Study Of The Effects And Mechanisms Of Active Constituents From Forsythiae Fructus On Immediate Hypersensitivity Reactions

Immediate hypersensitivity reactions(IHRs)refer to the typical clinical manifestations of bronchospasm,angioedema,urticaria and hypotension,which occur within a short period of time after receiving external stimuli.It can be induced by both immunological[triggered by antigen(Ag)/antibody(Ab)reaction,most commonly refer to IgE-mediated type I allergy]and non-immunological(triggered by Compound 48/80,small molecule drugs,complement,etc.,often refer to pseudo-allergy)-mediated mast cells(MCs)degranulation.And there has been an emerging trend to search for the promising anti-IHRs drugs from traditional Chinese medicine(TCM),since TCM has been therapeutic effective in treating IHRs.Forsythiae Fructus,the dried fruit of Forsythia suspensa(Thunb.)Vahl(Oleaceae),is a well-known herb(known as Lianqiao)that have heat-clearing and detoxifying effects on infectious diseases like epidemic pyrexia,carbuncle,scrofula and erysipelas.Although Forsythiae Fructus has also been widely used in treating IHRs-related non-infectious diseases,its main effective parts and the underlying mechanisms remain unclear.Based on our previous results,we focused on the anti-IHRs effects of forsythiasides,a class of components from Forsythiae Fructus.Firstly,we used shrimp protein(SP)and its antisera as well as Compound 48/80 as the stimulants of the immunological(IgE)and non-immunological IHRs experiment models,respectively.And we prepared the effective fractions of forsythiasides by macroporous resin based on the activity of dampening MCs degranulation,in which the contents of forsythiaside A and isoforsythiaside A were 27.47%,9.46%,respectively.The sum of these two components reached 36.9%,thus the obtained purified extracts can be called as Forsythiasides-rich extract(FRE).We evaluated the effect of FRE on MCs degranulation,and the results showed that FRE could dampen both Ag/IgE(immune)mediated and Compound 48/80(non-immune)-mediated MCs degranulation in vitro and in vivo.Even though Ag/IgE-mediated and Compound 48/80-mediated pathways are different,they both share a similar mechanism in elevating the cytosolic Ca2k(Ca2+[c])level.Therefore,we next examined the effect of FRE on Ag/IgE and Compound 48/80-induced elevation of Ca2+[c]level,respectively.The results showed that FRE could significantly reduce the Ca2+[c]of MCs.Moreover,FRE could directly reduce the Ca2+[c]of resting cells in a rapid and reversible manner.By screening the four typical routes of Ca2+[C]reduction in MCs one by one,it turned out that the effect of FRE-on Ca2+[c]reduction was independent of plasma membrane Ca2+-ATPase(PMCA),sarcoplasmic endoplasmic reticulum Ca2+-ATPase(SERCA)or Na+/Ca2+ exchanger(NCX),but via enhancing the mitochondrial Ca2+(Ca2+[m])uptake.Accordingly,FRE also significantly promoted the Ca2+[m]uptake in isolated mouse liver mitochondria,indicating that the effect of FRE on Ca2+[m]uptake was direct,rather than depending on membrane signal transduction.In Ag/IgE-mediated IHRs,MCs degranulation can be regarded as the late event,while Th2 response can be seen as an early event.Thus,can FRE also decrease the production of specific IgE(sIgE)by inhibiting the Th2 response?To answer this question,we established a murine model of Th2 response using SP and investigated the effect of FRE.The results showed that FRE could indeed significantly reduce the serum total IgE(tIgE),slgE,and mouse mast cell protease 1(MCPT-1),a sensitive biomarker of MCs degranulation.We further investigated the effect of FRE on the productions of Th2 cytokines.The results showed that FRE could inhibit the release of Th2 cytokines in mouse mesenteric lymph nodes and splenocytes directly,indicating that FRE could suppress the Th2 response.Given the migration of circulating basophils to lymph nodes and the release of early IL-4 are critical for initiating Th2 response,we next focused on the role of FRE on basophils migration.The results showed that the number of blood basophils of mice was significantly decreased after challenging with SP for four days,and administration of FRE could attenuate the decline of circulating basophils.Furthermore,we confirmed its effect in another murine model of basophil migration,and the obtained results suggested that the suppression of FRE on Th2 response was associated with preventing the migration of circulating basophils to lymph nodes.Taken together,this study revealed the material basis from Forsythiae Fructus and the underlying mechanism of its anti-IHRs effect.And in particular,we have elucidated the molecular mechanism of how FRE dampened MCs degranulation,which was to reduce the Ca2+[c]via directly increasing Ca2+[m]uptake.While,its effect on reducing IgE production was associated with inhibiting Th2 response.These results showed that FRE exerted a comprehensive effect on the development of IHRs,and provided a solid scientific experimental basis for the clinical application of Forsythiae Fructus as well as the discovery of new anti-IHRs drug.