| Background:Allergic diseases have been global public health issues for years,among which,allergic rhinitis(AR)has been concerned for patients suffering from AR are increasing.What's more,AR might increase the risk of asthma.Traditional Chinese medicine makes well-acceptance by the general population due to its meris of attenuating symptoms such as sneezing and rubbing,and protecting people from relapse.SXBQS,an empirical formula of Professor Wangqing guo,has satisfactory clinical effects on AR patients.Studying effect and mechanism of SXBQS on AR animal models can provide guidance for clinical treatment and lay foundation for drug development.Aim:To explore the core TCM patterns of "biqiu",known as "allergic rhinitis",and to study the effect and mechanism of SXBQS,as well as its main effective components,on allergic rhinitis.1.To collect patients suffering from "biqiu",analyze and refine main differentiation patterns.2.To prepare AR model of BN rats and Balb/C mice.3.To study Time-effect and Dose-effect relationship of SXBQS,as well as its effect and mechanism.4.To study pharmacokinetics of baicalin in SXBQS,5.To study the anti-allergic effect of baicalin.Methods:1.The TCM patterns'research of "biqiu" is a retrospective analysis.244 cases of Professor Wang's patients from Jan,2015 to Dec,2016 were collected and their information was sorted and inputted into the computer.TCM patterns were analyzed and refined according to the criterion of syndrome differentiation.2.Both BN rats and Balb/C mice were immunized with a mixture of ovalbumin(OVA)and alum into their peritoneal cavity and OVA into their nasal cavity to trigger an allergic reaction.Alterations of the nasal(number of nasal rubbing and sneezing),serum IgE and histopathological parameter were performed.3.After administrated orally,BN rats received intranasal OVA challenge at 1h,4h,8h,12h,respectively.Nasal sign assessment was performed to study Time-effect and Dose-effect relationship of SXBQS.The anti-allergic properties of SXBQS was evaluated by measuring biochemical makers(IgE,histamine,IFNy,IL-4,IL-5,IL-17)in serum and nasal perfusate,morphological and histopathological parameters.The expression of IL-4,IFNy protein in nasal mucosa were determined by Western blot.4.Tail blood of BN rats were collected at 30 min,1 h,2 h,3 h,4 h,6 h,8 h,10 h,12 h,respectively,immediately after SXBQS administration.Pharmacokinetics of BAL in SXBQS was studied based on Enzyme-Linked immunoassay method for BAL.Correlation of pharmacokinetics and pharmacodynamics of BAL was studied by curve fitting of BAL concentration and sneezing number.5.Alteration of the nasal,biochemical markers and histopathological parameter were performed to assess the anti-allergic effect of BAL.LDF was used to study nasal mucosal microcirculation of mice.Thl,Th2,Thl7,Treg cells were estimated by flow cytometry.The nasal vascular permeability of OVA-sensitized BN rats was evaluated by analyzing a brilliant blue concentration in nasal perfusate after exposure to OVA.Results:1.TCM patterns:Biqiu has a female dominance of about 1.6:1 and mainly afflicts people in midlife.Almost none suffered from single pattern.The majority were identified as dual deficiency of lung and kidney,along with three other patterns,namely wind,spleen deficiency and stagnated heat.2.For both BN rats and Balb/C mice,the AR groups showed significant phenotypic changes including increased sneezing and rubbing.As for BN rats,intranasal challenge with OVA and histamine except saline,both resulted in statistically significant increase of nasal rubbing and sneezing.The AR groups demonstrated elevated serum IgE and local mast cell infiltration than normal groups.3.As for OVA challenge after SXBQS administration for 1h,treatment with CTRZ and SXBQS(high and mid dose)showed significant attenuation in OVA-induced alterations of the nasal.As for OVA challenge after SXBQS administration for 4h,treatment with CTRZ and SXBQS(high dose)showed prevention of nasal symptoms.As for 8h,treatment with CTRZ and SXBQS(high dose)decreased nasal rubbing and sneezing.As for 12h,only SXBQS(high dose)attenuated frequency of scratching and sneezing.4.The OVA-induced sensitized rats showed significant body weight loss as compared with normal group.Treatment of SXBSQ and CTRZ did not have any effect on body weight as compared with AR control group.CTRZ and SXBQS treatment reduced infiltration of inflammatory cells into nasal cavity.As compared with AR control group,CTRZ and SXBQS pretreated rats showed statistically decrease in values of eosinophils in peripheral blood.CTRZ and SXBQS(high and mid)treatment show significant decrease in levels of serum IgE as compared with AR control mice.Treatment with CTRZ and SXBQS at all concentrations tested led to a decrease in the serum level of histamine.IL-4,IL-5,IL-17 levels were lower in SXBQS treated group than those from the OVA-induced group,while IFNy secretion was up regulated by SXBQS.CTRZ and SXBQS(high)treatment decreased concentration of total protein in nasal secretion.IgE,IL-4 levels in nasal perfusate were downregulated by SXBQS.There were no differences of histamine levels in nasal perfusate among the six groups.5.ELISA method for detecting BAL in rats blood was established with a linear range of 31.25 ngˇmL-1 to 1000 ngˇmL-1.The recovery rate is within 93.58 and 107.18%,with an average of 99.42%.RSDs were lower than 10%,meeting the requirements for biological samples.The first peak of BAL in SXBQS was observed at 2-3 h.The AUCtot of BAL after oral administration with high,middle,and low doses of SXBQS showed a non-linear relationship,which suggested that BAL may be the main active component of SXBQS attenuating nasal allergic symptoms.6.CTRZ and BAL(high and middle dose)pretreatment caused statistically prevention of OVA-induced elevated nasal rubbing and sneezing as compared with AR control mice.CTRZ and BAL pretreatment reduced infiltration of inflammatory cells into nasal cavity.CTRZ and BAL pretreatment show significant decrease in levels of serum IgE and histamine as compared with AR control mice.Thl cells percentage was decreased in AR control group,while BAL pretreatment,Thl cells was up regulated.The same tendency was observed for Treg cells percentage similarly.Both Th4 and Th17 cells percentage was increased in AR group as compared with normal group,while BAL suppressed their proliferation.LDF was used to study nasal mucosal microcirculation of mice.Provocation with OVA showed statistically significant decrease in blood flow,while CTRZ and BAL pretreatment inhibited this effect.Topical administration of OVA to the nasal cavity of anesthetized rats resulted in increased nasal vascular permeability.The increased nasal vascular permeability reached a maximum at 30 min after dye administration.Nasal vascular permeability in the BAL group was significantly lower than the AR control group.Conclusion:1.Description of TCM patterns.Core pattern of "biqiu" was refined as dual deficiency of lung and kidney,based on which,three main patterns were identified,namely wind,spleen deficiency and stagnated heat.2.SXBQS makedly alleviated nasal signs after administrated orally for 1h,and this effect last at least for 12 hours.SXBQS exerts dose-depended anti-allergic properties by suppressing serum IgE and histamine levels,and by decreasing infiltration of inflammatory cells in the nasal mucosa.SXBQS upregulated expression of IFN ?,and downregulated expression of IL-4?IL-5?IL-17,which suggested it showed prophylactic potential against OVA-induced alterations in BN rats probably through modulating the T cells levels.3.Negative correlation between BAL and sneezing number indicated BAL may be one of the main active anti-allergic components of SXBQS.4.BAL had the potential to protect from an allergic reaction,especially from OVA,by regulation of serum IgE and histamine,and inflammatory changes.Pretreatment with BAL upregulated Thl cells,Treg cells,and downregulated Th2 cells,Th17 cells,indicating BAL may suppress the progression of AR by inhibiting T cell immune response.The anti-histamine effect was further evaluated by nasal mucosal microcirculation and vascular permeability experiments. |
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