Injury Of Bisphenol A On Small Intestine And Effects Of Glutathione Synthesis In Small Intestinal Health

In this thesis,four experiments were conducted to investigate effects and mechanisms of bisphenol A(BPA)on small intestine injury and glutathione precursor glutamine to relieve BPA injury,and to study the effect of cysteine synthesizing glutathione on piglets growth and intestinal development under physiological conditions.(1)Exp.1 determined effects and mechanisms of BPA on rat intestinal epithelial cell line-6(IEC-6)and intestinal porcine epithelial cell-jejunal 2(IPEC-J2).Cell viability and cell proliferation were measured in IEC-6 cells treated with different concentrations of BPA(0,0.1,0.2,0.3,0.4 and 0.5 mmol/L)for 6 h or 24 h.The time and concentration of BPA were determined to be 0.4 mmol/L for 6 h and 0.2 mmol/L for 24 h.The results showed that 0.4 mmol/L BPA treatment for 6 h significantly increased the apoptosis of IEC-6 cells,increased the release of lactate dehydrogenase,and reduced the transmembrane resistance of monolayer cells(P<0.05).0.2 mmol/L BPA treatment for 24 h significantly increased the content of malondialdehyde and reactive oxygen species,decreased the activity of superoxide dismutase and the content of glutathione in cells(P<0.05).These results were verified on IPEC-J2 cells.Exp.1 showed that BPA caused oxidative stress,reduced the content of glutathione,and then damaged intestinal epithelial cells.(2)On the basis of experiment 1,the exp,2 determined the effect and mechanism of BPA on mice’s small intestine.The experiment used 40 6 week-old C57BL/6 male mice,which were divided into four treatments.The same amount of corn oil,5,50 or 500 mg/kg-BW/d BPA was continuously administered for 4 weeks.The results showed that the body weight,survival rate and the ratio of the small intestinale length to the body weight of the 500 mg/kg-BW/d BPA treatment group was significantly lower than that of the control group(P<0.05).And BPA caused an increase in mitochondrial fusion gene Mfn2 mRNA level(P<0.05).This indicted that BPA impaired mitochondrial function,and may lead to oxidative stress.BPA increased the proinflammatory cytokines IL-1β,IL-6 and TNFa mRNA levels(P<0.05)and decreased anti-inflammatory cytokine IL-10 mRNA level(P<0.05).The proteomics analysis of jejunal mucosal protein expression was performed in 500 mg/kg·BW/d BPA-treated and control groups.A total of 5,840 proteins were identified,of which 70 were differentially expressed(P<0.05&fold change<0.83 or>1.2).Functional enrichment and bioinformatics analysis of the differentially expressed proteins showed that BPA reduced the proliferation of intestinal cell,disrupted the immunoregulatory function of the jejunal mucosa leading to inflammatory responses,and increased glutathione metabolism.This was consistent with the results of experiment one.Exp.2 showed that BPA leaded to intestinal damage by causing the metabolism of glutathione.(3)Exp.3 used IEC-6 cells and C57BL/6 mice to investigate the alleviating effect of glutamine as a precursor of glutathione on BPA injury.IEC-6 cells were pretreated with BPA for 12 h and then treated with 4 mmol/L glutamine for 12 h.Mice were fed with 0,2%or 4%glutamine,and 500 mg/kg·BW/d BPA was given for 4 weeks to do proteomic analysis of jejunal mucosa.The results showed that glutamine significantly alleviated the decrease of IEC-6 cell viability and the transmembrane resistance of monolayer cells induced by BPA(P<0.05).In glutamine group,the body weight and small intestine weight of the mice were significantly higher than those of BPA group.And 4%glutamine significantly improved small intestine length and intestinal villus morphology(P<0.05).Proteomic analysis showed that glutamine changed proteins,signaling pathways,and biological processes related to mucosal immunity,absorption and transport of nutrients,cellular processes and glutathione metabolism to alleviate BPA-induced jejunal mucosal damage in mice.Glutamine synthesizing glutathione relieved BPA-induced intestinal damage.(4)Exp.4 used in vitro and in vivo assays to investigate effects of glutathione precursor cysteine on the growth and intestine development of piglets under physiological conditions.Firstly,IPEC-J2 cells were treated with cysteine-free medium,followed by addition of methionine,sodium pyruvate and glutathione,and then the cell viability and the transmembrane resistance of monolayer cells were determined.Secondly,96 piglets(10.98 ± 0.28 kg)were selected and divided into four treatments.The control group was feeded NRC(2012)recommended dietary with 0.48%methionine and 0.21%cysteine.The experimental group diet with SID methionine level was 0.36%,and the cysteine levels were 0.21%,0.32%(NRC 2012)and 0.43%.The trial period was 28 d.The results showed that cysteine deprivation caused significant decrease in IPEC-J2 cell viability and monolayer cell transmembrane resistance(P<0.05).The addition of glutathione reversed the decrease in cell viability caused by cysteine deprivation(P<0.05).Both the daily weight gain and the final weight were the highest(P<0.05),when methionine and cysteine satisfied NRC(2012)requirements,respectively.And a certain level of cysteine had the effect on increasing feed intake(P<0.05),but feed to gain ratio did not change between all the groups.Different dietary levels of cysteine caused changes in serum biochemical parameters(P<0.05).A sufficient dietary cysteine could significantly increase the content of glutathione,GSH/GSSG and the expression of tight junction proteins in jejunum(P<0.05),but not jejunal mucosal morphology.In summary,BPA damages the small intestine by causing the depletion of glutathione,and the addition of precursor of glutathione,glutamine and cysteine,could maintain the health of the small intestine under the conditions of injury and physiology,respectively.