The Effects Of HEP On The Apoptosis Of IPEC-J2 Cells Under Oxidative Stress

Intensive breeding mode causes the oxidative stress of the intestine to increase during the pig production process.Under oxidative stress,the intestinal epithelial absorptive function and barrier function are impaired,resulting in a significant reduction in herd health and a direct impact on production performance.Our previous research work showed that Hericium erinaceus polysaccharides(HEP)can significantly improve pathological changes such as structural damage and decreased permeability of porcine small intestine epithelium caused by oxidative stress.Based on this study,we further explored the effect of Hericium erinaceus polysaccharides on the apoptosis of pig intestinal epithelial cells(IPEC-J2)under oxidative stress,in order to initially clarify the molecular mechanism of HEP protecting small intestine epithelium from oxidative damage.The application in pig breeding provides scientific basis.In this study,we used Hericium erinaceus as material,used water extract-alcohol precipitation method obtained raw polysaccharide,and then used the DEAE-52 purified cellulose to obtained pure polysaccharide.We used a combination of chemical analyses,modern instrumental analysis techniques to identify the purity,monosaccharides,carbohydrate chain conformation and the clearance rate of Hericium erinaceus polysauharides(HEP)for free radical.In addition,we used swine intestinal epithelial cells(IPEC-J2 cells)as the research object,applied H2O2 construction model of oxidative stress,concentration of H2O2 and the best safety effect dose of HEP was determined by MTT assay.Used flow cytometry to determine the apoptosis rate,intracellular ROS level and cell cycle in IPEC-J2 cells.We measured Caspase-3,Caspase-8,Caspase-9,PARP,Cyt-C in oxidative stress-induced IPEC-J2 cells by Elisa Assay.Observed the effect of HEP on the expression of Bcl-2、Bax、Bad、TNFR1、FAS、FASL、protein in IPEC-J2 cells under oxidative stress by Western-blot.Results are as follows:(1)By the analysis of gel permeation chromatography(GPC),the HEP were composed of Mannitol,rhamnose,glucose,galactose and(2)fucose with a ratio of 0.8:0.1:9.7:10:1.6.Results of chemical analyses showed that HEP didn’t contain starch,contained a small amount of uronic acid and polyphenols.UV scanning showed that HEP didn’t contain nucleic acids and proteins.infrared scanning showed that HEP had the characteristic absorption peak of polysaccharide.In short,results indicated that HEP have high purity and good traits,therefore meet the requirements of the follow-up experiments.(3)Were measured the clearing effect of HEP on·OH、O2-·、DPPH、ABTS free radical and iron ion total reduction.The results showed that HEP had strong antioxidant capacity in vitro.(4)Determination of effects of different concentration of H2O2 and HEP on the proliferation rate of IPEC-J2 by MTT method,the optimal concentration of H2O2 concentration was 0.4 mM.10～350 μg/mL was the safe concentration of HEP.Finally,we selected 10 μg/mL,50 μg/mL and 100 μg/mL as the low,medium and high doses of group.(5)We used flow cytometry to detect the effect of HEP on the apoptosis rate,ROS content and cell cycle of IPEC-J2 cells under oxidative stress.The results showed that the apoptosis rate of IPEC-J2 cells was significantly decreased under the oxidative stress condition with HEP intervention(P<0.05),indicating that 10-100 μg/mL HEP can significantly inhibit the apoptosis of IPEC-J2 cells induced by H2O2.The content of ROS in the model group was significantly lower than that in the model group(P<0.01),indicating that HEP had obvious purging effect on ROS produced by H2O2 injuring cells.The number of cells in G2 phase was significantly lower than that in the model group(P<0.05),indicating that HEP has a mitigating effect on cell G2 arrest.It was shown that HEP has a significant effect on inhibiting the apoptosis of IPEC-J2 cells under oxidative stress.(6)Compared with the control group,H2O2 significantly up-regulated the expression of pro-apoptotic proteins:Bax,Bad,TNFRl,FAS and FASL(P<0.01),and significantly down-regulated the expression of anti-apoptotic protein Bcl-2(P<0.01).The concentration of HEP at 10-100 μg/mL can inhibit the protein secretion of Bax,Bad,TNFR1,FAS,and FASL under oxidative stress to a certain extent,and at the same time promote the expression of Bcl-2 protein with a certain amount,And there was a certain amount of relationship.(P<0.05).Therefore,HEP inhibited the expression of proapoptosis protein in IPEC-J2 cells under oxidative stress to some extent,alleviated the damage caused by apoptosis,and increased the cell survival rate.In conclusion,this study preliminary clarified the physicochemical properties of HEP such as monosaccharide composition and spectral absorption characteristics.It is clear that HEP has significant antioxidation in vitro and found that HEP can inhibit the apoptosis of IPEC-J2 cells caused by oxidative stress.The mechanism is closely related to the endogenous mitochondrial pathway and the exogenous death receptor pathway.