Protective Effect Of Alfalfa Saponins Against H₂O₂ Induced Oxidative Stress Injury In IEC-6 And IPEC-J2 Cells And Its Mechanism

In this study,we investigated the protective effect of alfalfa saponins on oxidative damage of small intestinal epithelial cells induced by H₂O₂ in rats and piglets and its mechanism.The test results are as follows:Exp 1: Effects of alfalfa saponins on H₂O₂ induced oxidative damage in IEC-6cells.The rats were divided into control group,25 μg/m L AS group,40 μg/m L AS group,model H group,25 and 40 μg/m L AS+H group.In the control group,25 and40 μg/m L AS group were incubated with 0,25 and 40 μg/m L alfalfa saponins for 24 h,model H group,25 μg/m L AS+H group,40 μg/m L AS+H group was treated with150 μmol/L H₂O₂ for 24 h after preincubation to simulate the oxidative stress injury of IEC-6 cells.The ratio of LDH,antioxidant enzyme and oxidized product MDA and GSSG/GSH were measured by microplate reader.The apoptotic cells of IEC-6 cells were detected by flow cytometry.Western blot was used to detect related protein expression of the mitochondrial apoptotic signaling pathway and MAPK signal pathway.The results showed that（1）Compared with the control group,the cell viability was significantly decreased and the release of LDH was significantly increased in the model group H;Compared with model group H,the cell survival rate and the release of LDH of 25 and 40 μg/m L AS+H group was significantly decreased（P<0.01）.（2）The content of MDA of 25 and 40 μg/m L AS group was significantly higher than that control group（P<0.01）,and the activity of antioxidant enzyme increased（P>0.05）;compared with the model group,the antioxidant activity of the cells in the 25 and 40 mg/m L AS+H group were significantly increasing（P<0.01）,the content of MDA in the oxidized product were decreased,the amount of GSH increased and the ratio of GSSG/GSH were significantly decreased（P<0.01）.（3）Compared with the control group,the intracellular apoptosis rate and pro-apoptotic Bax were decreased in 40 μg/m L AS group（P<0.05）,and Caspase-3 and Caspase-9 were significantly decreased,The expression of Bcl-2 in the model group H was significantly higher than that in the control group（P<0.01）.The expression of Bcl-2and the expression of Bcl-2 in the model group H were significantly higher than those in the control group（P<0.01）25 and 40 μg/m L AS+H group were significantly lower than those of model H group（P<0.05）.The apoptosis rate and the expression of Cleased caspase-3,Caspase-9 and Bax were significantly decreased and the expression of Bcl-2 were significantly increased（P? 0.01）.（4）Compared with the control group,the levels of ERK1/2,JNK and P38 MAPK phosphorylation in the 40 μg/m L AS group were significantly decreased,and the levels of ERK1/2,JNK and P38 MAPK phosphorylation in model group H were significantly increased（P<0.01）.The levels of ERK1/2,JNK and P38 MAPK in 25 and 40 μg/m L AS+H groups were significantly higher than those in model H group（P<0.01）.Conclusion Alfalfa saponins can protect oxidative stress injury induced by H₂O₂.The mechanism is that it can affect the mitochondrial apoptosis pathway and MAPK signal pathway,and realize the anti-oxidation and anti-apoptotic function.Exp 2: Protective effects of alfalfa saponins on oxidative damage induced by H₂O₂ in IPEC-J2 cells.The test group was divided into control group,200 μg/m L AS group,model H group and 200 μg/m L AS+H group.200 μg/m L AS group and 200 μg/m L AS+H group were preincubated with 200 μg/m L alfalfa saponin for 24 h,model H group and200 μg/m L AS+H group were treated with 300 μmol/L H₂O₂ 24 h,simulated oxidative stress injury of IPEC-J2 cells.The LDH release,antioxidant enzyme and oxidized product MDA content were measured by microplate reader.The results showed that（1）Compared with the control group,LDH release in the 200 μg/m L AS group decreased（P>0.05）,LDH release was significantly increased in model H group（P? 0.01）,LDH release of 200 μg/m L group AS+H was significantly higher than the model H group（P? 0.01）.（2）Compared with the control group,SOD,GSH-PX and CAT of 200 μg/m L AS group activity increased,the content of MDA of 200 μg/m L AS group decreased（P>0.05）;the expression of H cell SOD,GSH-PX model group and CAT were significantly lower than the control group,the content of MDA oxidation products was significantly lower than the control group（P? 0.01）the expression of SOD,GSH-PX and CAT;200 μg/m L AS+H group was significantly higher than that of H model group,MDA content of oxidation products was significantly lower than that of model group H（P? 0.01）.The results showed that alfalfa saponins could enhance the antioxidant effect of IPEC-J2 cells and protect the oxidative damage of cells.