Mechanism Of Oxidative Stress Induced By Hydrogen Peroxide On The Injury Of Breast Muscle In Broilers

In recent years,the genetic selection of modern broiler breeds and intensive production models have significantly increased the growth rate of broilers,but this has also led to an increasing range and complexity of triggering factors for oxidative stress in broilers,resulting in varying degrees of oxidative damage to the body,ultimately affecting the health and meat quality of broilers.Oxidative stress can reduce the performance of broilers and cause a decline in chicken meat quality,which can seriously endanger the healthy development of the broiler industry.Under oxidative stress,excess reactive oxygen species（ROS）can alter the redox state of broiler breast muscle,causing oxidation of proteins and lipids in muscle,and ROS can also modify the metabolic enzymes in breast muscle by carbonylation,leading to changes in their structure and function and causing disorders in breast muscle metabolism,which in turn affects the formation of meat quality traits.However,it is unclear how oxidative stress causes oxidative damage to broiler breast muscle and what biochemical metabolic pathways are affected by oxidative damage to regulate the formation of chicken meat quality.Therefore,the present study aims to establish an oxidative stress model by intraperitoneal injection of hydrogen peroxide（H₂O₂）in broilers to study the changes of redox status,energy metabolism,calcium ion homeostasis and apoptosis in breast muscle,and to elucidate the mechanism of oxidative stress on the physiological and metabolic functions of the body by studying the degree of carbonylation modification of key metabolic enzymes,so as to provide a theoretical basis for revealing the mechanism of oxidative stress causing the decline of chicken meat quality.1.Effects of intraperitoneal injection of different concentrations of hydrogen peroxide on the response of oxidative stress in broilersExperiment 1:320 1-day-old male Arbor Acres（AA）broilers were randomly divided into five treatment groups with eight replicates of eight chickens each.In the control group,broilers were fed and watered ad libitum without any treatment.In the Saline group,broilers were fed and watered ad libitum and were injected intraperitoneally with 0.75%saline at a dosage of 1.0 m L/kg body weight（BW）according to their body weight on the 16th and 37th of the experimental period.In the hydrogen peroxide group（H₂O₂）,broilers were fed and watered ad libitum and were injected intraperitoneally with 2.5%,5.0%and 10.0%H₂O₂ at a dosage of 1.0 m L/kg BW,depending on their body weight,on the 16th and 37th of the experimental period respectively.The period of this experiment was 42 days.The results showed that the levels of corticosterone（CORT）and the activities of creatine kinase（CK）in broiler serum were increased gradually with the increasing concentration of H₂O₂ compared to the control and saline groups,reaching a maximum in the 10.0%H₂O₂ treated group.The levels of ROS and the contents of malondialdehyde（MDA）,carbonyl,lactate and glycolytic potential in broiler pectoral muscle were increased significantly and linearly（P<0.05）with the increasing concentrations of intraperitoneally injected H₂O₂;the contents of sulfhydryl and glycogen,activities of catalase（CAT）,total antioxidant capacity（T-AOC）and glutathione peroxidase（GSH-Px）were decreased significantly and linearly（P<0.05）.Meanwhile,the m RNA expression of quinone oxidoreductase1（NQO1）and heme oxygenase 1（HMOX1）was significantly down-regulated in broiler breast muscle in the 2.5%,5.0%and 10.0%H₂O₂ groups（P<0.05）,nuclear factor E2-related factor 2（Nrf2）in the 2.5%and 10.0%H₂O₂ groups（P<0.05）,and the10.0%H₂O₂ group significantly down-regulated the m RNA expression of glutathione peroxidase（GPX）,CAT and superoxide dismutase（SOD）in broiler pectoral muscle（P<0.05）.In addition,immunohistochemical results showed that the number of brown Nrf2-positive cells was significantly reduced in the 5.0%and 10.0%H₂O₂ groups compared to the control and saline groups（P<0.05）.None of the above indicators were significantly different between the control and saline groups of broilers（P>0.05）.2.Effects of oxidative stress induced by hydrogen peroxide on growth performance,slaughter performance and meat quality of broilersExperiment 2:144 male Ross 308 broilers at 1 day of age were selected and randomly divided into three treatment groups with 6 replicates of 8 chickens each.In the control group,broilers were fed and watered ad libitum without any treatment.In the saline group,broilers were fed and watered ad libitum,and on the 16th and 37th of the experimental period,0.75%saline was injected intraperitoneally at a dosage of 1.0m L/kg BW based on the body weight of the broiler.In the H₂O₂ group,broilers were fed and watered ad libitum,and on the 16th and 37th of the experimental period,10.0%H₂O₂ was injected intraperitoneally at a dosage of the experimental period was 42 days.The results showed that the average daily gain（ADG）of broilers in the H₂O₂ group was significantly lower（P<0.05）compared to the control and saline groups during the first 21 d of the experimental period.After 21 d of the experimental period,the ADG of broilers in the H₂O₂ group was significantly lower（P<0.05）and the feed to weight ratio（F/G）was significantly higher（P<0.05）compared to the control and saline groups.Throughout the 42 d experimental period,broilers in the H₂O₂ group had significantly lower ADG（P<0.05）and higher F/G（P<0.05）compared to the control and saline groups.The organ indexes of glandular stomach and ventriculus muscularis were significantly higher in the broilers in the H₂O₂ group compared to broilers in the control and saline groups（P<0.05）.The p H_{24 h} and cohesion of broiler breast meat were significantly lower（P<0.05）and the hardness,drip loss and shear force of broiler breast meat were significantly higher（P<0.05）in the H₂O₂ group compared to broilers in the control and saline groups.In addition,H&E stained sections showed that myofibers in the control and saline groups of broiler pectoralis were tightly arranged and filled with a lattice of endomysium and fascia.The area occupied by muscle fibers and the diameter of muscle fibers were significantly reduced in the H₂O₂ group compared to the control and saline groups（P<0.05）,with a shift in muscle fiber size towards smaller diameter muscle fibers.There were no significant differences in the above indexes between the control and saline groups（P>0.05）.3.Effects of oxidative stress induced by hydrogen peroxide on redox state and energy metabolism of breast muscle in broilersThe experimental design is the same as experimentalⅡ.The results showed that the content of CORT and the activity of LDH in serum of broilers in H₂O₂ group were significantly higher than those in control group and saline group（P<0.05）.Compared with control group and saline group,the content of H₂O₂ and the activity of-OH in breast muscle of broilers in H₂O₂ group were significantly increased（P<0.05）,and the contents of ABTS and DPPH free radical scavenging ability were significantly decreased（P<0.05）.In H₂O₂ group,the contents of ROS,carbonyl,lactate,glycolytic potential and ADP and the ratio of AMP/ATP were increased significantly（P<0.05）,while the content of glycogen and the activities of CAT,T-AOC,SOD and GSH-PX were decreased significantly（P<0.05）.In H₂O₂ group,the activity of pyruvate dehydrogenase（PDH）was decreased significantly（P<0.05）,the activities of hexokinase（HK）and LDH were increased significantly（P<0.05）,and the m RNA expression of liver kinase B1（LKB1）,calcium/calmodulin dependent protein kinaseβ（Ca MKKβ）and adenosine monophosphate activated protein kinaseα2（AMPKα2）in broiler breast muscle were significantly up-regulated（P<0.05）.In addition,the protein expressions of HK1,PFK,Ca MKK1 and p-AMPK in breast muscle of broilers in H₂O₂ group were significantly higher than those in control group and saline group（P<0.05）.There was no significant difference in the above indexes between control group and saline group（P>0.05）.4.Effects of oxidative stress induced by hydrogen peroxide on calcium homeostasis of broiler breast muscleThe experimental design is the same as experimental II.The results showed that the levels of ROS,MDA and carbonyl in mitochondria of pectoral muscle of broilers in H₂O₂ group were significantly higher than those in control group and saline group（P<0.05）;the level of mitochondrial membrane potential（MMP）,the activities of mitochondrial complex I,mitochondrial complex III,T-AOC,T-SOD,CAT and GSH-PX were decreased significantly（P<0.05）.The content of Ca²⁺in cytoplasm and mitochondria of breast muscle in H₂O₂ group was significantly higher than that in control group and saline group（P<0.05）,while the m RNA and protein expression of mitochondrial calcium one-way transporter（MCU）and sarcoplasmic reticulum Ca²⁺-ATPase1（SERCA1）in H₂O₂ group were significantly lower than that in control group and saline group（P<0.05）,The m RNA and protein expression of cell membrane Ca²⁺-ATPase（PMCA）were significantly higher than that of control group and saline group（P<0.05）,while the carbonylation degree of MCU protein in breast muscle of H₂O₂group was significantly higher than that of control group and saline group（P<0.05）.In addition,in control group and saline group,the morphology and structure of the mitochondria in the pectoral muscle of broilers were intact,in the shape of short rod or ball,the structure of the outer membrane of mitochondria was complete,and the inner membrane of mitochondria wrinkled inward to form a rich number of cristae,while in H₂O₂ group,the mitochondria in the pectoral muscle of broilers were shrink,the interior of mitochondria showed vacuolization,and a large number of previously abundant cristae were disappeared.There was no significant difference in the above indexes between control group and saline group（P>0.05）.5.Effects of oxidative stress induced by hydrogen peroxide on ferroptosis in breast muscle of broilersThe experimental design is the same as experimental II.The results showed that the number of breast muscle cell apoptosis in H₂O₂ group was significantly higher than that in control group and saline group（P<0.05）.Compared with control group and saline group,the contents of MDA,lipid peroxide（LPO）,4-hydroxynonenal（4-HNE）,oxidized glutathione（GSSG）and Fe³⁺in breast muscle of broilers in H₂O₂ group were increased significantly（P<0.05）,and the content of reduced glutathione（GSH）and the ratio of GSH/GSSG were decreased significantly（P<0.05）.The m RNA and protein expression of transferrin receptor 1（TFR1）in breast muscle of broilers in H₂O₂ group were significantly higher than those in control group and saline group（P<0.05）,and the m RNA expression of iron response element binding protein 2（IREB2）and glutathione peroxidase 4（GPX4）were significantly lower than those in control group and saline group（P<0.05）.There was no significant difference in the above indexes between control group and saline group（P>0.05）.To sum up,this study draws the following conclusions:（1）Intraperitoneal injection of different concentrations of H₂O₂ could trigger the stress response of broilers.The degree of stress response intensified with the increase of H₂O₂ concentration.Nrf2 signal pathway in pectoral muscle was inhibited,resulting in the weakening of antioxidant function and the increase of oxidation products in pectoral muscle.The ideal concentration for establishing the oxidative stress model of broilers by intraperitoneal injection of H₂O₂ was 10.0%.（2）Oxidative stress induced by H₂O₂ could reduce the growth performance,breast muscle texture characteristics and meat quality of broilers,and oxidative damage occurred to the fiber structure of breast muscle.（3）Oxidative stress induced by H₂O₂ could cause oxidative damage to broiler pectoral muscle,increase the level of ROS in pectoral muscle,affect the antioxidant function of pectoral muscle,and then increase the content of oxidation products.Oxidative stress enhanced the catabolism of energy substances and inhibited the aerobic respiration of muscle cells.The body enhanced glycolytic metabolism by activating AMPK signal pathway and carbonylation to modify glycolytic enzymes.（4）Oxidative stress induced by H₂O₂ could damage the mitochondria of broiler pectoral muscle,destroy the structure and function of mitochondria,change the redox state in mitochondria and aggravate the oxidative damage of muscle cells.When intracellular Ca²⁺was overloaded,the activity of Ca²⁺channel protein in cell membrane and sarcoplasmic reticulum changed to maintain intracellular calcium homeostasis.ROS modified mitochondrial MCU protein through carbonylation,reduced its activity,and then alleviated mitochondrial Ca²⁺overload.（5）Oxidative stress induced by H₂O₂ could cause apoptosis of muscle cells,increase the content of lipid oxidation products,increase the concentration of iron ion and weaken the activity of GSH.ROS could induce ferroptosis of breast muscle cells by activating TFR1/Fe²⁺signal pathway and inhibiting System X_C^-/GPX4 signal pathway.