Effects Of Terpen-4-ol On Growth Performance And Intestinal Damage In Weaned Piglets With Oxidative Stress

The development of animal husbandry is facing the background of feed prohibition and resistance,environmental factors,feed raw materials and weaning stress all put piglets to a great challenge in healthy production.Meanwhile,early weaning makes piglets more vulnerable to oxidative stress,and ROS content increases significantly,resulting in oxidative damage,which greatly reduces breeding benefits.Therefore,intestinal health of piglets has become a common concern and needs to be solved urgently.At present,a variety of bioactive substances in plant extracts have been proved to have significant effects on antibacterial,antiinflammatory,antioxidant and intestinal health,and are expected to be used as substitute antioxidant substances.Due to their complex components,the specific mechanism of action is still unclear,which makes it a problem for plant extracts to be used as substitute antioxidant substances to promote the development of animal husbandry.Our previous studies have shown that Terpen-4-ol can alleviate intestinal oxidative damage in piglets,but the specific mechanism of action remains unclear.Therefore,in this study,H2O2 was used to induce oxidative stress in weaned piglets,to explore the regulatory effects of Terpen-4-ol on growth performance and intestinal damage of weaned piglets with oxidative stress,and to explore the potential mechanism of action of Terpen-4-ol on intestinal damage of weaned piglets with oxidative stress,so as to provide theoretical guidance for the application of Terpen-4-ol in the production of piglet.It provides a new target for nutritional regulation of intestinal oxidative damage in piglets.Experiment 1 Effects of Terpen-4-ol on Growth Performance and Serum Antioxidant Function of Weaned piglets with oxidative stressThis experiment was conducted to investigate the effects of Terpen-4-ol on growth performance and serum antioxidant function of weaned piglets with oxidative stress.Thirtytwo(8.53±0.42kg)piglets weaned at 21 days of age(Duroc×(Landrace × large white pig)with similar body weight were randomly divided into four treatment groups:(1)normal control group(CON group);(2)Group H2O2(group H2O2);(3)H2O2+TER group(H+T group);(4)TER group.CON group and H2O2 group were fed a basal diet,and TER group and H+T group were fed a basal diet supplemented with 60mg/kg TER.the pre-test period was 3 days and the formal period was 21 days.On the 15th and 18th days,the H2O2 group and the H+T group were intraperitoneally injected with 10%H2O2 respectively(30%H2O2 was diluted to 10%H2O2 with sterilized 0.9%normal saline before injection,and the volume ratio of dilution was 1:2).CON group and TER group were intraperitoneally injected 0.9%sterile normal saline.Growth performance,serum reactive oxygen species,lactate dehydrogenase activity and serum antioxidant enzyme activity were measured.The results showed as follows:(1)TER significantly increased the average body weight at 14 and 21 days of oxidative stress(P<0.05),and increased the average daily feed intake at 21 days(P<0.05).The average body weight and average daily feed intake of H2O2 group were significantly decreased(P<0.05),and the addition of 4-terpenol could effectively reduce the diarrhea rate.(2)Compared with CON group,the serum ROS level of piglets in TER group was significantly decreased(P<0.05),the serum ROS level in H2O2 group was significantly increased(P<0.01),and the serum ROS level in H+T group was significantly lower than that in H2O2 group(P<0.01).(3)The activities of serum antioxidant enzymes CAT and GSH-Px in TER group were significantly increased(P<0.05),the activities of MDA were significantly decreased(P<0.05),and the activities of CAT and SOD in H2O2 group were significantly decreased(P<0.05).The activities of CAT,SOD and GSH-Px in H+T group were significantly increased(P<0.05),and the activities of MDA were significantly decreased(P<0.05).(4)Compared with CON group,serum LDH activity in H2O2 group was significantly increased(P<0.01).Compared with H2O2 group,the serum LDH activity in H+T group was significantly decreased(P<0.05).In conclusion,Terpen-4-ol can significantly improve the growth performance of weaned piglets with oxidative stress,reduce the levels of reactive oxygen species and lactate dehydrogenase in serum,and increase the activity of antioxidant enzymes,so as to improve the antioxidant function and oxidative stress of weaned piglets.Experiment 2 Effects of Terpen-4-ol on intestinal damage in weaned piglets under oxidative stressThis experiment was conducted to investigate the regulatory effects of Terpen-4-ol on intestinal damage in weaned piglets under oxidative stress.The design of the experiment is the same as that of experiment 1.The results showed as follows:(1)Compared with CON group,ROS content in the three intestinal segments(duodenum,jejunum and ileum)of TER group was significantly decreased(P<0.05),while ROS content in H2O2 group was significantly increased(P<0.05).Compared with H2O2 group,ROS contents in the three intestinal segments of H+T group were significantly decreased(P<0.05).(2)Compared with CON group,LDH activity in duodenum and jejunum TER groups was significantly increased(P<0.05).Compared with H2O2 group,LDH activity in jejunum and ileum H+T groups was significantly increased(P<0.05).(3)Compared with CON group,the intestinal activities of T-AOC,GSHPx and CAT enzymes in TER group were significantly increased(P<0.05),while the intestinal activities of SOD and GSH-Px enzymes in H2O2 group were significantly decreased(P<0.05).Compared with H2O2 group,the activities of SOD and GSH-Px in intestinal tract of H+T group were significantly increased(P<0.05),while the activities of MDA enzyme were significantly decreased(P<0.05).(4)Compared with CON group,Gpx1 and SOD1 gene expressions in TER group were significantly increased(P<0.05),while Gpx1 and CAT gene expressions in H2O2 group were significantly decreased(P<0.05).Compared with H2O2 group,intestinal CAT gene expression in H+T group was significantly increased(P<0.05).(5)Compared with CON group,the height of intestinal villus in TER group was significantly increased(P<0.01),the depth of intestinal crypt was significantly decreased(P<0.01),the ratio of intestinal villus to crypt was significantly increased(P<0.01),and the height of intestinal villus in H2O2 group was significantly decreased(P<0.01),and the depth of intestinal crypt was significantly increased(P<0.01).The lint ratio was significantly decreased(P<0.01).Compared with H2O2 group,the height of intestinal villus in H+T group was significantly increased(P<0.01),the depth of crypt was significantly decreased(P<0.01),and the ratio of villus to crypt was significantly increased(P<0.01).(6)Compared with the CON group,the intestinal villi of the H2O2 group were more seriously damaged,with breakage and shedding,and the mitochondria showed obvious swelling and mitochondrial ridge breakage.The villi of the TER group and the H+T group were well developed,neatly arranged and relatively robust,and the mitochondria showed normal condition and better development.(7)Compared with CON group,intestinal GPX4 and SLC7A11 gene expressions in TER group were significantly increased(P<0.05),and ACSL4 gene expression in H2O2 group was significantly decreased(P<0.05).Compared with H2O2 group,intestinal GPX4 gene expression in H+T group was significantly increased(P<0.05),while ACSL4 gene expression was significantly decreased(P<0.05).(8)Compared with CON group,the relative expression levels of ACSL4 protein in duodenum of H2O2 group were significantly increased(P<0.05),and the relative expressions of GPX4 and SLC7A11 protein in ileum of TER group were significantly increased(P<0.05).Compared with H2O2 group,Duodenal ACSL4 protein relative expression level in H+T group was significantly decreased(P<0.05).The relative expression level of GPX4 protein in ileum was significantly increased(P<0.05).In conclusion,H2O2 stimulation can lead to the increase of ROS level,the decrease of antioxidant enzyme activity,the damage of intestinal villus morphology and structure,and the increase of iron death level in piglets.Terpen-4-ol can alleviate intestinal damage induced by oxidative stress,promote intestinal villus development and maintain the normal mitochondrial structure,and inhibit the expression of iron-death related genes and proteins.Thes alleviating intestinal damage of piglets.Experiment 3 Potential mechanism of Terpen-4-ol on iron death in intestinal epithelial cells of weaned piglets under oxidative stressThis experiment was conducted to investigate the effect of Terpen-4-ol on iron death in intestinal epithelial cells of weaned piglets under oxidative stress.The design of the experiment is the same as that of experiment 1.The expression levels of intestinal autophagy,mitochondrial membrane potential and mitochondrial autophagy were determined.The results showed as follows:(1)Compared with CON group,Beclin,PINK1(PTEN Induced Putative Kinase 1,PINK1),LC3(Lightchain Sociated Protein,LC3)and Parkin(Parkinson,The expression of Disease Gene 2,Parkin gene was significantly increased(P<0.05).Compared with H2O2 group,Beclin and LC3 gene expressions in H+T group were significantly increased(P<0.05),while P62(Sequestosom-1,P62)gene expressions were significantly decreased(P<0.05),while P62 gene expressions were significantly decreased(P<0.05).(2)Compared with the CON group,the mitochondrial membrane potential in the three intestinal segments of the TER group was significantly increased(P<0.01),and the mitochondrial membrane potential in the H2O2 group was significantly decreased(P<0.01).Compared with H2O2 group,mitochondrial membrane potential was significantly increased in H+T group(P<0.05).(3)H2O2 group significantly decreased the mitochondrial membrane potential of the three segments of intestinal tract(P<0.01),H+T group and TER group significantly increased the mitochondrial membrane potential of duodenum and jejunum(P<0.01),and TER significantly increased the mitochondrial membrane potential of ileum(P<0.01).(4)Compared with CON group,the expressions of intestinal autophagy genes Beclin,PINK1,LC3 and Parkin in TER group were significantly increased(P<0.05).Compared with H2O2 group,the expression of intestinal autophagy gene of P62 in H+T group was significantly decreased(P<0.05).In conclusion,H2O2 stimulation can reduce intestinal epithelial autophagy,reduce intestinal mitochondrial membrane potential and reduce intestinal mitochondrial autophagy of piglets,while Terpen-4-ol can increase intestinal epithelial cells and mitochondrial autophagy of piglets,which may inhibit iron death of intestinal epithelial cells under immune stress through mitochondrial autophagy pathway,thus alleviating intestinal oxidative damage of piglets.In summary,the addition of Terpen-4-ol to the basal diet of weaned piglets can effectively increase the level of growth index of weaned piglets,and the level of reactive oxygen species induced by H2O2 and the related damage caused by the increase of lactate dehydrogenase content are inhibited by the addition of Terpen-4-ol.It can effectively improve the damage and swelling of intestinal villi and intestinal mitochondria,increase the relative expression of antioxidant enzymes and antioxidant genes and proteins,reduce the level of ferroptosis induced by H2O2,and effectively increase the level of mitochondrial membrane potential.To a certain extent,it can effectively reduce the oxidative damage caused by the increase of reactive oxygen species,so that the body can alleviate the oxidative damage through the mitochondrial autophagy pathway.It can be seen that the addition of Terpen-4-ol can greatly improve the actual benefits in the production practice,and can act on the damage target at the molecular gene level and quickly alleviate the protection of the intestine from oxidative damage.