Regulation Of Th17,Th1 Polarization,and Neutrophil Apoptosis By Apigenin In Neutrophilic Asthma

ObjectiveTo investigate the inhibitory effects of apigenin on neutrophilic asthma in vivo,and dissect its possible mechanisms.MethodsFemale BABL/c mice were randomly divided into 6 groups as follows:Control,OVA+LPS,OVA+LPS+Apigenin(A5,5 mg/kg apigenin;A10,10 mg/kg apigenin),and OVA+LPS+DMSO(dimethylsulfoxide).Mice in the neutrophilic asthma groups were intranasally sensitized with 75 μg OVA plus 10 μg LPS on days 0,1,2,and 7，and then intranasally challenged with 50 μg OVA on days 14,15,21,and 22.Apigenin and DMSO were intraperitoneally injected at 30 min before challenge.The Control subjects were sensitized and challenged with same volume of phosphate-buffered saline(PBS)alone.BALF and lung tissue were examined for inflammation.Airway hyperresponsiveness(AHR)to aerosolized acetylcholine chloride(Ach)was measured.The release of cytockines in the BALF,the differentiation of helper T type(Th)cells,airway neutrophilia and neutrophil(Neu)apoptosis in the lung were determined.Then,human neutrophil apoptosis in response to apigenin and some mediators was investigated by cellular morphology,flow cytometry,western blots and pharmacological caspase inhibitors Q-VD-OPh and z-VAD-fmk.Results(1)In vivo,apigenin attenuated neutrophilic allergic airway inflammation with decreased infiltration of inflammatory cells especially neutrophil in BALF(P<0.05).Moreover,apigenin significantly abolished AHR and the level of OVA-sIgE(P<0.05).(2)In neutrophilic asthma,apigenin administration decresed the secretion of Th17-and Thl-related cytockines such as interleukins(IL)-6,IL-17A and interferon-gamma(IFN)-γ in the BALF(P<0.05),reduced the differentiation of Th17 and Thl cells(P<0.05),attenuated neutrophilia in airway,and induced neutrophil apoptosis in the lung(P<0.05).(3)Apigenin induced time-,concentration-,and caspase(cysteine aspartic specific proteases)-dependent neutrophil apoptosis and overrided powerful anti-apoptosis signaling from survival factors such as LPS(Lipopolysaccharide),CpG-DNA(CpG containing DNA),and IFN-y(P<0.05).Furthermore,apigenin-induced apoptosis was related with the downregultion of anti-apoptosis protein Mcl-1(Myeloid cell leukemia 1)and the upregulation of pro-apoptosis protein Bax(B-cell lymphoma 2(Bcl-2)associated X)(P<0.05),and the induction of apoptosis was blocked by the pharmacological caspase inhibitors Q-VD-OPh and z-VAD-fink(P<0.05).ConclusionsApigenin regulates the polarization of Th17,Th1,and induces neutrophil apoptosis,suggesting its therapeutic potential for treating severe asthma in human.