Protective Effect Of Kaempferol Against Oxidative Damage Of Extravillous Trophoblast Cell Line Caused By BaP

PAHs are common air pollutants, mainly from tobacco smoke, coalburning, vehicle exhaust and indoor cooking fumes, easily in water, soil andcrop residues accumulate[1]. Benzo (a) pyrene is the most representative ofPAHs in the environment of organic pollutants. Epidemiological investigationsand animal experiments show that benzo (a) pyrene and the occurrence ofadverse pregnancy outcomes, such as miscarriage, premature birth, fetalgrowth retardation and birth defects, but also decrease the fertility andinfertility related[2-5], which may affect the metabolism of mechanism ofaction include cytochrome P450enzymes, oxidative damage, apoptosis, DNAdamage and anti-estrogenic effects, etc.[6-11].Kaempferol are flavonoids, yellow needles, as Equisetaceaecharacteristic ingredient, is also present in the roots of Zingiberaceaekaempferol and Berberis plants, Euphorbiaceae cat’s eye grass aboveground infruits, vegetables, beans and tea. It has been found that kaempferol has avariety of biological functions such as anti-inflammatory, tumor growthinhibition, inhibition of platelet aggregation and adhesion, antibacterialprotection of liver cells pharmacological effects[12-18]. Anti-oxidationpathways of kaempferol are: free radical scavenging (ROS), by regulatingNOS (nitric oxide synthase), NO (nitric oxide) levels to suppress thegeneration of ROS, inhibition of lipid peroxidation (MDA), enhancedactivity of antioxidant enzymes (superoxide dismutase SOD, glutathioneperoxidase GSH-Px), inhibition the oxidative damage of LDL, inhibition ofmitochondrial membrane potential and release of cytochrome C, the impact ofnuclear transcription factor κB (NF-κB) signaling pathways[19-21]. However,the potential protection effect of kaempferol on embryo damage caused by airpollutants such as BaP had not be reported or researched till now. In this study, we used human chorionic trophoblast cells as foreign objects to explore theprobable effect of kaempferol on inhibiting oxidative injury caused bybenzopyrene.Objective: To explore the inhibited effect and potention mechanism ofkaempferol against oxidative damage caused by BaP, based on HTR8-SVneocells.Methods:1Construction of BaP injury model: MTT assay0.1~50μmol/Lbenzopyrene for24hours and48hours of cell survival respectively. Select theconcentration and time of cell survival effect around30%.2Experimental groups: control group, BaP exposure group, BaP exposureto different concentrations of kaempferol (0.01μmol/L~1μmol/L) group.3Using colorimetric measured each experimental group SOD, MDA andNO content.4Using Hochest33342and Giemsa staining to observe apoptoticmorphological change; using flow cytometry to observe apoptosis rate in eachgroup.5Measurement of caspase-3、iNOS、AhR mRNA expression by RT-PCR.Results:1Inhibited effects of kaempferolon on the viability of HTR8-SVneo cellsexposed to BaPThe study found0.1~50μmol/L BaP at24and48hours a decrease incell survival, and cell survival with increasing concentrations of benzopyreneand the role of extension of time decreases. According to the exposureintensity and cell survival, we selected10μmol/L48hours as exposureintensity to establish the injury model of BaP. We found that compared withthe BaP group, kaempferol significantly inhibited the damage of HTR8-SVneocell lines induced by BaP.2Measurement of SOD, MDA and NO releaseThe result showed that compared with the BaP group, kaempferol cansignificantly increase the activity of SOD and decrease the activities of MDA and NO.3Flow cytometry determine the apoptosis rate and the morphologicalchanges of apoptosisThe result showed that the inhibitory effects of kaempferol inBaP-induced apoptosis rate of HTR8-SVneo cells. It suggested thatkaempferol posses the inhibition function of apoptosis.4The expression of Caspase-3mRNA, iNOS mRNA and AhR mRNAExpression of Caspase-3mRNA, iNOS mRNA, AhR mRNA inkaempferol inhibited BaP-induced HTR8-SVneo. We found that comparedwith the BaP group, kaempferol significantly inhibited the expression ofCaspase-3mRNA, iNOS mRNA, AhR mRNA.Conclusions:In vitro, kaempferol can significantly inhibite the oxidative damage ofHTR8-SVneo cells induced by BaP.The potential mechanism was related toenhance the activity of antioxidant enzymes SOD, reduce lipid peroxidesMDA, production of NO, reduce apoptosis rate of BaP injury model. We alsofound that kaempferol can reduce the genes expression of apoptosis and arylhydrocarbon receptor.These results indicated that kaempferol possess excellent antioxidant. Ourresearch has provided strong evidence for the value of kaempferol as anantioxidant agent.