Study On The Antioxidant Mechanism Of Conjugated Linoleic Acids In Laying Hens

Conjugated linoleic acids （CLA）, a mixture of linoleic acid, have received much attention recentlyfor their multiple beneficial properties, such as antiobesity, antidiabetic, anticancer, antiatherogenic,Improving immunity. It has been well demonstrated that levels of CLA in egg yolks increase in adose-dependent manner with dietary CLA addition （Shang et al.2004; Yin et al.2008）, but the effects ofhigh levels of dietary CLA on the metabolism of laying hens, including lipid metabolism and theantioxidant capacity of hens, have not been reported. The effects of dietary CLA on lipid metabolismand antioxidant capacity in laying hens were mainly studied in the current research. We further studiedthe mechanism of CLA affecting the antioxidant enzyme activities in the cellular level. The performance,egg quality, lipid metabolism, the composition of egg-yolk fatty acid, the activities of antioxidantenzymes, oxygen free radical, gene expression of antioxidant enzymes, protein phosphorylation andgene expression of MAPK-Nrf2/ARE signaling pathway and so on. The entire research includes sixmain parts as followed:Experiment1. Effect of Dietary Conjugated linoleic acid on Performance, Egg Quality, andEgg-yolk fatty acid composition of Laying HensThis experiment was designed to examine the effects of dietary CLA on performance, egg quality,and egg-yolk fatty acid composition of laying hens. Hy-Line Brown layers （n=384,52weeks old） wererandomly allocated to one of four dietary treatments. Each treatment had six replicates of16hens each.All birds were assigned to a corn-soybean meal-based diet containing a mixture of CLA at0%,1%,2%or4%for six weeks. The results showed that egg weight was not affected （P>0.05） when dietary CLAsupplementations were increased from0to2%, and decreased significantly （P <0.05） relative to theother treatments when hens were fed4%CLA. With an increase in dietary CLA, feed intake decreasedlinearly （P <0.05）. Feed efficiency improved significantly （P <0.05） as dietary CLA was increasedfrom0%to2%, and decreased significantly （P <0.05） relative to the other groups when hens were fed4%CLA. There were no differences （P>0.05） between the control and the CLA-added groups inalbumen height, Haugh unit, eggshell strength, or egg-shape index, although yolk color was darkened（deep yellow） linearly （P <0.05） with dietary CLA supplementation. The fatty acid composition of eggyolk, which was expressed as the percentage of total fatty acids in egg yolk, was significantly altered bydietary CLA supplementation after42days of the feeding trial （Table5）. The deposition of two majorisomers of CLA （c9,t11; t10,c12） in yolk lipids increased linearly with dietary CLA supplementation （P<0.05）. The relative proportion of the c9,t11CLA isomer in egg yolk was much higher than that of thet10,c12isomer in all treatments. Feeding CLA-enriched diets resulted in a significant （P <0.05）increase in saturated fatty acid （SFA） proportion, whereas the proportion of monounsaturated fatty acids（MUFA） declined significantly （P <0.05）. The proportion of polyunsaturated fatty acids （PUFA）increased quadratically （P <0.05） with dietary CLA addition and reached a plateau with1%CLAaddition. Non-CLA PUFA was reduced linearly （P <0.05） with dietary CLA supplementation. In addition, the proportions of linoleic acid, arachidonic acid （AA）, and docosahexaenoic acid （DHA） inegg yolk lipids were reduced （P <0.01） as dietary CLA increased. The results demonstrated that adding2%dietary CLA did not affect the performance, but deepened the egg yolk color. Meanwhile, thecontents of PUFA （including CLA） were also increased. The appropriate adding amount of dietary CLAis2%.Experiment2. Effect of Dietary Oxidized Oil on Serous and Hepatic Redox Status and the mRNAExpression of Antioxidant Enzymes of Laying HensThis experiment mainly investigated the effects of dietary conjugated linoleic acids on lipidmetabolism and antioxidant capacity in laying hens. Hy-Line Brown layers （n=384,52weeks old） wererandomly allocated to one of four dietary treatments. Each treatment had six replicates of16hens each.All birds were assigned to a corn-soybean meal-based diet containing a mixture of CLA at0%,1%,2%or4%for six weeks. The results showed that serum TC, LDL-C, HDL-C, TG levels and egg yolk lipidcontent were significantly affected by dietary CLA supplementation （P <0.05）. An increase in dietaryCLA supplementation from0to4%linearly decreased （P <0.05） the TG concentration by31.17%and56.49%on days21and42, respectively. There were no differences in the TC level （P>0.05） betweenthe control and the CLA-added groups on day21. However, a linear decrease in TC levels from3.74to3.19mmol/L （P <0.05） was observed on day42as dietary CLA levels increased, representing adecrease of14.71%. HDL-C levels were increased linearly （P <0.05） by adding dietary CLA on days21and42. On the contrary, LDL-C was significantly reduced （P <0.05） in the CLA added groups. Withthe increasing of dietary CLA addition, total lipid contents of dried egg yolk decreased linearly （P <0.01） compared to the control. The results demonstrated that dietary CLA meliorated serum lipidprofiles and enhanced the antioxidant capacity of laying hens.Experiment3. Establishment of Oxidative Stress Model Induced by Hydrogen Peroxide inPrimary Cultured Chicken HepatocytesThis experiment was conducted to establish the oxidative stress model induced by hydrogenperoxide in primary cultured chicken hepatocytes. Different concentrations of hydrogen peroxide wereused to treat primary cultured chicken hepatocytes for1,2,4and24h to establish the oxidative stressmodel. Methyl thiazolyltetrazolium （MTT） assay was used to assess cell viability. The relativeformation of Intracellularmalonaldehyde （MDA）, reactive oxidative species （ROS）, the activities ofsuperoxide dismutase （SOD） and catalase （CAT） were measured respectively. The results showed thattreatment of chicken hepatocytes with H₂O₂at4mmol/L for4h, the cell viability was significantlylower than that of the control group （P <0.05）, the intracellular formation of MDA and ROS increased（P <0.05）, while the activities of SOD and CAT also increased （P <0.05）. In conclusion, treatment ofchicken hepatocytes with H₂O₂at4mmol/L for4h can be used to establish the oxidative stress model. Experiment4. Effects of different concentrations of c9,t11-CLA and t10,c12-CLA on the activitiesof antioxidant enzymesThis experiment was designed to investigate the effects of different concentrations of c9,t11-CLAand t10,c12-CLA on the activities of antioxidant enzymes to determine the optimal concentration ofCLA for the next study. Trial1, fatty acid toxicity test. Hepatocytes were assigned to one of followedtreatments: normal control, c9,t11-CLA or t10,c12-CLA or LA （25、50、100、200、400、800μmol/L）.Cell viability was determined by MTT method. And cell morphology was conducted. Trial2,Pretreating the hepatocytes with different concentrations of c9,t11-CLA or t10,c12-CLA or LA （25,50,100μmol/L） for24h. Media were then replaced with the growth medium containing freshly made4mMH₂O₂for another2h. After treatment, the hepatocytes were harvested for subsequent analysis. Theresults showed that compared with the control group, cell morphology was not affected significantly inthe treatment c9,t11-CLA or t10,c12-CLA or LA （25、50、100、200μmol/L）. But in the conditions of400and800μmol/L, the effects on cell morphology were remarkably. Cell viabilities were more than90%in the concentrations of25,50,100, and200μmol/L in all treatments. However, at400and800μmol/L,cell viability was decreased significantly, less than80%; In the group of c9,t11-CLA and t10,c12-CLA,with increased concentrations of CLA isomers, the activities of SOD and CAT were significantlyincreased （P <0.05）. When the concentration came up to100μmol/L, the activities of antioxidantenzymes were the highest. In the LA group, with increased concentrations of LA, the activity of CATwere significantly increased （P <0.05）; In all the treatment groups （c9,t11-CLA or t10,c12-CLA or LA）,with increased treating concentrations, SOD1and CAT mRNA expression were markedly improved （P<0.05）, and reached a maximum at100μmol/L. The results demonstrated that cell morphology andviability were not affected significantly in the groups of c9,t11-CLA or t10,c12-CLA or LA below200μmol/L. The optimal concentration of CLAisomers for the next study was100μmol/L.Experiment5. Effects of c9,t11-CLA and t10,c12-CLA on the activities and gene expression ofantioxidant enzymes in Primary Cultured Chicken HepatocytesThis experiment mainly investigated the effects of c9,t11-CLA and t10,c12-CLA on the activitiesand gene expression of antioxidant enzymes in primary cultured chicken hepatocytes to determinewhich isomer of CLA play a major role. Prior to H₂O₂insult, monolayers were pre-incubated in growthmedium （supplemented or not with c9,t11-CLA, t10,c12-CLA or LA at final concentration of100μmol/L） for24h. Media were then replaced with the growth medium containing freshly made4mMH₂O₂for another2h. After treatment, the hepatocytes were harvested for subsequent analysis. Theresults showed that pretreatment for24h of hepatocytes with100μM c9,t11and t10,c12-CLAsignificantly reduced cellular oxidative damage. It is worth noting that the viability in t10,c12-CLAgroup was higher （P <0.05） than other groups except the control one. Pretreatment with t10,c12-CLAmarkedly attenuated the increase of ROS. There was no significant differences （P>0.05） betweenc9,t11and t10,c12treatment in preventing the generation of ROS. Pretreatment with c9,t11or t10,c12-CLA counteracted the lipid peroxidation induced by H₂O₂, but no statistical difference （P>0.05）was found compared to the H₂O₂group. Pretreatment with c9,t11or t10,c12-CLA counteracted the lipidperoxidation induced by H₂O₂, but no statistical difference （P>0.05） was found compared to the H₂O₂group. Only pretreatment with t10,c12-CLA significantly elevated the activities of T-SOD and CAT,compared to the H₂O₂treatment （P <0.05）. A24h pretreatment of hepatocytes with100μMt10,c12-CLA evoked an higher increase （P <0.05） of mRNA levels for SOD1and CAT than any othertreatments. Interestingly, no remarkable differences （P>0.05） among all the treatments were found inthe level of SOD2mRNA. Pretreatment with t10,c12-CLA or LA but not c9,t11-CLA evoked a markedincrease in level of Nrf2mRNA with respect to the H₂O₂group. Compared with the H₂O₂group, t10,int10,c12-CLA group, the protein expression of Nrf2in the cytoplasm of hepatocytes was decreased. Butno change occurred in the c9,t11-CLA group. In the nucleus, t10,c12-CLA or c9,t11-CLA or LAincreased the protein expression of Nrf2, but t10,c12-CLA induced a highest expression of Nrf2protein. The results demonstrated that t10,c12-CLA but not c9,t11-CLA or LA significantly improved the geneexpression of antioxidant enzymes via activating ERK or JNK-Nrf2/ARE signaling pathway, and furtherimprove the antioxidant enzyme activities.Experiment6. Effect of t10,c12-CLA on MAPK-Nrf2/ARE signaling pathway in PrimaryCultured Chicken HepatocytesMAPKs signaling pathway were investigated to screen which one （ERK, JNK, p38） plays a keyrole in regulation of gene expression of antioxidant enzymes. Hepatocytes were assigned to one of sixtreatments: Control （A）, H₂O₂（B）, t10,c12-CLA+H₂O₂（C）, inhibitor p38+t10,c12-CLA+H₂O₂（D）,inhibitor ERK+t10,c12-CLA+H₂O₂（E）, inhibitor JNK+t10,c12-CLA+H₂O₂（F）. The inhibitortreatment group were treated with inhibitor p38, ERK or JNK respectively for30min. Then, the groupC, D, E, F were treated with t10,c12-CLA for24h. Media of group B, C, D, E, F were then replacedwith the growth medium containing freshly made4mM H₂O₂for another2h. The results showed thatcompared with B, C significantly increased the cell viability （P <0.05） and decreased the ROSproduction （P <0.05）. There was no difference between the two groups in the content of intracellularMDA （P>0.05）. The activities of SOD and CAT were increased significantly （P <0.05）. The geneexpression of SOD1, JNK, Nrf2were also improved remarkably （P <0.05）. But no difference in thegene expression of SOD2, CAT, ERK, p38（P>0.05）. t10,c12-CLA did not affect the phosphorylatedproteins of p38and ERK1/2, but increased the phosphorylated proteins of JNK; Compared with C, inthe group of D and E, the effects on cell viability, ROS and MDA production, the activities of SOD andCAT, the gene expression of SOD1and SOD2were not significant （P>0.05）. F significantly decreasedthe cell viability （P <0.05）, increased the MDA and ROS production （P <0.05）, decreased the activitiesof SOD and CAT and the gene expression of SOD1, SOD2, CAT and Nrf2（P <0.05）.Using specificinhibitor of MAPK pathway inhibited the p38, ERK and JNK respectively, there was no difference inthe protein expression of Nrf2in the cytoplasm. Also, no change occurred in the nuclear protein expression of Nrf2when inhibiting the p38and ERK pathway. But inhibiting the JNK pathway, thenuclear protein expression of Nrf2decreased significantly.The results demonstrated that t10,c12-CLAregulated the gene expression of antioxidant enzymes mainly via JNK-Nrf2/ARE signaling pathway.