Mechanism Of Nrf2/HO-1 Inhibiting Cellular Inflammatory Response Induced By E. Tenella Infection By Regulating ChTLR15/ChNLRP3

Coccidiosis is an avian infectious disease caused by Eimeria of the genus Eimeria.E.tenella is the most pathogenic species of coccidia and primarily parasitizes the cecum of chickens,causing severe cecal lesions.Coccidiosis occurs worldwide and causes significant economic losses to the poultry industry.The invasion of host cells by chicken coccidia is the first step in establishing infection.The discovery of new biological products or drugs for prevention and controlling of avian coccidiosis requires a thorough exploration of the mechanisms by which the parasite invades host cells.Our previous research has shown that E.tenella sporozoites specifically activate the Ch TLR15/NFκB/NLRP3/IL-1β pathway,suggesting that Ch TLR15 plays an important role in coccidial infection-induced intestinal inflammatory damage.Ouri previous study found that lactic acid bacteria can significantly activate Nrf2 antioxidant pathway and downregulate IL-1β expression.Oxidative stress is closely related to inflammation,thus it is speculated that activation of Nrf2 pathway can mediate the crosstalk between Nrf2 antioxidant pathway and Ch TLR15/Ch NLRP3,thereby negatively regulating Ch TLR15 inflammatory pathway and reducing inflammatory damage.Specific strains of lactic acid bacteria and natural polyphenols from plants have the ability to activate the Nrf2 pathway.In this study,Lactobacillus brevis 23017 and Corilagin,a polyphenolic compound that has been proved to activate Nrf2/HO-1 pathway in our previous experiments,were both selected as activators of Nrf2/HO-1 pathway.An in vitro infection model of E.tenella sporozoites was constructed using chicken fibroblast cell line DF-1,and RNAi technology was applied to explore the regulatory relationship between Nrf2/HO-1 antioxidant pathway and Ch TLR15/NLRP3 inflammatory signaling pathway.This research provides new insights into the mechanisms of Eimeria invasion into and interaction with host cells.The specific content of this study includes several aspects:To clarify the positive activation effect of Corilagin and L.brevis 23017 on the Nrf2/HO-1antioxidant signaling pathway in DF-1 cells,in this study,we firstly detected the dose and time of Corilagin and L.brevis 23017 acting on DF1 cells using CCK-8 and antioxidant enzyme kits.Then,DF-1 cells were pretreated with appropriate doses/bacterial counts and the m RNA transcription levels of related factors in Nrf2/HO-1 antioxidant signaling pathway and Ch TLR15/NLRP3 inflammatory signaling pathway were detected.The expression levels of p-Nrf2,Nrf2,and HO-1proteins were detected by Western Blot.The results showed that pretreatment of DF-1 cells with Corilagin and L.brevis 23017 had a positive regulatory effect on the antioxidant pathway Nrf2/HO-1.The significant antioxidant effect was observed on cells pretreated with 15 μM Corilagin(Cori-15 μM)and bacterial supernatant of L.brevis 23017.To further determine whether Nrf2/HO-1 antioxidant pathway activated by Corilagin and L.brevis 23017 can regulate Ch TLR15/Ch NLRP3 inflammatory pathway stimulated by E.tenella sporozoites,in this study,an E.tenella sporozoites invasion model based on DF-1 cells was established.Corilagin and L.brevis 23017 were used to pretreat DF-1 cells,and the m RNA transcription levels of related factors in Nrf2/HO-1 and Ch TLR15/NLRP3 signaling pathway were detected after sporozoites stimulation.The expression levels of p-Nrf2,Nrf2,HO-1,Ch TLR15,and Ch NLRP3 proteins were detected by Western Blot.The expression level of malondialdehyde(MDA)and reactive oxygen species(ROS)was detected by lipid peroxidation kit and ROS detection kit,respectively.The results showed that the expression levels of related moleculs in Ch TLR15/NLRP3 inflammatory signaling pathway that activated by E.tenella sporozoites during invasion into DF-1cells pretreated with Cori-15 μM and L.brevis 23017 supernatant were significantly downregulated,and the levels of MDA and ROS alos displayed the same change trend.The resluts indicates that Cori-15 μM and L.brevis 23017 supernatant can regulate Ch TLR15/NLRP3 inflammatory pathway that activated by sporozoites through activating Nrf2/HO-1 antioxidant signaling pathway in DF-1cells.To further clarify the crosstalk mechanism between the Nrf2/HO-1 antioxidant pathway that activated by Corilagin and L.brevis 23017 and Ch TLR15/NLRP3 inflammatory pathway,in this study,RNAi technology was used to interfere with the expression of Nrf2 and Ch TLR15 in DF-1cells.Corilagin and L.brevis 23017 were used to pretreat DF-1 cells with knockdown of Nrf2 and Ch TLR15,respectively.After sporozoites stimulation,the expression levels of related molecules in Nrf2/HO-1 and Ch TLR15/NLRP3 pathway were detected by real-time quantitative PCR(q RT-PCR)and Western Blot.The findings demonstrate that the activation of the Nrf2/HO-1 antioxidant pathway by Corilagin and L.brevis 23017 inhibits the Ch TLR15/Ch NLRP3 inflammatory pathway activated by E.tenella infection,thereby exhibiting anti-inflammatory effects.In summary,this study showed that Nrf2/HO-1 antioxidant signaling pathway activated by Corilagin and L.brevis 23017 can regulate Ch TLR15/NLRP3 inflammatory pathway and reduce the inflammatory damage caused by Eimeria invasion into host cells.This research provides new insights for the development of new products forprevention and controlling of chicken coccidiosis.