Effects And Mechanism Of Anthocyanins From Dioscorea Alata L. On Antioxidant Activity Of Goat Mammary Epithelial Cells

Perinatal female goats are usually in a state of negative energy balance,and a large number of reactive oxygen species(ROS)produced can cause oxidative damage or even immunosuppression to mammary gland,thus affecting their lactation ability and production performance.Therefore,some antioxidants are often used to maintain the oxidative homeostasis of the body by domestic and foreign scholars.Anthocyanin,as a water-soluble natural pigment,has become the focus of antioxidant research in recent years because of its high safety and strong ability to scavenge free radicals.Dioscorea alata L.has great potential to be used as special animal feed due to its tubers containing abundant anthocyanins and dietary fiber.In order to explore the pre-protective effect and mechanism of Dioscorea alata L.anthocyanin(DAC)on oxidative damage of goat mammary epithelial cells(GMEC),and the application effect in actual production,this experiment was conducted through four parts.Test 1:The relative survival rate of cells,the activities of lactate dehydrogenase(LDH),catalase(CAT),superoxide dismutase(SOD)and total antioxidant capacity(T-AOC)in culture medium,the activity of glutathione peroxidase(GSH-Px),the relative content of ROS and the content of malondialdehyde(MDA)in GMEC were determined.The optimal effect conditions of DAC on GMEC were explored.Test 2:H2O2 was selected as the oxidant,and the optimal condition of GMEC oxidative damage induced by H2O2 was explored through the same method and detection index.Test 3:Then by measuring the clearance rate of 1,1-diphenyl-2-trinitrophenylhydrazide(DPPH),the treatment concentration of positive control VC with the same in vitro antioxidant capacity as DAC was obtained,and on this basis,six treatment groups were designed,respectively:control group,injury group,DAC group,VC group,DAC pre-protection group and VC pre-protection group.The pre-protective effect of DAC on H2O2-induced oxidative damage of GMEC was investigated by measuring the same antioxidant index in test 1,so as to explore the protective effect of DAC on H2O2-induced oxidative damage of GMEC and its mechanism.Test 4:Feeding experiment was carried out.Twenty-four Hainan black goats were randomly divided into control group and experimental group.The experimental group was fed the diet with Dioscorea alata L.powder replacing 30%corn.Rumen fluid fermentation parameters of ewes at the 14 d of the experiment period and 21 d after delivery were determined.The blood and milk samples of postpartum ewes at 1,2,3,7,14 and 21 d and the blood samples of newborn lambs at 7,14 and 21 d were gathered.To investigate the effect of anthocyanin containing Dioscorea alata L.powder on the antioxidant capacity of perinatal ewes and lambs in actual production.The results showed that:(1)20 ug/mL DAC was the best effect condition for GMEC treatment for 9 h;(2)GMEC treated with 430 μmol/L H2O2 for 8 h was the best oxidation damage condition;(3)DAC pretreatment could up-regulate mRNA expression levels of Nrf-2,HO-1 and NQO-1(P<0.05),and increase activities of CAT,SOD,GSH-Px and T-AOC(P<0.05);Meanwhile,the mRNA expression levels of IL-1β and TNF-α were up-regulated and JNK phosphorylation was promoted(P<0.05),and the expression levels of Cytochrome C,Caspase-3 and Caspase-9 and LDH activity were significantly increased(P<0.05),while ROS and MDA contents were decreased.Thus,DAC can play a pre-protective role against oxidative stress;(4)Feeding Dioscorea alata L.powder instead of 30%corn diet can significantly reduce the total volatile fatty acid concentration on day 14 of the experiment period,significantly reduce propionic acid and butyric acid in rumen of Hainan black goats on day 21 after delivery,and increase the ratio of acetic acid to propionic acid and pH value.Meanwhile,the activities of CAT,SOD,GSH-Px and T-AOC in blood and milk of ewes were increased,and the content of MDA was decreased,so as to improve the antioxidant capacity of lambs and enhance their resistance to oxidative stress.In conclusion,anthocyanins from Dioscorea alata L.have a good pre-protective ability against H2O2-induced GMEC oxidative stress.The mechanism may be that by activating Nrf-2/HO-1 and MAPK-JNK signaling pathways,the activity of intracellular antioxidant enzymes can be improved,apoptosis can be promoted,cell renewal and metabolism can be accelerated,and accumulation of peroxide products can be reduced.In addition,the substitution of Dioscorea alata L.powder for 30%corn diet in actual production can change the rumen fermentation pattern of goats,and effectively improve the activity of antioxidant enzymes in blood and milk of Hainan black goats during perinatal period,so as to improve the antioxidant capacity of lambs,which has certain practical production value.