The Mitigation Role Of Dietary Oregano Essential Oil In Intestinal Oxidative Stress Induced By Transportation

Pig transport is an indispensable part in pork production process.Pigs are exposed to different stressors when transported to the slaughter house,increasing the risk of reduced welfare for the animals and economic losses related to carcass damage,inferior meat quality and even mortality.Previous studies from our laboratory have found that dietary oregano essential oil(OEO)supplementation improved pig transport stress through protection intestine oxidative stress.However,how OEO acts in the intestine oxidative stress of pig is still unclear.In the study,we aimed that OEO promotes intestine oxidative status.We firstly to evaluate the antioxidative effects of OEO and their underlying molecular mechanisms in porcine small intestinal epithelial(IPEC-J2)cells and RAW264.7 cells.Subsequently,we investigate the effects of OEO on transport stress in pigs after transportation,and we further studied the potential mechanisms by which OEO modulates the oxidative stress of the intestinal in pigs.The main contents and results are as fllows:In the first part,we investigate the oxidative damaging effects of hydrogen peroxide(H2O2)on growth of porcine small intestinal epithelial(IPEC-J2)cells in vitro,determine whether OEO has an antioxidative effect(restores the cell redox state during oxidative stress)on IPEC-J2 cells,and,if OEO does have an antioxidative effect,to elucidate the underlying molecular mechanisms.The main results are as follows: We found that OEO(2.5-10 ?g/ml)treatment prior to H2O2 exposure increased the cell viability and prevented lactate dehydrogenase(LDH)release into the medium.H2O2-induced ROS and MDA were remarkably suppressed by OEO.OEO(2.5-10 ?g/ml)dose-dependently increased mRNA and protein levels of the nuclear factor-erythroid 2-related factor-2(Nrf2)target genes Cu/Zn-superoxide dismutase(SOD1)and g-glutamylcysteine ligase(GCLC,GLCM),as well as intracellular concentrations of SOD1 and GSH.OEO(2.5-10 ?g/ml)also increased intranuclear expression of Nrf2 and the activity of an antioxidant response element reporter plasmid in IPEC-J2 cells.To investigate OEO-mediated nuclear translocation of Nrf2,the amount of cytosolic and nuclear Nrf2 protein was determined in the respective fractions of IPEC-J2 cells.The results showed increased nuclear Nrf2 and decreased cytosolic Nrf2 with 10 ?g/ml of OEO alone.For further confirmation,nuclear import of Nrf2 in control and treated cells was monitored by immunofluorescence.The OEO(2.5-10 ?g/ml)induced expression of Nrf2-regulated genes and increased SOD1 and glutathione concentrations in IPEC-J2 cells were reduced by Nrf2 small interfering(si)RNAs,counteracting the protective effects of OEO against oxidative stress in IPEC-J2 cells.In the second part,although intestinal microbiota disorders have been implicated connection with intestine oxidative stress,the underlying molecular mechanisms remain to be elucidated.Here,we studied the effects of ROS derived from the RAW 264.7 cells on LPS induced oxidative stress in IEC-18 cells.The main results are as follows: RAW264.7 cells reduced the trans-epithelium electrical resistant(TEER)and increased the fluorescein permeability of IEC-18 cells which was induced by LPS.Coincidently,RAW264.7 cells also inhibited the tight junction protein of ZO-1 and Occludin expression in IEC-18 cells gene and protein levels induced by LPS.For further confirmation,the ZO-1 and Occludin protein in control and treated cells was monitored by immunofluorescence.LPS induced generation of the ROS and MDA in IEC-18 cells,and were enhanced by the co-culture with RAW264.7 cells,while a decrease in the level of glutathione/striatal oxidized glutathione(GSH/GSSG)was also observed.The IEC-18 cells Caspase-9,Caspase-3 and Bax protein improved by LPS were further ehbanced by co-culture with RAW264.7 cells.Subsequently,we confirmed that the apoptosis of IEC-18 cells by PI staining flow cytometry method and PI fluorescence intensity method.RAW264.7 cells increased the abundance of NF-?B p65 protein in the nucleus of IEC-18 cells induced by LPS.This results is accordance with the greater increase expression of inflammatory cytokines in IEC-18 cells co-cultured with RAW264.7 cells which was activated by LPS.NADPH oxidase catalytic subunit(NOX2)of RAW264.7 cells were inhibited by transfection with NOX2 siRNA.The enhancement of RAW264.7 cells on oxidative stress of IEC-18 cells induced by LPS were reduced by NOX2 siRNA,counteracting the damage effects of RAW264.7 on oxidative stress in IEC-18 cellsIn the third part,we investigate the oxidative stress and inflammation response effects of LPS on IEC-18 cells in vitro,determine whether OEO has an antioxidative and antiinflammation effect on IPEC-J2 cells,and,if OEO does have an antioxidative and antiinflammation effect,to elucidate the underlying molecular mechanisms.The main results are as follows: The production of Interleukin-1?(IL-1?),Interleukin-6(IL-6)and Tumor necrosis factor-?(TNF-?)in RAW264.7 cells were markedly increased in response to LPS compared with the control group,whereas pretreatment with OEO(2.5-10 ?g/ml)significantly inhibited IL-1?,IL-6 and TNF-? secretion.Furthermore,OEO inhibited the mRNA levels of IL-1?,IL-6 and TNF-? in RAW 264.7 cells stimulated with LPS.OEO(2.5-10 ?g/ml)suppressed LPS-induced phosphorylation of AKT and MAPKs,while decreasing the NF-?B p65 concentration in a dose-dependent manner in the nucleus).Pretreatment with OEO(2.5-10 ?g/ml)inhibited ROS and MDA levels in RAW264.7 cell induced by LPS,as well as increases the GSSG: GSH ratio in the cells.Treatment with NOX2 siRNA suppressed the LPS-induced increase NADPH oxidase activity and ROS production compared with the control cells.Moreover,Treatment with NOX2 siRNA inhibited the mRNA expression of IL-1?,IL-6 and TNF-?.Pretreatment with OEO significantly inhibited NOX2 mRNA and protein expression induced by LPS in RAW 264.7 cells.Moreover,pretreatment with OEO also resulted in a reduction in NADPH oxidase activity in response to LPS in RAW 264.7 cells.In the fourth part,we compared the effects of dietary oregano essential oil(OEO),quercetin or vitamin E(vit E),on the tansport stress,antioxidant status and intestine injury of pigs after transportation.The effects of dietary OEO on growth performance were also determined.A total of 255 finishing pigs were randomly assigned to one of three treatment groups.Pigs consumed the basal diet(control)or the basal diet supplemented with vit E(200 mg/kg),or OEO(25 mg/kg)4 weeks.After this period,108 pigs were transported for 5 h before slaughter.The main results are as follows: The OEO-treated pigs showed decreased endotoxin level in serum,and increased villus height and expression of Occludin and zonula occudens-1 in the jejunum.The OEO-treated pigs had a lower population of Escherichia coli in the jejunum,ileum and colon than the control.Pigs offered diets containing OEO had lower levels of ROS and higher levels of T-SOD in the jejunum compared with the control group.The live body weight loss was less in the OEO group after 5 h transportation than in the control group.The hot carcass weight and dressing percentage were higher in the OEO group after 5 h of transportation than in the control group.After slaughter,the pH value at 45-min postmortem and Opto-star value(meat color)at 24-h postmortem increased in the vit E or OEO groups.The muscle of pigs from the OEO groups produced lower 24-h drip loss values than that of pigs from the control group.The OEO groups had reduced levels of MDA(malondialdehyde)and ROS(reactive oxygen species)in serum,muscle and liver,while the vit E group had reduced levels in serum only.The OEO groups also had increased levels of Gpx(glutathione peroxidase)and T-SOD(total superoxide dismutase)activity in serum and liver.Conversely,there were no differences between the vit E and control groups in Gpx or T-SOD activities.Furthermore,the OEO groups had a higher average daily gain,and the OEO group also had a lower feed intake/gain.In conclusion,our findings provide direct evidence of the effects of RAW 264.7 derived ROS on LPS induced oxidative stress in IEC-18 cells,which is transduced in part by NOX2/ROS signaling.The OEO protects against oxidative stress by inducing Nrf2 and related antioxidant enzymes,or through inhibition of NOX2 expression.On the other hand,under the transportation procedures used in this study,pigs fed OEO exhibited reduced live weight shrinkage and higher hot carcass weights and dressing percentages after transportation than pigs fed a control diet.The benefical effect of OEO on transport stress of pigs might be achieved by the OEO modulate intestinal oxidative stress.