Protective Effect Of Quercetin On Intestinal Porcine Enterocyte Cells Exposure To Hydrogen Peroxide And Lipopolysaccharide

Weaning stress,severe abnormal-environmental stimulation or poisoning,etc.during the breeding process have important influences on porcine intestinal health,such as inducing intestinal oxidative damage and inflammatory reaction,then to reduce the growth performance of pigs,which brings huge economic losses to the pig industry.Therefore,it is of great significance to explore the natural products with low toxicity and no harmful residues,as veterinary drugs or feed additives,to protect pig's intestines.Quercetin?Que?,a common flavonoid,shows antioxidant,anti-inflammatory activities in a variety of model cells from human and mice,et al.,but there is no detailed report about regulative effect of Que on the health of pig intestines.Thus,the effect of Que on the proliferation of intestinal porcine enterocyte cells?IPEC-J2?,and the protective effect and possible mechanism of Que on the porcine intestinal epithelial cells under oxidative damage and inflammatory response model establised by hydrogen peroxide?H2O2?and lipopolysaccha-ride?LPS?in vitro,respectively,were investigated.The content of Que was confirmed by UV spectrophotometry and the morphology of IPEC-J2 was preliminarily identified by cell morphology comparison and immuno-fluorescence method.The effect of Que on proliferation of IPEC-J2 was detected by MTT,flow cytometry and Real-time PCR,and the effect of Que on IPEC-J2 repair was determined by cell scratch test.MTT assay was applied to detect the effect of Que on viability of cells exposed to H2O2,the activity of LDH in cell culture supernatant was measured by 2,4-dinitrophenylhydrazine method,and the morphology of cells was observed under microscope.Flow cytometry was used to detect the apoptosis rate,cell cycle,intracellular ROS,and??m in H₂O₂-exposed cells after Que treatment and the expression levels of apoptosis-related proteins were detected by Western-blot.The effects of Que on MDA content and SOD and CAT activities in cells with low-degree oxidative damage were assayed by colorimetry,and the effect of Que on expression of Claudin-1 and Occludin in cells with low-degree damage was also detected by Western-blot.MTT assay was conducted to determine the viability of cells exposure to LPS after Que treatment,the morphology of cells was also observed under microscope,NO content in culture supernatant was detected by Griess assay,and Real-time PCR was used to detect the mRNA expression levels of IL-6 and IL-8.The results showed that the average content of test drug Que was 97.97%,the cell morphology was round-fusiform and oval,which was basically same to that of IPEC-J2 cells reported,and the expression of CK-18 protein was found in the cells.And 1.25 to 5?g/mL Que significantly increased the viability of IPEC-J2 cells within 24 h,and the most obvious proliferation was observed at 9 h.Que with 5?g/mL treating cells for 24 h propelled cells into S phase.Que inhibited the relative expression levels of P27 and P21,but could not promote the repair of cell injury in scratch test.Pretreatment with Que for 2 h then incubation with 375?M H₂O₂ for 22 h,pretreatment with Que for 20 h then culture with 750?M H₂O₂ for 4 h,co-treatment with Que and H₂O₂ for 24 h,pretreatment with Que for 3 h then treatment with 750?M H₂O₂ for 1 h,2 h and 4 h,as well as pretreatment with Que for 3h then incubation with 1000?M H₂O₂ for 4 h,all could significantly increase the viability of IPEC-J2 reduced by H₂O₂.Pretreatment with Que could dramatically improve the abnormal morphological changes induced by 750?M H₂O₂ and inhibit the release of LDH from cells.Pretreatment with Que markedly reduced the apoptosis rate,the ROS level,and the proportion of G0/G1 cells,increased the total proportion of S phase and G2/M phase cells,??m,and alleviated Bax/Bcl-2 ratio,of IPEC-J2 treated with H₂O₂.Pre-incubation with Que for 3 h markly decreased the intracellular MDA content and SOD activity under the condition of low degree of oxidative damage?after exposure to 750?M H₂O₂ for 2 h?,and had no obvious effect on the activity of CAT and the expression of Claudin-1 and Occludin,but it showed a slight up-regulative effect on the protein expression of Claudin-1 and Occludin.The treatment of LPS of 1 to 10?g/mL had no significant effect on the viability of IPEC-J2,while Que obviously enhanced the cell viability under LPS exposure,and Que or LPS also showed no effect on NO content in the supernatant of IPEC-J2,but Que could suppress the mRNA expression of IL-6 and IL-8,under LPS exposure.The investigation suggests that content of Que test drug is high,the cells in this study is epithelial cells and morphology of cells is basically consistent with the reported IPEC-J2.Low concentration of Que can promote the proliferation of IPEC-J2,and its proliferation effect is related to the entry of G1 phase into S phase and the inhibition of P21 and P27expression,but Que does not have the function of scratch injury repair.Que at different treatment time can decrease cell death caused by H₂O₂.In addition,Que relieved the apoptosis,which is related to the inhibition of the endogenous apoptosis pathway,and inhibited cell arrest in G0/G1 of IPEC-J2 induced by H₂O₂.Besides,Que can regulate the redox system of IPEC-J2 under low degree injury caused by H₂O₂.There is no obvious change in expression of Claudin-1 and Occludin of the cells with low degree damage,while Que may have a potential up-regulative effect on barrier proteins Claudin-1 and Occludin.Moreover,Que inhibits LPS induced IPEC-J2 inflammatory response,which suppresses the expression of pro-inflammatory genes in IPEC-J2 exposed to LPS.The mechanism is not associated with the NO pathway,but may be related to other pathways which need further study.Therefore,Que promotes the proliferation of porcine intestinal epithelial cells in vitro,and alleviates the oxidative damage and inflammatory response of porcine intestinal epithelial cells.