| Objective:1.Observe theYupIFNeng granule on the treatment of allergic rhinitis nasal mucosa of rats effect,through the detection of peripheral blood lymphocyte subsets(CD3+CD4+CD8+T cells and CD4+CD25+Foxp3+cells),the proportion of the nasal mucosa of rats in protein involved in the NF-κB pathway(p65,p-p65,I kappa B predominate,p-I kappa B predominate)levels,and nasal mucosa of rats in cytokine interleukin 4(IL-4),interleukin 17(IL-17 alpha)and interferon gamma(IFN-y)protein levels preliminary clarify jade screen particles mechanism for the treatment of allergic rhinitis.2.Observe the poly(I:C)real and histamine induced allergic HaCaT cell model,through the observation of cell proliferation and cell interleukin 1 beta,interleukin 6(IL-6),interleukin8(IL-8),tumor necrosis factor alpha(TNF-α),mitochondrial pyruvate transport carrier 1(MPC-1)gene expression level of jade screen particles improve allergic disease treatment effect of cell damage and reduce the release of IFNlammatory mediators.Methods:1.60 SD rats were randomly divided into the normal group(n=10)and the modeling group(n=50).Only the other groups were provided with an OVA model for allergic rhinitis.Starting from day 15,nasal drops containing 10%OVA(50μl on each side)were administered for 7 consecutive days.Normal rats were given normal saline instead of OVA suspension and OVA saline solution.Observe behavior score 30 min after intranasal rats,made after the success of the module module according to the score is divided into six groups at random,each group of 10,model group,loratadine group respectively(1.8 mg/kg),better stop particles group(5.4 g/kg),jade screen particles high-dose group(5.4 g/kg),jade screen particles dose(2.7 g/kg),jade screen particles low dose group(1.35 g/kg).Each dose group and the positive control group were gavaged at 10ml/kg once a day for 4 weeks.The normal group and the model group were given equal volume distilled water.During administration,the model group and the administration group were given 10%OVA of normal saline solution with a nasal drip of 50 μl on each side every other day,while the normal group was given normal saline for parallel operation.After 4 weeks of administration,fasting and dehydration were observed for 12 hours before the last administration,followed by nasal drops of 10%OVA of normal saline solution for 1 hour after the last administration.The rats were anesthetized within 30 minutes after nasal drops.Blood was collected and severed,and peripheral blood and nasal mucosa of the rats were collected for subsequent flow cytometry and western blog experiments.2.HaCaT was cultured in vitro with high glucose DMEM medium containing 10%fetal bovine serum/in a cell incubator at 37℃ and 5%CO2.When the cell growth exceeded 70%-90%,the cells were digested with 0.25%trypsin until floating.After digestion,the cells were added into the medium and centrifuged.After the supernatant was discarded,the cells were re-suspended in the medium.(1)the effect of yupingfeng granules on HaCaT cell proliferation was detected by MTT method.According to the result of cell count by 1×104/hole cell number 96-well plates,cultivating after 24 hours,jade screen particles according to the concentration of 25,50,100,12.5,6.25,3.125,1.5625,0.78125,0μg/ml,respectively,to join to the hole,foster after 24 h,abandoned to the upper medium,join determined by MTT solution,cultivating 6 h,each hole after joining DMSO to dissolving the crystallization,enzyme league immune detector for detecting under 570 nm absorbance in each group.According to poly(I:C)(20 μg/ml)and Histamine(40 μmol/ml)to join again after induction respectively according to the concentration of 25,50,100,12.5,6.25,3.125,1.5625,0.78125,0 μg/ml to join the jade screen particles,foster after 24 h,abandoned to the upper medium,join determined by MTT solution,cultivating 6 h,each hole after joining DMSO to dissolving the crystallization,enzyme league immune detector for detecting under 570 nm absorbance in each group.(2)the effect of yupingfeng granules on the mRNA level of cytokines in HaCaT cell allergy model was detected by RT-qPCR.According to the result of cell count by 1×105/spread 6 orifice hole cell number,divided into normal group,model group,the jade screen particles high-dose group(100 μg/ml),jade screen particles dose group of(20 μg/ml),jade screen particles low dose group(4 μg/ml),poly(I:C)real except the normal group each group to join 40 μg poly(I:C)real building,Histamine in addition to the normal group each group to join 80 μmol,Histamine mode,cultivate 24 h,to each according to the concentration in the jade screen particles,each group of three holes in parallel,The mRNA levels of il-1 IL,il-6,il-8,TNF-group and MPC-1 were detected by RT-qPCR.Results:(1)Effect of Yupingfeng Granule on the animal behavior of rats with allergic rhinitis.Yupingfeng granule can effectively restrain the allergic symptoms of rats with allergic rhinitis.After 4 weeks of treatment by Yupingfeng granule,through the observation of animal behavior,we found that the rats in each group had normal morphology,neat and smooth hair,normal activities and diet before modeling.After the establishment of the model,the rats were scratched,fidgety and restless,more likely to gather,their hair was disordered and fell off,the frequency of runny nose and sneezing became higher,and even had dyspnea and mental depression.After administration,the frequency and intensity of sneezing and the behavioral score of rats in each group decreased.Compared with the normal control group,the behavioral score of rats in the model control group was significantly different(P<0.05);compared with the model control group,the behavioral score model of rats in each group was significantly lower(P<0.05)after one week of administration and four weeks of administration.(2)Effect of Yupingfeng Granule on lymphocyte subsets in peripheral blood of rats with allergic rhinitis.1.The effect of Yupingfeng Granule on the subsets of CD3+CD4+CD8+cells in the peripheral blood of rats with allergic rhinitis.The results of flow cytometry showed that the ratio of CD3+,CD3+CD4+,CD3+CD8+cell subsets in the peripheral blood of the model control group increased significantly(P<0.05)compared with the normal control group after 4 weeks of administration;compared with the model control group,the ratio of CD3+cells in the high dose group of Yupingfeng granules decreased significantly(P<0.05),and the ratio of CD3+CD4+cells in the high,medium and low dose groups of loratadine and Yupingfeng granules increased significantly(P<0.05)The proportion of CD3+CD8+cells in the high dose group of Yupingfeng granules decreased significantly(P<0.05).2.Effect of Yupingfeng Granule on CD4+CD25+Foxp3+cell subsets in peripheral blood of rats with allergic rhinitis.The results of flow cytometry showed that compared with the normal control group,the CD25+Foxp3+cell subsets in the peripheral blood of the model control group increased significantly(P<0.05);compared with the model control group,the proportion of CD25+Foxp3+cells in the Xuhanting granule group decreased significantly(P<0.05),and the proportion of CD4+Foxp3+and CD25+Foxp3+cells in the high dose Yupingfeng granule group decreased significantly(P<0.05).(3)Effect of Yupingfeng Granule on NF-κB pathway related proteins and cytokines in nasal mucosa of rats with allergic rhinitis.1.Yupingfeng regulates the expression of NF-κB signal protein in the nasal mucosa of allergic rhinitis rats.The results of Western bolt showed that The expression of p-p65,p-IκB was significantly increased(P<0.05).Compared with the model control group,the content of p65 in Yupingfeng high,medium and low dose group was significantly decreased(P<0.05).The content of IκB a in Yupingfeng high,medium and low dose group was significantly decreased(P<0.05).The content of IκBα in Yupingfeng high,medium and low dose group was significantly increased(P<0.05)The content of p-IκB in the dose group decreased significantly(P<0.05).2.Yupingfeng regulates the expression of cytokines in the nasal mucosa of rats with allergic rhinitis.Western The results showed that there was no significant difference in the expression of IL-4 in the nasal mucosa between the model control group and the normal control group(P>0.05),the expression of IL-17α decreased significantly(P<0.05),the expression of IFN-γ increased significantly(P<0.05),and the level of IL-4 protein in the high,middle and low dose groups of loratadine and Yupingfeng granules decreased significantly(P<0.05)compared with the model control group The content of IL-17 α in loratadine group,Xuhanting granule group,Yupingfeng granule high and middle dose group increased significantly(P<0.05),while the content of IFN-y in loratadine group,Xuhanting granule group,Yupingfeng granule high,middle and low dose group decreased significantly(P<0.05).(4)Effect of Yupingfeng Granule on cell proliferation and cytokines in HaCaT cell allergic model.1.The effect of Yupingfeng Granule on the proliferation of HaCaT cell allergic model.MTT assay showed that Yupingfeng granules promoted the proliferation of normal HaCaT cells(P<0.05)in 100,50,25,12.5,6.25,3.125mg/ml,inhibited the proliferation of HaCaT cells(P<0.05)in poly(I:C)induced HaCaT cells(P<0.05),and Yupingfeng granules in 100,50,25,12.5,6.25,1.5625,0.78125mg/ml Under the concentration of 100,50,25 mg/ml,Yupingfeng granule had a protective effect on HaCaT cells(P<0.05).2.The effect of Yupingfeng Granule on the expression of cytokines in HaCaT cell allergic model.(1)The effect of Yupingfeng Granule on the level of IFNlammatory factor mRNA in poly(I:C)induced HaCaT cell model by RT-qPCR.RT-qPCR results showed that IL-1 β,IL-8,TNF-α and MPC-1 in HaCaT cells after poly(I:C)induction were significantly higher than those in the control group(P<0.05).Compared with the model group,the expression of IL-1β,IL-6,IL-8,TNF-α,MPC-1 mRNA in the high dose group,the middle dose group and the low dose group of Yupingfeng decreased significantly(P<0.05).(2)Detection of the effect of Yupingfeng Granule on the level of IFNlammatory factor mRNA in histamine induced HaCaT cell allergic model by RT-qPCR.The results of RT-qPCR showed that IL-1 β,IL-6,IL-8,TNF-α and MPC-1 in HaCaT cells induced by histamine were significantly higher than those in the control group(P<0.05).After administration,the expression of IL-1 β,IL-6,IL-8,TNF-α,MPC-1 mRNA in Yupingfeng high dose group,Yupingfeng middle dose group,Yupingfeng low dose group were significantly decreased(P<0.05).Conclusion:The antiallergic effect of Yupingfeng granules is achieved by regulating lymphocyte differentiation,inhibiting NF-κB pathway activation and reducing the expression and secretion of cytokines in nasal mucosa.1.Yupingfeng granule can obviously improve the allergic symptoms of allergic rhinitis animals.2.Yupingfeng granule can reduce the subsets of CD3+CD4+,CD3+CD8+,CD4+Foxp3+,CD25+Foxp3+cells in the peripheral blood of rats with allergic rhinitis and inhibit the immune response.3.Yupingfeng granule inhibited the synthesis and activation of p65,p-p65,IκB-α,p-IκB pathway and the expression of downstream protein in the nasal mucosa of allergic rhinitis rats.4.Yupingfeng granule can inhibit the secretion of IL-4,IL-17 and IFN-γ in the nasal mucosa of rats with allergic rhinitis and alleviate the allergic symptoms of the nasal mucosa.5.Yupingfeng granule can down-regulate the expression of IL-1β,IL-6,IL-8,TNF-α and MPC-1 in HaCaT allergic model.In conclusion,Yupingfeng granule can through regulating the NF-κB signaling pathways,reduce exudation of IFNlammatory factor and suppress the nasal mucosa of allergic reactions,regulate differentiation of T-lymphocyte subgroup in mice,play an important role in prevention and treatment to allergic rhinitis and its development.Then instructions of HaCaT allergic cell model illustrate that Yupingfeng granule can effectively reduce allergic condition of cell death,and inhibit the expression of IFNlammatory cytokines,its signal transduction mechanisms and intervention effect remains to be further research. |
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