The Effects Of Xiao-Qing-Long-Tang On Allergic Rhinitis Mice And Its Mechanism

Objective:Xiao-Qing-Long-Tang(XQLT)is the classical prescription for treating respiratory diseases such as allergic rhinitis,which is widely used clinically,but its mechanism is not clear.The research validates the effects of XQLT on allergic rhinitis by the mouse model of allergic rhinitis,and the anti-allergic effects and anti-inflammatory effects were investigated to reveal the mechanism on the treatment of allergic rhinitis.Methods:1.The fingerprint of XQLT:The fingerprint of XQLT was detected by HPLC,the fingerprint of XQLT was built by the optimization of chromatographic conditions,and the reliability of the methodology were determined.The similarity of 8 batches XQLT was tested to determine the stability of XQLT;2.The role of XQLT on allergic rhinitis:Balb/c female mice(18-22g)were randomly divided into normal,model,dexamethasone and XQLT low,medium and high dose group.Allergic rhinitis mice model was built by intraperitoneal inject 300?L PBS containing 50pg OVA,1mg aluminum hydroxide adjuvants on day 0,7,14 to sensitize.then challenged by 500?g/side OVA through nasal drip for 7 days.Control group was given the same amount of PBS.XQLT(low,medium,high dosages were 30,60,120g/kg)and dexamethasone(5mg/kg)were given on the 14th day,and lasting for 14 days.(1)The number of scrathing and sneezing was observed for 10 minutes after challenge.(2)The leukocytes of peripheral blood was detected;(3)The thymus and spleen were taken for testing the immune functions;(4)The nasal mucousa and lung were taken for the pathological test;(5)The inflammation factor were tested by nasal mucosa and lung homogenate;(6)The expression of TLR4 on nasal mucosa were investigated by immunohistochemistry technology.3.The anti-inflammatory effects of XQLT:The inflammation model was built in RAW264.7 macrophage induced by LPS.XQLT were incubated with RAW264.7 cell for 12 hour,then incubated with 1?g/mL LPS for 24 hours,the NO content were tested by Griess reagent kit,and cytokines such as IL-6,TNF-?,were tested by CBA inflammatory reagent kit.The influence of XQLT for the activation of macrophage were tested by the expression of CD86,MHCII molecule,and the number of fluorescent microspheres were observed to investigate the phagocytosis function of macrophage.The expression of iNOS,TLR4,NF-?Bp65 were determined by western blot,which determined the effects of XQLT on TLR4/NF-?B inflammation signaling pathway.4.The antagonism on histame H1 receptor of XQLT:The concentration-response curves of histamine were performed by the contraction of guinea pig ileum induced by histamine in vitro,and XQLT,diphenhydramine were given to test the influence of the contraction of ileum induced by histamine.Then determine the influence of XQLT for histamine H1 receptor.5.The material basis of XQLT:XQLT serum were tested by HPLC-MS,and compared with standard to evaluate the effective constituents,which is the material base of XQLT.The antagonism of effective components on histamine was tested by intestinal contraction induced by histamine in vitro,and the anti-inflammatory effects of active ingredients was determined on RAW264.7 cells induced by LPS.Results:1.The fingerprint of XQLT was completed,and the similarity of various batches of XQLT was above 0.96,which means the quality of XQLT is reliable and controllable.2.After OVA challenged,the number of snezzing and scratching of mice were significantly higher than normal group,and the inflammatory cells infiltrate in the nasal mucousa and lung.When treated with XQLT and dexamethasone,the number of snezzing and scratching reduced significantly compared with model group.And the thymus and spleen index reduced also.And the level of white blood cell in peripheral blood was reduced significantly.The pathological results show that XQLT and dexamethasone can reduce the inflammatory cells infiltrated in the nasal mucous membrane and lung.The change of cytokines in the nasal mucosa homogenate is not obviously.But the content of MCP-1 of model group lung homogenate rised.XQLT and dexamethasone reduced the content of MCP-1.Immunohistochemistry detected the expression of TLR4 in the nasal mucosa,the expression of TLR4 in model group is higher than normal group,and the expression of TLR4 reduced when treated with XQLT and dexamethasone.3.Guinea pig ileum contraction in vitro was induced by histamine,and the pressure value rised.Diphenhydramine shifted the concentration-response curve of histamine to the right parallelly,XQLT shifted the concentration-response curve to the bottom right,antagonisted histamine H1 receptor noncompetitively,which is one of the mechanism of anti-allergy.4.XQLT inhibited the release of NO,IL-6,IL-10,TNF-? in RAW264.7 cells induced by LPS,reduced the expression of CD86,MHCII,and TLR4,reduced the phagocytosis function of macrophage.XQLT reduced the expression of iNOS,and NF-?Bp65 in nuclear,which means XQLT inhibit inflammatory by TLR4/NF-?B signaling pathway.5.The component of XQLT in blood were:ephedrine,cinnamic acid,catechin hydrate,schisandrol A,6-gingerol,Liquiritin,glycyrrhizic acid,Paeoniflorin.among them,catechin hydrate and ephedrine shifted the concentration-response curve to the bottom right,antagonisted histamine H1 receptor noncompetitively;catechin hydrate and schisandrol A inhibited the release of NO,IL-6 in RAW264.7 cells induced by LPS,reduced the expression of CD86,MHCII,reduced the phagocytosis function of macrophage.And reduced the expression of iNOS in cytoplasm,and TLR4 on cytomembrane,suppressed NF-?Bp65 expression in nuclear.Conclusion:XQLT can treat allergic rhinitis mice by anti-allergy and anti-inflammatory.The effective components:catechin hydrate and glycyrrhizic acid can oppose the function of histamine;and catechin hydrate and schisandrol A can suppress the inflammatory,which are the mechanism of the role of XQLT on allergic rhinitis.