| The intestine is an important nutrient digestion and absorption organ in piglets.In addition,it also plays a defensive role to the body and piglet’s health.The piglets’gut is particularly vulnerable to oxidative stress during weaning.Therefore,the prevention and treatment of intestinal epithelial cells’ oxidative stress and the improvement of their antioxidant capacity are important to solve the intestinal oxidative stress and improve the health of piglets.Tea tree oil is a natural plant essential oil with anti-oxidant effect.Most of it is used in medical treatment and cosmetics.It has the ability to improve the antioxidant capacity of weaned piglets.This experiment first used H2O2 to stimulate porcine ileal epithelial cells to establish an oxidative stress model,then explored the effect of tea tree oil on the regulation of oxidative stress in piglets ileal epithelial cells,and further affected its Nrf2 signaling pathway.The result is as follows:Experiment 1:Establishment of oxidative stress model of porcine ileal epithelial cells IPI-2IThe establishment of cellular oxidative stress model is the basis for studying the antioxidant mechanism of cells.Therefore,the mature oxidative stress model of porcine ileal epithelial cells was stimulated with H2O2 to establish oxidative stress model.Different concentrations of H2O2 were used to stimulate IPI-2I to detect cell viability at different times.After preliminary screening of the stimulation concentration and time,the relative production of oxygen free radicals(ROS),malondialdehyde(MDA)content and antioxidant enzymes in IPI-2I cells were detected vitality.The results showed that:1)With the increase of H2O2 stimulation time and the increase of concentration,the viability of IPI-2I cells decreased significantly(P<0.05),and the production of ROS and MDA increased significantly compared with the control group(P<0.05).The activities of superoxide dismutase(SOD),total antioxidant capacity(T-AOC),catalase(CAT),and glutathione peroxidase(GSH-Px)decreased significantly(P<0.05).Compared with the control group,200 μmol/L H2O2 stimulated IPI-2I cells for 6 hours,and the cell viability decreased significantly(P<0.05);the contents of ROS and MDA increased significantly(P<0.05);SOD,T-AOC,CAT,GSH-Px enzyme activities decreased significantly(P<0.05).It was determined that 200 μmol/L H2O2 was used to stimulate IPI-2I cells for 6 h to establish an oxidative stress model.Experiment 2:Effect of tea tree oil regulation of oxidative stress in IPI-2I cellsExplore to understand that tea tree oil can alleviate the oxidative damage of porcine ileal epithelial IPI-2I cells.Different concentrations of tea tree oil were used to treat IPI-2I cells,and the safe concentration of tea tree oil on IPI-2I cells was determined to be less than 0.05%.Based on the previously established oxidative stress model of IPI-2I cells,the study was divided into five groups,namely the control group(CON group,without H2O2 and tea tree oil),and the oxidative stress model group(H2O2 group,with 200 μmol/L H2O2),low-concentration tea tree oil group(HLT group,0.025%TTO+200 μmol/L H2O2),medium-concentration tea tree oil group(HMT group,0.0375%TTO+200 μmol/L H2O2),high-concentration tea tree oil group(HHT group,0.05%TTO+200 μmol/L H2O2).The results showed that the cell viability of the HLT and HMT groups was significantly higher than that of the H2O2 group(P<0.05),the relative production of ROS and the content of MDA were significantly lower than that of the H2O2 group(P<0.05),and the content of SOD,T-AOC and GSH-Px were significantly higher than that of the H2O2 group(P<0.05),the content of CAT in HLT group was significantly higher than that in H202 treatment group(P<0.05).The expression of Nrf2 in HLT and HMT group was significantly higher than that in H2O2 group(P<0.05),the expression of Nrf2 in HLT treatment group was significantly higher than that in HMT group(P<0.05);the expression of CAT in HLT,HMT and HHT group was significantly higher than that in H2O2 group(P<0.05);the expression of SOD in HLT and HMT group was significantly higher than that in H2O2 group(P<0.05).These prove that tea tree oil can increase cell viability in IPI-2I cells under oxidative stress,reduce the relative production of ROS in cells,reduce MDA content,increase antioxidant enzyme activity,and increase the expression of some antioxidant-related genes,indicating that tea tree oil can alleviate the oxidative stress induced by H2O2 in IPI-2I cells,and the concentration of 0.025%TTO is the best.Experiment 3:The mechanism of tea tree oil on the regulation of oxidative stress in IPI-2I cellsTea tree oil can alleviate the oxidative stress injury of IPI-2I cells.In order to further understand its mechanism of action,then examined the effect of tea tree oil on the Nrf2-ARE antioxidant pathway in pig ileal epithelial cells.The study was divided into 4 groups:control group(CON group,without adding H2O2 and tea tree oil),oxidative stress model group(H2O2 group,adding 200 μmol/L H2O2),tea tree oil group(TTO group,0.025%TTO)and low concentration tea tree oil group(HLT group,0.025%TTO+200 μmol/L H2O2).The results showed that the expression of Nrf2 gene in intestinal epithelial cells of the TTO group was significantly higher than that of the H2O2 group and the HLT group(P<0.05);the expression of Nrf2 in the HLT group was significantly higher than that of the H2O2 group(P<0.05).The expression of HO-1 and NQO1 in the TTO group were significantly higher than those in the CON and H2O2 groups(P<0.05);the expressions of HO-1,NQO1,and GCLC in the HLT group were significantly higher than the H2O2 group(P<0.05),and the expression of GCLM was significantly higher than the CON group(P<0.05).The protein expressions of NQO1,HO-1 and Nrf2 in HLT and TTO group were significantly higher than those in H2O2 group(P<0.05),and the protein expressions of NQO1,HO-1 and Nrf2 in HLT group were significantly lower than that in CON group(P<0.05).In summary,the results showed that tea tree oil can alleviate the problem of decreased expression of antioxidant genes and protein caused by oxidative stress,thereby alleviating the damage caused by oxidative stress. |
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