| Intrauterine growth retardation(IUGR)is an important issue in animal production.It is usually caused by multiple factors,such as insufficient food intake,disease,environmental stress or uterine dysfunction.The intestinal tissue is the first organ that is affected when oxidative damage occurs,and oxidative damage of intestine exerts adverse effect on animal’s health.Recent research indicated that intrauterine growth retardation showed a significant negative effect on the structure and function of intestine in piglets.The intestine tissue is responsible for the digestion,absorption and metabolism of nutrients in diet,and is crucial in invasion of pathogenic bacteria,secretion of digestive enzymes,and body health.Therefore,this paper aims to study the antioxidant function of dimethylglycine sodium salt(DMG-Na)and its antioxidant mechanism in jejunum of IUGR weaned piglets through the following experiments.Experiment 1 Free radicals scavenge capacity of DMG-Na in vitroThis study investigated free radicals scavenging capacity of DMG-Na to verify its antioxidant properties in vitro.In this study,water-soluble vitamin E(Trolox)was selected as a standard(the value was set to 100%).The different concentrations(0.08 M,0.16 M,0.24 M,0.32 M,0.40 M,0.48 M,0.56 M,and 0.64 M)of DMG-Na and Trolox were used to measure 1,1-diphenyl-2-trinitropheny-lhydrazine free radical(DPPH),2,2’-Biazine-bis-3-ethylbenzothiazolin-6-sulfonic acid free radical(ABTS+),superoxide anion free radical(O2-),hydrogen peroxide free radical(H2O2)scavenging capacity,and their ferric-reducing antioxidant power(FRAP)ability respectively.The results showed that DMG-Na exerted strong ability to scavenge free radicals(DPPH,ABTS+,O2-,and H2O2)and FRAP ability in vitro(0.08 M-0.64 M),and exerted its best performance at 0.32 M.Experiment 2 DMG-Na relieves the oxidative damage of cellsThis study investigated the effects of DMG-Na on 2,2-azobis(2-methylpropylimidyl)dihydrochloride(AAPH)induced porcine erythrocytes oxidative damage and OAHPx-induced porcine intestinal epithelial cells(IPEC-J2)oxidative damage.In the study of AAPH-induced porcine erythrocytes oxidative damage,2%porcine erythrocytes were divided into three groups:PBS-treated porcine erythrocytes group(CON),AAPH-treated porcine erythrocytes group(CA),and DMG-Na and AAPH co-treated porcine erythrocytes group(DA).Samples were collected from each group after culturing 5 h,and related indicators were measured.In the OAHPx-induced porcine intestinal epithelial cell oxidative damage test,IPEC-J2 cells were divided into three groups:PBS-treated IPEC-J2 cell group(T),OAHPx-treated IPEC-J2 cell group(TO),and DMG-Na and OAHPx co-treated IPEC-J2 cell group(DTO).Samples were collected from each group after culturing 24 h,and related indicators were determined.The results of AAPH-induced porcine erythrocytes oxidative damage test showed that with the extension of time(1-5 h),the porcine erythrocytes hemolysis rate of treatment group(CA group and DA group)showed an upward trend;the porcine erythrocytes superoxide dismutase(SOD)activity,glutathione peroxidase(GSH-Px)activity,and reduced glutathione(GSH)content of treatment group(CON group,CA group,and DA group)showed a downward trend;the porcine erythrocytes malondialdehyde(MDA)content and reactive oxygen(ROS)level of treatment group(CA group and DA group)exerted an upward trend.At the same time point,compared with the CA group,the DA group significantly reduced(P<0.05)porcine erythrocytes hemolysis rate;significantly increased(P<0.05)porcine erythrocytes SOD activity,GSH-Px activity,and GSH content;significantly reduced(P<0.05)MDA content and ROS level of porcine erythrocytes,indicating an improved porcine erythrocytes antioxidant capacity.The results of OAHPx-induced oxidative damage of porcine intestinal epithelial cells study showed that at the concentration of 32 μM DMG-Na,IPEC-J2 cells showed a better cell viability;Compared with the TO group,the DTO group significantly increased(P<0.05)SOD activity(53.09%),GSH-Px activity(36.87%),GSH content(85.69%)and GR activity(90.51%)of IPEC-J2 cell culture,and significantly reduced(P<0.05)MDA content(32.25%)of IPEC-J2 cell culture;significantly increased(P<0.05)SOD activity(31.24’%),GSH-Px activity(52.92%),GSH content(63.03%)and GR activity(69.81%)in IPEC-J2 cells,and significantly reduced(P<0.05)MDA content(36.02%)of IPEC-J2 cells;significantly increased(P<0.05)MnSOD activity(31.12%),GPx activity(121.97%),GSH content(100.00%),GR activity(52.07%),and y-GCL activity(52.73%)of IPEC-J2 cells mitochondrial;significantly reduced(P<0.05)MDA content(36.02%),ROS level(35.20%),PC content(34.72%),8-OHdG content(35.29%),apoptosis level(26.11%)and necrosis level(30.84%)of IPEC-J2 cells,and significantly increased(P<0.05)MMP level(164.29%)of IPEC-J2 cells;significantly improved(P<0.05)gene expression level and their protein content of antioxidant-related genes in IPEC-J2 cells.Experiment 3 DMG-Na relieves the oxidative damage of miceThis test aimed to study the effects of DMG-Na on lipopolysaccharide(LPS)-induced oxidative damage in mice and indomethacin-induced intestinal oxidative damage in mice.In the study of DMG-Na-gavaged dose groping:40 male Kunming mice were randomly divided into 4 groups and 10 mice in each group:saline-gavaged group(CON),9 mg DMG-Na-gavaged group(D-1),12 mg DMG-Na-gavaged group(D-2),15 mg DMG-Na-gavaged group(D-3).Gavaged study was maintained for 28 days.Samples were collected after the gavaged study and related indicators were measured.In the study of LPS-induced mice oxidative damage:30 male Kunming mice were randomly divided into 3 groups(n=10):saline-gavaged+saline-injected group(CS),saline-gavaged+LPS-injected group(CL),and DMG-Na-gavaged+LPS-injected group(DL).Gavaged study was maintained for 28 days.Samples were collected after LPS-injected and related indicators were determined.In the study of indomethacin-induced mice intestinal oxidative damage:80 male Kunming mice were randomly divided into 4 groups of 20 mice each group:saline-gavaged group(CON),indomethacin-injected+saline-gavaged group(IN),DMG-Na-gavaged+indomethacin-injected group(DIN),and indomethacin-injected+DMG-Na-gavaged group(IND).Samples were collected after indomethacin injection and related indicators were measured.The results of DMG-Na-gavaged dose exploration test showed that compared with the CON group,the DMG-Na-gavaged group(D-1 group,D-2 group,and D-3 group)significantly reduced(P<0.05)mice serum alanine aminotransferase(ALT)activity and aspartate aminotransferase(AST)activity;significantly increased(P<0.05)mice serum SOD activity,CAT activity,GSH-Px activity and T-AOC ability,and significantly reduced(P<0.05)mice serum MDA content,This showed that anti-oxidation ability of mice serum was improved;significantly increased(P<0.05)SOD activity,CAT activity,GSH-Px activity and T-AOC ability of mice liver,and significantly reduced(P<0.05)MDA content of mice liver,indicating that the antioxidant capacity of mice liver has been improved.Among the above results,the mice in the D-2 group exerted the best antioxidant capacity.For this reason,we selected 12 mg DMG-Na as the optimal gastric administration dose for subsequent experimental research.The results of LPS-induced mice oxidative damage showed that compared with the CL group,the DL group significantly reduced(P<0.05)mice serum ALT(21.83%)activity and AST level(20.05%)activity;significantly increased(P<0.05)mice hepatic mitochondrial MnSOD activity(91.30%),GSH content(52.00%),GPx activity(31.43%),and GR activity(86.30%);significantly reduced(P<0.05)mice hepatic ROS level(36.38%)and PC content(20.12%),and significantly increased(P<0.05)mice hepatic MMP level(76.74%),indicating that the antioxidant capacity of mice hepatic mitochondria had improved.The results of indomethacin-induced mice intestinal oxidative damage showed that compared with the IN group,the DIN group and IND group significantly improved(P<0.05)mice jejunum morphology and structure;significantly increased(P<0.05)SOD activity(65.63%,53.13%),GSH-Px activity(48.39%,41.94%),GSH1 content(80.65%,77.42%)and GR activity(52.60%,50.00%)of mice serum,and significantly reduced(P<0.05)mice serum MDA content(10.01%,8.40%);significantly increased(P<0.05)mice jejunum SOD activity(81.25%,68.75%),GSH-Px activity(61.11%,55.56%).GSH1 content(81.82%,72.73%)and GR activity(100.00%,81.93%),and significantly reduced(P<0.05)mice jejunum MDA content(16.85%,15.18%),indicating that the antioxidant capacity of mice serum and jejunum improved;significantly increased(P<0.05)mice jejunum MnSOD activity(75.00%,66.67%),GPx activity(66.67%,60.00%),GSH2 content(68.42%,52.63%),GR activity(150.98%,139.22%),and γ-GCL activity(73.10%,68.97%);significantly reduced(P<0.05)mice jejunum ROS level(32.82%,25.13%),PC content(14.76%,12.26%),8-OHdG content(26.34%,23.70%),apoptosis level(26.11%,22.29%)and necrosis level(30.84%,26.51%),and significantly increased(P<0.05)mice jejunum MMP level(100.00%,94.44%),adenosine triphosphate level(ATP)(54.90%,41.18%)and mitochondria copy number(mtDNA)content(63.41%,53.66%);significantly improved(P<0.05)the gene expression level and their protein content of antioxidant-related genes in mice jejunum.There was no significant difference between the DIN group and the IND group among the above indicators,but apparently the DIN group was better than the IND group.Experiment 4:Comparative study on oxidative damage between normal birth weight(NBW)newborn piglets and IUGR newborn pigletsThis study investigated the differences of oxidative damage between NBW newborn piglets and IUGR newborn piglets.Sows with the same lactation parity were selected,and 10 NBW newborn piglets(NBW group)and 10 IUGR newborn piglets(IUGR group)were selected according to their birth weight.Without breastfeeding,samples were collected after coma by electric shock and related indicators were measured.The results indicated that in comparison with the NBW group,the IUGR group significantly reduced(P<0.05)serum SOD activity(27.00%),GSH-Px activity(33.00%)and GSH content(39.00%)of newborn piglets,and significantly increased(P<0.05)serum MDA content(77.00%)of newborn piglets;significantly reduced(P<0.05)jejunum SOD activity(32.00%),GSH-Px activity(45.00%)and GSH content(48.00%)of newborn piglets,and significantly increased(P<0.05)jejunum MDA content(88.00%)of newborn piglets,indicating that the antioxidant capacity of serum and jejunum of newborn piglets decreased;significantly reduced(P<0.05)MnSOD activity(22.00%),GPx activity(38.00%),GSH content(41.00%),and y-GCL activity(29.00%),indicating that the jejunum mitochondrial antioxidant capacity of newborn piglets decreased;significantly increased(P<0.05)jejunum PC content(66.00%),8-OHdG content(71.00%),ROS level(115.00%),apoptotic level(99.00%)and necrosis level(23.00%)of newborn piglets,and significantly reduced(P<0.05)jejunum MMP level(36.00%)of newborn piglets.Test 5 Dietary effects of DMG-Na on growth performance,intestinal structure,intestinal physiological and biochemical of weaned pigletsThis study investigated the dietary effects of DMG-Na on growth performance,intestinal structure,intestinal physiological and biochemical of weaned piglets in order to further confirm that the DMG-Na could help to alleviate oxidative damage in animals.10 NBW newborn piglets(NBW)and 20 IUGR newborn piglets(IUGR)according to their birth weight were obtained after farrowing,and divided into 3 groups after weaning:the NBW weaned piglets fed with basal diet group(N),the IUGR weaned piglets fed with basal diet group(1),and the IUGR weaned piglets fed with basal diet+0.1%DMG-Na group(ID).Feeding was continued for 4 weeks,and samples were collected and relevant indicators were measured after being shocked by electric shock.The results suggested that compared with the I group,the ID group significantly improved(P<0.05)ADG(24.04%),G:F(20.75%),and FBW(15.50%)of weaned piglets;significantly improved(P<0.05)jejunum microvilli structure of weaned piglet;significantly improved(P<0.05)jejunum autophagosomes and mitochondrial morphology of weaned piglets;significantly improved(P<0.05)jejunum villi length,crypt depth,villi width,and villous area of weaned piglet;significantly increased(P<0.05)serum IgA content(40.58%),IgG content(76.52%),and jejunum sIgA content(49.90%)of weaned piglets;significantly increased(P<0.05)jejunum amylase(101.20%),lipase(157.14%),trypsin(110.00%),maltase(17.87%),and lactase activity(29.63%)of weaned piglets.Experiment 6 Dietary effects of DMG-Na on antioxidant function and its antioxidant mechanism in intestine of weaned pigletsThis study investigated the dietary effect of DMG-Na on intestinal antioxidant function and the specific mechanism,thus to improve the intestinal oxidative damage of weaned piglets.In dietary effects of DMG-Na on weaned piglets intestinal antioxidant function study:sows with the same lactation were selected,and 10 NBW newborn piglets(NBW)and 20 IUGR newborn piglets(IUGR)were divided into 3 groups after weaning:NBW weaned piglets fed with basic diet group(N),IUGR weaned piglets fed with basic diet group(1),and IUGR weaned piglets were fed with basal diet+0.1%DMG-Na(ID).Feeding was maintained for 4 weeks,and samples were collected after being shocked by electric shock and the relevant indicators were measured.In the study of antioxidant mechanism:sows with the same lactation were selected,and 10 NBW newborn piglets(NBW)and 10 IUGR newborn piglets(IUGR)were obtained after birth.Samples were collected after coma by electric shock and related indicators were measured without breastfceding.The results of rasing experiment showed that compared with the I group,the ID group significantly increased(P<0.05)serum SOD activity(12.84%),GSH-Px activity(21.71%),GSH content(54.97%),GR activity(106.27%)and CAT activity(163.24%)of weaned piglets,and significantly reduced(P<0.05)serum MDA content(28.93%)of weaned piglets;significantly increased(P<0.05)jejunum SOD activity(43.79%),GSH-Px activity(102.17%),GSH content(88.03%),GR activity(74.15%)and CAT activity(112.31%)of weaned piglets,and significantly reduced(P<0.05)jejunum MDA content(28.92%)of weaned piglets,indicating that the antioxidant capacity of serum and jejunum of weaned piglets improved;significantly increased(P<0.05)jejunum MnSOD activity(50.21%),GPx activity(75.15%),GSH content(102.70%),GR activity(100.96%),and y-GCL activity(52.85%)of weaned piglets;significantly reduced(P<0.05)jejunum ROS level(36.60%),PC content(23.56%),8-OHdG content(29.38%),apoptotic level(38.29%)and necrosis level(29.34%)of weaned piglets,and significantly increased(P<0.05)jejunum MMP level(46.67%),ATP level(80.65%),and mtDNA content(54.90%)of weaned piglets;significantly improved(P<0.05)the gene expression level and their protein content of antioxidant-related genes of weaned piglets jejunum.The results of microRNA sequencing test showed that the IUGR group significantly up-regulated(P<0.05)six intestinal microRNAs and significantly down-regulated(P<0.05)two intestinal microRNAs.Then,we were able to confirmed that microRNA149 acts on PARP2,which in turn affects SIRT1/PGC1 activity and affects related antioxidant enzyme activity and mitochondrial function by transfecting the empty vector or the over-expressed microRNA149 vector into IPEC-J2 cells.On basis of abovementioned,it is concluded that:(1)DMG-Na exerts a strong ability to scavenge free radicals in vitro,and improves the hemolysis of porcine erythrocytes,improves the antioxidant capacity of porcine erythrocytes and IPEC-J2 cells,and relieves oxidative damage.(2)DMG-Na can effectively alleviate the jejunum oxidative damage suffered by oxidative damage in mice.Both the preventive and therapeutic doses have the effect of scavenging excessive free radicals produced in the body,repairing mitochondrial dysfunction,and inhibiting the abnormal expression of stress-related factors,thereby reducing the apoptosis caused by oxidative damage.(3)IUGR induces the activation of PARP2 and affects the antioxidant capacity and mitochondrial function of jejunum in newborn piglets by inhibiting microRNA149/SIRT1/PGC1a network activity.(4)DMG-Na can directly neutralize the excessive free radicals produced in IUGR newborn piglets,and indirectly improve the body’s antioxidant capacity through the microRNA 149/SIRT1/PGC1α network,inhibit the abnormal expression of stress-related factors,and thus regulate the intestine health. |
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