Inflammation In Allergic Rhinitis By Inhibiting NF-κB/TSLP Signal Pathway

Objective:Allergic rhinitis was an inflammatory disease caused by an intense immune response to external allergens for specific individuals.Thymic stromal lymphopoietin was one of the key inducement to Th2 inflammation in allergic rhinitis.So,it was significance to further explore the mechanism under the effect of TSLP.Our research team found that catechin as an active ingredient in xiao qing long decoction,the effective compound of allergic rhinitis,showed a well anti-inflammatory activity in vitro and vivo.For them,our research speculated that catechin might be one of the important pharmacodynamic substances of xiao qing long decoction.We try to research the treatment of catechin for allergic rhinitis and found out possible mechanism under this effect,which was based on TSLP factor.Then provide a basis for the clinical application of catechin in the end.Methods:1.The pharmacodynamics study of catechin in allergic rhinitis mice.1.1 The therapeutic effect of catechin on allergic rhinitis mice:Firstly,we established an allergic rhinitis mouse model induced by OVA.The SPF female balb/c mice in eight weeks aged were assigned to the normal group,the model group,the loratadine group(2mg/kg)and the catechin group(75,150,300 mg/kg)randomly,each group with 8 samples.In addition to the normal group,the mice in the other groups were treatment with the 300μl PBS solution with 25μg OVA and 1mg aluminum hydroxide in the 0st,1,and 14 days respectively by intraperitoneal injection,and the normal group was injected with the same amount of PBS.From 21 to 27 days,30 min after oraling RO water or drugs,the mice in the normal group were challenged nasal with 40μl PBS,others were challenged nasal with 1 mg OVA.And then record the numbles of nasal rubbing and sneezing in 10 min after nasal challenge.In the29th day,the mice were quickly executed and the mouse serum,nose and lung tissue samples were obtained.The changes of white blood cells in peripheral blood were detected by the animal blood cell analyzer.Some of nasal and lung tissues were fixed with 4%polyformaldehyde,which was used for pathological section.1.2.Effects of catechin on Th2 inflammation in allergic rhinitis mice:The content of IL-5 and IL-13 in the serum and lung tissues of allergic rhinitis mice were detected by ELISA.The ratio of Th1 and Th2 in peripheral blood CD4~+Tcell was detected with flow cytometry,and the fluorescence intensity was recorded after PE and APC fluorescein marking with the corresponding factor IL-4 and INF-gamma.1.3.The influence of catechin to the expression of TSLP and its regulation factor in allergic rhinitis mice:The level of TSLP in lung tissue homogenate for AR mouse were assaied in ELISA kit.Immunohistochemical method was used to detect the expression of TSLP and NF-κBp65 protein in mouse nasal mucosa.The contents of IκB,NF-κBp65 protein in the AR mouse lung tissues were detected in western blot.Meanwhile,we also used tissue immunofluorescence to assay the transcription of NF-κBp65 in the mouse lung tissue.2.Effects of catechin on the expression of TSLP and its regulatory factors in human nasal mucosal cell lines(HNEp Cs)Firstly,our study investigated the cytotoxic of catechin to HNEp Cs by MTT method,in order to find the non-toxic dose.For establishing a TSLP release model in vitro,we used TLR3 agonist poly(I:C)in 20μg/m L does to stimulates HNEp Cs,and then treated the cells with catechin.The level of TSLP in HNEp Cs cellular supernatant was assaied in ELISA,and the expression of TSLP,Ikappa B,p-NF-kappa Bp65 and NF-kappa Bp65 in HNEp Cs after poly(I:C)stimulation were detect by westron blot.Results:1.The pharmacodynamics study of catechin in allergic rhinitis mice.1.1 The therapeutic effect of catechin on allergic rhinitis mice.After the based sensitization with OVA/aluminium adjuvants and OVA stimulating,the mice showed a series of allergic symptom like the continuous nasal rubbing and the repeated nasal sneezing.At the same time,the level of OVA-specific Ig E in serum had corresponding risen.After the treatment of catechin,the allergic symptoms and the level of OVA specific Ig E of allergic rhinitis mice were significantly alleviated.The results of leukocyte classification showed that the proportion of white blood cells,including lymphocytes,monocytes and granulocytes showed a rising trend in AR mouse peripheral blood,which shows a state of inflammation,and catechin could inhibits leukocyte increasing especially in high dose.In addition,the results showed that the nasal mucosa in the AR group apparented a tissue thickening,and there is a large number of lymphocytes clustered around in the secretory cavities of the mucosa.In lung tissue,lymphocytes were mainly concentrated around in the trachea.At the same time,we found that catechin can also relieve the infiltration of local inflammatory cells in allergic rhinitis mice and inhibit the inflammatory response of diseased region.1.2.Effects of catechin on Th2 inflammation in allergic rhinitis mice:The level of Th2cytokines in serum and lung tissue were detected by ELISA.The resoults show that the Th2factor like IL-5 and IL-13 in serum or lung tissue homogenate had risen and catechin could reduce the level of Th2 factor obviously with a dose dependent.The results of flow experiment showed that Th1 and Th2 imbalance occurred in AR mice,while catechin could significantly restore the immune balance between Th1 and Th2.1.3.The influence of catechin to the expression of TSLP and its regulation factor in allergic rhinitis mice:Immunohistochemical detection showed that the expression of the NF-κBp65 and TSLP in allergic rhinitis mouse nasal mucosa change synchronously.Catechin not only can inhibit the expression of them obviously,but also can reduce the level of TSLP in lung tissue homogenate.Catechin also could affect the upstream regulatory factors of NF-κB/TSLP inflammatory path.The result of westeron blot indicated that catechin could inhibit the activation of NF-kappa B pathway in the lung tissues of allergic rhinitis mice by relieving the degradation of the IκB protein in the cytoplasm,restoring the content of NF-κBp65 in the cytoplasm and reducing the protein content of NF-κBp65 in the nuclear.In addition,tissue immunofluorescence also further demonstrated the effect of catechin on the activation of NF-κBp65 protein.The results showed that catechin inhibited the nuclear transcription of the NF-κBp65 in mouse lung tissue.2.Effects of catechin on the expression of TSLP and its regulatory factors in human nasal mucosal cell lines(HNEp Cs).Our studies found that the level TSLP in culture supernatant of poly(I:C)group,was higher than the control group,after poly(I:C)stimulated HNEp Cs for 24h,and catechin could reduce the secretion of TSLP,especially in high dose.To this end,we detected the content of TSLP protein in HNEp Cs cytoplasm protein by the westron blot method,and the results proved that poly(I:C)could induce HNEp Cs to express TSLP protein,and catechin also showed the inhibiting effect of TSLP expression.The result of westeron blot indicated that catechin could reduce p-NF-κBp65 and activation,NF-κBp65 levels in the nucleus,and the degradation of the IκB protein.The immunofluorescence results showed that catechins inhibited the nuclear transcription of the NF-κBp65 induced by poly(I:C)stimulation,which further demonstrated the effect of catechins on the activation of NF-κBp65 protein.ConlusionCatechin could relieve allergic symptoms and inflammatory reactions in allergic rhinitis mice.The underlying mechanism was that catechin inhibited the expression of TSLP in epithelial cells by influencing NF-κB/TSLP path.