The Effect Of Galangin On Airway Remodeling In Asthma And Its Mechanism

Part One The effect of galangin on airway remodeling in OVA-induced chronic asthma miceObjective: To elucidate the effect of galangin on airway remodeling in ovalbumin(OVA)-induced chronic asthma mice and illuminate the underlying mechanisms.Methods: 48 female BALB/c mice were randomly divided into the following 6 groups: control,OVA,OVA+GL(Galangin 0.1 mg/kg),OVA+GH(Galangin 0.5 mg/kg),OVA+DEX(1 mg/kg),and OVA+DMSO(vehicle).The mice were sensitised on days 0,7 and 14 by intraperitoneal injection of OVA emulsified in aluminum hydroxide gel.These sensitised mice were exposed to aerosolised 5% OVA in sterile saline for 8 weeks beginning on day 16 for 30 min three times a week.The mice were sacrificed 24 h after the last challenge.Bronchoalveolar lavage fluid(BALF),serum,and lung tissue were collected for analysis.The total number of inflammatory cells in the BALF was determined by counting and differential cell counts were performed with Wright's staining.The levels of TGF-?1 in BALF and OVA-specific Ig E in serum were measured using commercial enzyme-linked immunosorbent assay(ELISA)kits.Lung sections were stained with haematoxylin and eosin staining(H&E)to assess inflammatory cell infiltration,Periodic acid-Schiff(PAS)to quantify airway global cells and mucus production,Masson's trichrome to visualise collagen deposition and fibrosis,and immunohistochemical staining to examine smooth muscle actin alpha chain(?-SMA)and matrix metallopeptidase-9(MMP-9)distribution.The expression of ?-SMA,MMP-9 and vascular endothelial growth factor(VEGF)in lungs was evaluated by western blot.Results: Our results showed that the OVA-induced mice developed severe airway inflammatory responses,including extensive inflammatory cells trafficking into BALF and infiltrating around respiratory tract and vessels.Meanwhile,significantly airway remodeling such as goblet cell hyperplasia,collagen deposition/fibrosis and increased ?-SMA expression were also observed by morphology analysis(P<0.05).Treatment with GH and DEX significantly inhibited total cell counts and eosinophil counts and obviously suppressed the infiltration of inflammatory cells(P<0.05).Galangin also markedly attenuated the extend of airway remodeling,reduced the mucus hyper-secretion,fibrosis and ?-SMA expression,compared with the vehicle group(P<0.05).Additionally,GH and DEX treatment could reduced TGF-?1 levels in BALF(P<0.05)and suppressed the protein expression of VEGF and MMP-9 in lung tissue(P<0.05),implying that the anti-remodeling activity of galangin might be associated to these factors which were known as key modulators involved in airway remodeling.Conclusions: Taken together,these data suggested that galangin could attenuate OVA-induced chronic airway inflammation as well as airway remodeling probably by inhibiting TGF-?1 production and decreasing the synthesis of VEGF and MMP-9,providing a potential and promising therapeutic option for asthma patients.Part Two The effect of galangin on TGF-?1-induced proliferation of human airway smooth muscle cellsObjective: To investigate whether galangin can reduce TGF-?1-induced proliferation of human airway smooth muscle cells(ASMCs)and its mechanism involved.Methods: Human primary ASMCs were cultured in vitro.The effect of TGF-?1 on ASMCs and the cytotoxicity of Galangin on ASMCs were measured by cell counting kit-8(CCK-8)assay.The effect of galangin on TGF-?1 induced ASMC proliferation was determined using CCK-8 assay and the EDU assay.Intracellular reactive oxygen species(ROS)were measured using the DCFH-DA assay and using a confocal laser scanning microscope.Western blot was used to detect the expression of NADPH oxidase 4(Nox4),catalase and superoxide dismutase(SOD).The expression of p-JNK,p-ERK,p-Akt were also evaluated by western blot.Results: The proliferation of human ASMCs was promoted by 1 ng/m L TGF-?1 stimulation(P<0.05).The cell viabilities were 90% and 86% in 10 ?M galangin at 24 h and 72 h.Treatment with 10 ?M galangin decreased TGF-?1-induced ASMC proliferation from 124% ± 8% to 101% ± 2%(P<0.05);no significant effect was observed by 0.1 or 1 ?M galangin.Furthermore,the ROS level in ASMCs was also promoted by 1 ng/m L TGF-?1(P<0.05).Flow cytometry analysis showed that pretreatment with 10?M galangin,1 m M N-acetyl cysteine(NAC)or 10 m M NAC decreased the intracellular levels of ROS to 79%,82% or 62%,respectively,compared to the vehicle control(P<0.05).TGF-?1 up-regulated the expression of Nox4 while down-regulating the expression of SOD and catalase(P<0.05).We found that TGF-?1 could activate ERK,JNK and Akt in ASMCs within 1 h after stimulation(P<0.05).This activation was partially blunted in the group pretreated with galangin(10?M)compared to the vehicle control(P<0.05).Conclusions: These results may highlight that a novel role for galangin as a promising anti-proliferation agent in human ASMCs,which likely involves the TGF-?1-ROS-MAPK pathway.