The Role And Mechanisms For Hydroxyproline In Ameliorating Intestinal Inflammation Induced By Dextran Sulfate Sodium In Mice

Intestinal inflammation may lead to reduction in digestive and absorptive capacity,affecting the growth and production performance of weaned piglets and resulting in considerable economic losses to the pig industry.The mechanisms responsible for intestinal inflammation is closely related to the excessive activation of immune responses and the overproduction of reactive oxygen species(ROS).Improvement of intestinal health through the regulation of immune response and ROS production has become one of the most goals in animal nutrition in recent years.Hydroxyproline(Hyp)is an amino acid derivative that is rich in collagen and swine’s milk.Studies have shown that Hyp may exert a protective effect against inflammation and oxidative stress.In this study,animal experiments in vivo and cell culture experiments in vitro were conducted to evaluate the anti-inflammatory and anti-oxidative effects of Hyp on the gut by using techniques in histological,cellular and molecular biology.In Experiment 1,dextran sulfate sodium(DSS)-induced colitis in male C57BL/6 mice were used to investigate the protective effects of Hyp supplementation against inflammation.The results showed that 2.5%DSS-treated mice showed weight loss,increased disease activity index(DAI)and histological score,shortened the length of the colon,and elevated myeloperoxidase activity,which were significantly alleviated by 1%Hyp supplementation.Compared to the DSS-treated group,Hyp reduced the percentage of monocytes in the lamina propria,including neutrophils,macrophages,dendritic cells,NK cells,T helper cells,and B lymphocytes.In addition,supplementation of Hyp significantly attenuated the excessive production of ROS and malondialdehyde and augmented glutathione content as well as the activity of superoxide dismutase in DSS-treated animals.The DSS-induced increase in mRNA and the content of pro-inflammatory cytokines IL-6 and TNF-a in the colon were significantly suppressed by Hyp treatment.The enhanced mRNA expression of anti-inflammatory cytokines,IL-10,chemokine CXCL1 and CXCL2 in the DSS-treated mice were also attenuated by Hyp.Importantly,the DSS-induced increase in the phosphorylation of nuclear factor kappa B(NF-κB)and signal transducer and activator of transcription 3(STAT3)was significantly mitigated by Hyp.The results indicated that Hyp alleviates colonic mucosal injury via enhancing anti-oxidative capacity and reducing leukocyte subtype infiltration and inflammatory reaction,which may be related to the inhibition of NF-κB and STAT3 activation.Experiment 2 is an in vitro study that using the cultured mouse macrophage cell line(RAW264.7)to explore whether Hyp contributes to inhibit lipopolysaccharide(LPS)-induced inflammatory responses.Medium supplemented with 0.5,1 or 2 mM Hyp dose-dependently reduced the expression of iNOS,PTGS2,IL-6,IL-1β and CXCL1 mRNA in the presence of LPS(100 ng/mL).Meanwhile,Hyp inhibited the phosphorylation of NF-κB p65 and STAT3 as well as the nuclear translocation of NF-κB p65,in comparison with the cells treated with LPS.Inhibition of NF-κB activation by Bay11-7085 suppressed LPS-induced mRNA expression of iNOS,PTGS2,IL-6,IL-1β,and CXCLl as well as the phosphorylation of STAT3 in a dose-dependent manner,suggesting that the protective role of Hyp against LPS-induced inflammation in macrophage is related to the lowered activity of NF-κB p65.In Experiment 3,the protective effect of Hyp on apoptosis and DNA damage induced by 4-hydroxy-2-nonenal(4-HNE)in porcine intestinal epithelial cells(IPEC-1)was studied.The results showed that 5 mM Hyp significantly inhibited cell apoptosis,and the generation of ROS triggered by 4-HNE(40 μM).Additionally,Hyp significantly alleviated 4-HNE-induced DNA damage,phosphorylation of H2AX,JNK and p38 MAPK,and the increase in the mRNA level of GADD45a/b.The addition of Hyp significantly enhanced the 4-HNE-induced increase in Kruppel-like factor 4(KLF4)levels.Suppression of KLF4 expression by kenpaullone exacerbated the 4-HNE-induced apoptosis and DNA damage,indicating the resistance to 4-HNE-induced cell apoptosis and DNA damage in IPEC-1 cells treated with Hyp may be related to KLF4.The above evidence indicates that Hyp can enhance the antioxidative capacity of intestinal epithelial cells and relieve the inflammatory responses in gut,thereby improving gut health.These findings provide a theoretical foundation for the antioxidative and anti-inflammatory effects of Hyp in the intestinal tract.