Effects Of TLR9/NF-κB In Dapulian Pigs On Oxidative Stress And Inflammation In IPEC-J2 Cells

Oxidative stress is one of the most important factors affecting large-scale breeding,especially the performance of pigs.Oxidative stress plays a role by affecting various genes in pigs,which can cause serious body damage,functional degradation and reduce production performance.TLR9 plays an important role in regulating the oxidation level and maintaining the normal physiological activities of the body.In this study,Diquat was used as a source of oxidative stress to study the effects of oxidative stress on Dapulian pigs by detecting relevant indicators.Then the porcine jejunal epithelial（IPEC-J2）cells were selected to construct an oxidative stress model in vitro to verify the role of TLR9 signaling pathway in oxidative stress.（1）Six weaned piglets with an average weight of 13.16 kg were randomly divided into control group and oxidative stress group,with 3 piglets in each group.Each piglet in oxidative stress group was injected with 10 mg/kg diquat,and the piglets appeared vomiting,loss of appetite and other symptoms;the control group was injected with the same amount of normal saline.At the end of the experiment,the average body weight and average daily gain of the oxidative stress group were significantly lower than those of the control group（P<0.05）,the content of superoxide dismutase（SOD）in jejunum was significantly lower than that of the control group（P<0.05）,and the content of malondialdehyde（MDA）was significantly higher than that of the control group（P<0.05）.In the oxidative stress group,the levels of IL-6,IL-18,IL-1βand TNF-αwere significantly increased,and the expression of TLR9 was significantly increased（P<0.05）.（2）After H₂O₂ treatment,reactive oxygen species（ROS）in cells increased significantly,so it can be used to study the effects of oxidative stress on gene expression and antioxidant enzyme activity.Compared with H₂O₂ group,the expression of GSH and SOD in H₂O₂-pc DNA3.1（+）-TLR9 group was significantly increased（P<0.05）,TLR9 and its downstream pathway genes MyD88,NF-κB and TLR9 were significantly up-regulated（P<0.05）,while in H₂O₂-si-TLR9 group,the expression of GSH and SOD were significantly decreased（P<0.05）,and the pathway genes were also significantly down regulated and inflammatory factors such as IL-6,IL-18,IL-1βand TNF-αwere significantly decreased in H₂O₂-pc DNA3.1（+）-TLR9（P<0.05）,while MDA and inflammatory factors were significantly increased in H₂O₂-si-TLR9 group（P<0.05）.（3）We constructed MyD88 overexpression vector and synthesized si RNA to verify whether MyD88 is involved in the regulation of oxidative stress and whether TLR9 regulates NF-κB signaling pathway through MyD88.The results showed that compared with H₂O₂group,the expression of MyD88 and NF-κB in H₂O₂-pc DNA3.1（+）-MyD88 group was significantly up-regulated（P<0.05）;compared with H₂O₂ group,the expression of MyD88and NF-κB in H₂O₂-si-MyD88 group was significantly down regulated（P<0.05）,indicating that MyD88 itself is also involved in the regulation of oxidative stress.When TLR9 and MyD88 were co transfected in oxidative stress model,the expression of NF-κB was significantly lower than that of TLR9 overexpression alone,which indicated that TLR9indeed regulated the expression of NF-κB through MyD88.（4）At the same time,based on the oxidative stress model of IPEC-J2 cells,immunofluorescence assay confirmed that NF-κB in H₂O₂ group was transferred from extranuclear to intranuclear compared with the control group.In addition,Western blot analysis using nuclear protein also confirmed that the expression of NF-κB in H₂O₂ group was significantly up-regulated compared with the control group（P<0.05）,which proved that NF-κB was regulated and activated in oxidative stress.In conclusion,we successfully constructed the oxidative stress model,and found that oxidative stress led to the inhibition of body weight of weaned piglets,high expression of TLR9 gene in oxidative stress-related pathway,decreased antioxidant capacity of jejunum,and produced inflammatory response;secondly,in vitro verification in IPEC-J2 cells showed that TLR9 pathway had a significant positive effect on antioxidant.These results provide a theoretical basis for further study on the molecular mechanism of oxidative stress and tissue damage induced by diquat and H₂O₂.