| The present study was conducted to investigeate the effects of N-acetylcysteine (NAC) on growth and viability of intestinal porcine epithelial cells and therelated mechanisms.1Effect of NAC on the growth and vitality of IPEC-1.(1) The IPEC-1cells were cultured for4days in cysteine-free Dulbecco’smodified Eagle’s-F12Ham medium (DMEM-F12) containing0,100,200,400μMNAC or50μM Cystine (Cyss).(2) Cells were treated with cysteine-freeDMEM-F12containing0,50,100,200μM Cyss or NAC. Cell numbers andproliferation were determined for1,2,3or4days. The results revealed:(1) the cellnumbers treated with NAC (100μM,200μM,400μM) or50μM Cyss weresignally increased compared with the control (P<0.01), and up to a maximumnumber after3days.(2) The cell viability of NAC (100μM,200μM,400μM)groups or50μM Cyss group was significantly increased compared with the control(P<0.01), and the group of cells treated with NAC was significantly increased alsothan Cyss treatment (P<0.01).2The effects of NAC on the genes expression relative to the function ofIPEC-1and cell growth.IPEC-1cells were treated with the cysteine-free DMEM-F12containing0,50μM,100μM,200μM Cystine (Cyss) or100μM NAC. The medium was changedeveryday.3days later, expression and activation of mammalian target of rapamycin(mTOR), and epidermal growth factor receptor (EGFR) signaling and some othermolecules were determined by RT-PCR. As well as4E-BP1protein expression onthe control and100uM NAC group. Result: compared with the control group, theexpression of AR、Ras、ERK、Akt、ZO-1、SGLT、CFTR were significantly increasedin IPEC-1cells of100μM NAC group (P<0.01). While the expression of4E-BP1were significantly reduced (P<0.01).(2) no effect on the4E-BP1protein expressionon the100mM NAC treatment compare with the control (P<0.05).3. Effect of GSH on the regulation of NAC on IPEC-1cell growth.(1) IPEC-1cells were incubated with the cysteine-free DMEM-F12containingL-buthionine sulfoximine (BSO), or diethyl maleate (DEM). Cell numbers andviability were determined.(2) IPEC-1cells were treated with cysteine-freeDMEM-F12containing BSO, DEM,100μM NAC, Cys, Cyss, GSH, or GSH-EE.Cell numbers and viability were determined.(3) IPEC-1cells were treated withOPA, and the concentrations of GSH and Cys in IPEC-1cells were measured byHPLC. Results:(1) Compared with the control group, BSO and DEM treatmentsinhibited the cells growth and viability effectively (P<0.01), and the half-number-inhibiting dosage of BSO and DEM regarding IPEC-1cells were10μM and20μM.(2) NAC, Cys, Cyss, GSH and GSH-EE administration couldpromote the growth and proliferation of IPEC-1cells, and the IPEC-1cells numberand cell viability in NAC, Cys and Cyss groups were higher than GSH and GSH-EEgroups.(3) Compared with the control group, the concentration GSH in IPEC-1were significantly increased by NAC or GSH-EE supplementation (P<0.01).(4)GSH-EE increased the concentration of Cys, whereas NAC or DEM treatmentdecreased the Cys concentration (P<0.01).4Effect of NAC and LPS on cell growth and cell viability of IPEC-1.The IPEC-1cells were treated with0,10μg/mL LPS,100μM NAC or10μg/mLLPS+100μM NAC. Cell numbers and viability were determined on1,2,3, or4days of the trial. The genes expression was also determined after3days. Resultsshowed that:(1) compared with the control group, the addition of NAC or both ofNAC and LPS increased the cell numbers and improved cell viability (P <0.01).The effect of NAC on the simulation of IPEC-1cells growth and cell viability.Compared with LPS group, the NAC+LPS treatment inreased the cell numberssignificantly (P<0.01).Conclusion:(1)100μM NAC can improve the the growth and cell viability ofIPEC-1cells by EGFR signaling pathway and improve the concentration of GSH inIPEC-1partly.(2) The optimum volume of NAC was100μM in IPEC-1cellsculture.(3) There was no effect on LPS (O55: B5) separately addition in IPEC-1cells culture. |
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