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

Part One The effect of diosmetin on airway remodeling in OVA-induced chronic asthma miceObjective:Bronchial asthma,one of the most common allergic diseases,is characterized by airway hyperresponsiveness(AHR),inflammation and remodeling.The anti-oxidant flavone aglycone diosmetin ameliorates the inflammation in pancreatitis,but little is known about its impact on asthma.In this study,the effects of diosmetin on chronic asthma were investigated with an emphasis on the modulation of airway remodeling in BALB/c mice challenged with ovalbumin(OVA).Methods:BALB/c mice were sensitised on days 0,7 and 14 by intraperitoneal injection of OVA emulsified in aluminum hydroxide gel.Starting on Day 16,mice were challenged with phosphate-buffered saline or OVA by nebulization three times per week for 8 weeks.Diosmetin(0.05,0,1,0.5,or 1 mg/kg)were given intraperitoneally 30 min before each inhalation challenge.24 hours after the last challenge,AHR was assessed,and the cell composition of bronchoalveolar lavage fluid(BALF),serum,and lung tissue were collected for analysis.The levels of transforming growth factor-?1(TGF-pl)in BALF and OVA-specific IgE in serum were measured using commercial enzyme-linked immunosorbent assay(ELISA)kits.Hematoxylin-eosin(H&E),periodic acid Schiff(PAS),and Masson's trichrome to detect inflammatory cell infiltration,goblet cell hyperplasia,and collagen deposition around the airway,respectively.Immunohistochemistry was used to evaluate the expression of smooth muscle actin alpha chain(a-SMA)and matrix metallopeptidase(MMP)-9.The expression of a-SMA,MMP-9 and vascular endothelial growth factor(VEGF)in lungs was evaluated by western blot.Results:It was found that OVA induced increases in AHR,the total cells and eosinophils in BALE,and the level of OVA-specific immunoglobulin E in serum was attenuated by diosmetin administration(P<0.05).OVA-challenged mice displayed inflammatory cell infiltration,extensive mucus secrection and collagen deposition,diosmetin siginifantly attenuated inflammation infiltration,goblet cell hyperplasia and collagen deposition around the peribronchial and peri-vascular areas(P<0.05).Furthermore,diosmetin remarkably suppressed the expression of a-SMA,indicating a potent anti-proliferative effect of diosmetin on airway smooth muscle cells.MMP-9,TGF-?1,and VEGF levels were also alleviated by diosmetin,suggesting that the remission of airway remodeling might be attributed to the decline of these proteins.Conclusions:Taken together,our findings showed that diosmetin alleviated AHR,airway inflammation and remodeling and suppressed the levels of TGF-?1,?-SMA,MMP-9,and VEGF,highlighting a novel profile of diosmetin with therapeutic benefits in chronic asthma.Part Two Diosmetin prevents TGF-?1-induced epithelial-mesenchymal transitionObjective:Epithelial-mesenchymal transition(EMT)plays a critical role in airway remodeling in many respiratory diseases such as asthma and pulmonary fibrosis.The flavone aglycone,diosmetin,possesses anti-remodeling activity in a murine model of chronic asthma,but little is known about its effects on EMT.Herein,we investigated whether diosmetin inhibits TGF-?1-induced EMT with underlying mechanisms.Methods:Human primary bronchial epithelial(HBE)cells were cultured in vitro.The viability of diosmetin on HBE cells were measured by cell counting kit-8(CCK-8)assay.Cell migration was assessed by cell scratch and migration assays.The level of reactive oxygen species(ROS)was measured following co-incubated with dichlorodihydrofluorescein diacetate assay by FACScan and confocal laser scanning microscope.The protein expressions such as N-cadherin,E-cadherin,a-SMA were assessed by Western blot and immunofluorescent.NADPH oxidase 4(NOX4),superoxide dismutase(SOD),catalase,as well as Akt,Erk,p38 and phosphorylation of Akt,Erk,p38 were evaluated by Western blot analysis.Results:The cell viabilities were 98%,96%and 92%in 10,20 and 40 ?M diosmetin at 24 h.TGF-?1 promoted EMT in HBE cells and generation of ROS compared with control group(P<0.05).Diosmetin significantly suppressed TGF-?1-induced increases of cell migration and altered N-cadherin,E-cadherin and?-SMA(P<0.05).In addition,diosmetin prevented TGF-?1-induced intracellular ROS generation,down-regulated NOX4,and up-regulated SOD and catalase expression(P<0.05).Furthermore,TGF-?1 induced p38,Erk,JNK and Akt activation,diosmetin remarkably inhibited activation of phosphoinositide 3-kinase(PI3K)/Akt and mitogen activated protein kinase(MAPK)signaling pathways to reduce the phosphorylation level of p38,Erk and Akt induced by TGF-?1 in HBE cells(P<0.05).Conclusions:Our results demonstrated for the first time that diosmetin alleviates TGF-?1-induced EMT via inhibiting ROS generation as well as inactivating PI3K/Akt and MAPK pathways,hightlighting a new profile of diosmetin in remitting airway remodeling and fibrogenesis.