| Oxidative stress model of broiler breast muscle satellite cells (SCs) and animal chronic mild stress model were optimized to demonstrate the main MAPK pathway in oxidative stress. Four experiments were conducted to study the mechanism of MAPK pathway on meat quality. Summarized as follows:Experiment One:Optimization of oxidative stress model from broiler breast muscle satellite cellsObjective:Broiler breast muscle satellite cells (SCs) were treated with dexamethasone phosphate solution (DEX). DEX concentration and treatment hours were optimized to establish the oxidative stress model of broiler breast SCs. Methods: Seven concentrations of DEX (0, 0.03, 0.06, 0.12, 0.24, 0.48 and 0.96 mg/ml) were used to treat broiler breast muscle SCs. Methyl thiazolyl tetrazolium(MTT)assay was used to assess the survival rate of SCs at different times (6 h, 12 h, 24 h and 48 h). The relative production and activities of malonaldehyde (MDA), reactive oxidative species (ROS), the superoxide dismutase (SOD) and glutathione S-transferase (GST) of cells and medium were measured respectively. Results: With the increasing of DEX level, the concentrations of MDA and ROS increased (P<0.01), while activities of SOD and GST decreased (P<0.01). Cell livability and the activities of SOD and GST decreased with the prolonging of DEX treated, while the production of MDA and ROS increased. Treatment of broiler breast muscle SCs with DEX at 12 mg/ml for 24 h, the cell livability was significantly lower than that of the control group (P<0.01). Conclusion: Treatment of broiler breast muscle SCs with DEX at 12 mg/ml for 24 h could be used in the oxidative stress model of broiler breast SCs.Experiment Two:Screening of MAPK signaling pathway in broiler satellite cell under oxidative stressObjective: The satellite cells oxidative stress model was used to screen the key mitogen-activated protein kinases (MAPKs) signaling pathways during oxidative stress. Methods: The broilers breast muscle SCs were assigned to one of the ten treatments, which included DEX, Inhibitor p38MAPK, DEX + Inhibitor p38MAPK, Inhibitor JNK, DEX + Inhibitor JNK, Inhibitor ERK5, DEX + Inhibitor ERK5, Inhibitor ERK1/2, DEX + Inhibitor ERK1/2 and control. All the inhibitor treatments were treated with inhibitors for 30 min, and then treated with DEX or normal medium for 24h. Then the relative production and the activities of malonaldehyde (MDA), reactive oxidative species (ROS), superoxide dismutase (SOD) and glutathione S-transferase (GST) of medium were measured. Reverse transcriptional polymerase chain reaction (RT-PCR) was employed to assess the gene expression of key protein of MAPK pathway and SOD, GST. Results: Compared with DEX treatment, the MDA and ROS concentrations were decreased (P<0.05) in all the inhibitor-treated groups. The MDA and ROS concentrations of DEX + Inhibitor ERK5 and DEX + Inhibitor ERK 1/2 treatments were significantly higher than those of Inhibitor ERK5 and Inhibitor ERK1/2 treatments (P<0.05). However, there were no significant differences in MDA and ROS concentrations among DEX + Inhibitor p38MAPK, DEX+ Inhibitor JNK, Inhibitor p38MAPK, Inhibitor JNK treatments (P>0.05). The activities of SOD and GST of all the Inhibitor-treated groups were significantly higher than those of DEX groups (P<0.01). The activities of SOD and GST of DEX + Inhibitor ERK5 and DEX + Inhibitor ERK 1/2 groups were significantly lower than those of Inhibitor ERK5 and Inhibitor ERK1/2 groups (P<0.01), while there were no significant differences among DEX + Inhibitor p38MAPK, DEX + Inhibitor JNK and Inhibitor p38MAPK, and Inhibitor JNK groups (P >0.05). DEX inhibited the expression of SOD and GST gene, and the inhibition rate was more than 37%. DEX treatment had no significant differences on the expression of SOD and GST gene while p38MAPK and JNK pathway were inhibited. Conclusion: p38MAPK and JNK pathway played a key role in broiler breast muscle SCs under oxidative stress induced by DEX.Experiment Three:The effect of antioxidants on p38 and JNK pathway under oxidative stressObjective:Oxidative stress model of broiler breast muscle SCs were used to investigate the effects of antioxidants (VC, VE, tea polyphenol (TP), daidzein(DZ), pyrro-quinoline quinone(PQQ)) on p38MAPK and JNK pathway. Methods: The broilers breast muscle SCs were assigned to one of the twelve treatments: control, DEX, Inhibitor p38MAPK, DEX + Inhibitor p38MAPK, Inhibitor JNK, DEX + Inhibitor JNK, DEX + VC and VE,DEX + TP, DEX + VC, DEX + VE, DEX + DZ, DEX + PQQ. The relative production and the activities of malonaldehyde (MDA), reactive oxidative species (ROS), superoxide dismutase (SOD) and glutathione S-transferase (GST) of medium were measured. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to assess MEK3, MEK4 and phaseⅡenzyme gene expression. Results: Compared with DEX, the MDA and ROS concentrations were decreased in all the inhibitors-treated and antioxidants groups, especially TP + DEX and VC + DEX groups (P <0.01). The activities of SOD and GST of all antioxidants groups were significantly higher than those of DEX groups (P <0.01), and treatments of TP + DEX and VC + DEX groups were higher than treatments of DZ + DEX, VE + DEX and PQQ + DEX groups. Antioxidants could decrease the expression of MEK3 and MEK4, and increase the expression of SOD and GST. Conclusion: Antioxidants could regulate p38MAPK and JNK pathway to decrease the oxidative injury of broiler breast muscle SCs induced by DEX.Experiment Four:Effect of VC and TP on growth performance, antioxidative activity of breast muscle and MEK3, MEK4 expression during chronic stress on broilersObjective:Oxidative stress induced by DEX was used to investigate the effects of VC and TP on broiler performance, antioxidative activity of breast muscle and MEK3, MEK4 expression of boilers. Method: Seven-hundred 1 day-old Arbor Acres male broilers were raised for 7 days together, and then divided into six treatments (Control,DEX,VC,VC + DEX,TP,TP + DEX) by average weight (±5%). The corresponding treatment diets were fed for 7～21 d and 22～42 d. The amount of 4 mg/kg BW DEX was applied to the broilers via drinking water for 7 days from 22 d to 28d. At 28 d and 42 d, the production of MDA, the activity of SOD and GST of broiler breast muscle were measured, reverse transcriptional polymerase chain reaction (RT-PCR) was employed to assess the key protein of MAPK pathway and SOD, GST gene expression. Result: There were no differences on ADFI and ADG between TP and VC treatments (P >0.05). Compared with the control, there were no difference on the performance of chicks from TP + DEX, VC + DEX and DEX treatment in later period (28～42 d) (P >0.05), but there were significant differences between stress groups (supplement with DEX) and the non-stress groups (without DEX) (P <0.01). At 28 d, the activities of SOD and GST in the breast muscle of VC and TP treatments were significantly higher than those of DEX treatment (P <0.01), the MDA concentrations were on the contrary (P <0.01). There were no differences of the activities of SOD and GST and the production of MDA among TP + DEX, VC + DEX and DEX treatments (P >0.05). TP and VC decreased the expression of MEK3 and MEK4, while SOD and GST on the contrary. At 42 d, the activities of SOD and GST of VC and TP treatments were higher than those of DEX treatment (P <0.05), there were no differences of MDA among all treatments (P >0.05). The activities of SOD and GST of TP + DEX, VC + DEX treatments were higher than those of DEX treatment,but the differences were not significant (P >0.05). TP and VC decreased the expression of MEK3 and MEK4, and increased the expression of SOD and GST. Conclusion: There were no significant effects on broiler performance with supplements of TP and VC to diet, but TP and VC could regulate p38MAPK and JNK pathways to decrease the oxidative injury on broiler breast muscle induced by DEX. |
