| Isoflavone Genistein is one of the secondary metabolic compounds in soybeans.It prevents diseases and exerts healthy benefits by the cellular and molecular modification on animals and humans.The genetic modification by screening key enzymes in isoflavone biosynthetic pathwys is effective to increase isoflavone concentrations in non-leguminous plants thus provide health benefits.The objectives of the current experiments are to investigate the effects of Genistein on preventing neuronal damage and neurological dysfunction in in vivo as well as in vitro models,to verify its neuroprotection and mechanism of action.The non-leguminous plant tobacco is introduced as the bioreactors for the expression of genes affecting the synthesis of Genistein and its accumulation.The main findings are as follows:1.We investigated the potential neuroprotective effects of Genistein and its possible mechanism of action in a cerebral ischemia mouse model by transient middle cerebral artery occlusion(MCAO).Mice were pretreated with Genistein(2.5,5,and 10 mg/kg)or vehicle orally once daily for 14 consecutive days before the surgery was performed.Genistein at doses of 2.5-10 mg/kg significantly reduced the infarct volume,improved the neurological deficits and prevented cell apoptosis after ischemia.In addition,Genistein pretreatment was shown to inhibit the ischemia-induced reactive oxygen species(ROS)production,enhance the activities of antioxidant enzymes superoxide dismutase(SOD)and glutathione peroxidase(GPx),and decrease the levels of malondialdehyde(MDA)in stroke mice.Moreover,Genistein reversed the mitochondria dysfunction after ischemia,as evidenced by preventing cytochrome C release from mitochondria to the cytoplasm and inhibiting caspase-3 activation.Western blot showed ischemia activated the ROS-dependent nuclear factor-?B(NF-?B)signaling pathway,and Genistein suppressed phosphorylation and expression of the NF-?B p65 subunit,as well as the phosphorylation and degradation of the inhibitor protein of ?Ba(I?B?).Our findings suggested that Genistein has a neuroprotective effect in transient focal ischemia,which may involve direct and indirect anti-oxidation,regulation of mitochondria dysfunction and suppression of ROS-induced NF-?B activation.2.We investigated the potential cytoprotection of Genistein and its molecular mechanism against exogenous hydrogen peroxide(H2O2)-induced cell death in primary rat cortical neurons.By morphological observation,immunocytochemistry identification and western blot assay,we demonstrated that Genistein(0.01-1?M)pretreatment for 24 h attenuated H2O2(500 ?M)-mediated neuronal viability loss and over production of intracellular ROS.In addition,Genistein exerted the anti-apoptotic effects by reversing the apoptotic factors Bcl-2 and Bax ratio,along with a suppression of caspase-9 and caspase-3 activities.Similar with the observation in in vivo study,Genistein down-regulated the expression of NF-?B/p65,and suppressed the phosphorylation of p65 and I?Ba.Genistein also inhibited H2O2-induced activation of the MAPK signaling pathway including c-Jun N-terminal kinase(INK)and extracellular signal-related kinase(ERK).These results indicated that Genistein effectively protects cortical neurons against oxidative stress via inhibition of ROS production,regulation of apotosis-related proteins,and inactivation of NF-?B as well as MAPK signaling pathways.3.Two soybean materials,Beifeng 17 and ZYD4368 were selected for cloning the cDNA sequences of two isoforms of soy isoflavone synthase(IFS),IFS1 and IFS2.Expression vectors were constructed as pMDC83-Beifengl7IFS1,pMDC83-ZYD4368IFS1,pMDC83-Beifengl7IFS2 and pMDC83-ZYD4368IFS2,which were than transformed into the model plant tobacco by Agrobacterium infection.A total of 128 tobacco tranfomants were obtained by PCR detection,with the positive rate of 87.1%.The relative expression and copy number of the target genes were analysed by real-time quantitative PCR.Results showed that IFS1 expression in leaves of tobacco transformants varied from 6 to 50,with the copy number of 1-9.And the expression of IFS2 in leaves was between 2 and 25,with the copy number of 1-7.The expression of IFS1 and IFS2 in tobacco petals was not significantly different in that of leaves.Genistein content in the transgenic plants was determined using the high performance liquid chromatography(HPLC)method.Significant accumulation of Genistein in petals was observed in all transformants detected,at levels 5.41-6.28 ?g/g fresh weight in trans-IFS1 plants and ranging from 19.65 to 23.7 ?g/g fresh weight in trans-IFS2 plants.A total of 6 tobacco transformants containing Genistein in leaves were obtained,at levels 3.06 ?g/g and 6.27 ?g/g fresh weight in Beifengl7IFS2 transformants,and 3.31-18.78 ?g/g fresh weight in ZYD4368IFS2 transformants.In trans-IFS1 plants leaves,expressors failed to produce Genistein at detectable levels.Constitutive expression of IFS1 or IFS2 leading to Genistein production in tobacco was not associated with major phenotypic effects on vegetative development.One effect that was observed,however,within multiple,independent tobacco transformants was a clearly discernible reduction in floral pigmentation.These results suggested that IFS2 plays a more critical role in the Genistein synthesis pathways,and Genistein accumulation in flowers are higher and more stable than that in leaves.Novel strategies for genetic modification to increase the accumulation of Genistein need further investigation and may be used as a possible plant bioreactor in actual production.As indicated above,Genistein elicited excellent outcomes in cerebral ischemia.There is a direct neuroprotective approach of Genistein with regard to anti-oxidative mechanism.It may be used as the food additive strategy to prevent the occurrence of acute neurological diseases.The introduction of IFS,the key enzyme gene into tobacco to induce Genistein accumulation in non-leguminous plants,may become an effective way to improve the source and yield of the bioactive compounds. |
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