COX-2 Mediates Giardia Duodenalis Induced Inflammatory Response In J774A.1 Macrophages

Giardia duodenalis(G.duodenalis)is an anaerobic protozoan,and its life cycle has two forms:the trophozoite and the cyst.Giardia commonly causes diarrheal disease throughout the world.Clinical features may range from diarrhea to constipation,nausea,headache,and flatulence.Illness can last several months if untreated.At present,there are few effective drugs and methods for the prevention and treatment of giardiasis,thus it is urgent to reveal the pathogenesis of Giardia.Studies have found that cycloxygenase-2(COX-2)expression is related to the development of many protozoan diseases.However,the role of COX-2 in the infection of G.duodenalis is still unclear.The purpose of this study was to explore the role of COX-2 in specific mechanisms of Giardia induced J774 A.1 macrophage inflammation.For the infection challenge,mice were infected by Giardia through gavage with cysts in sterile phosphate buffered saline(PBS).Real-time fluorescent quantitative PCR(RT-q PCR)and Western blot(WB)were used to detected the expression of COX-2 in the intestine.The results suggested that COX-2 expression was increased(p value of density < 0.01).Giardia and J774 A.1macrophages were co-cultured to establish a Giardia infection model in vitro.After Giardia treatment for different times,the level of prostaglandin E2(PGE2)was detected by the PGE2 kit.We further detected COX-2 expression in J774 A.1 cells by WB,RT-q PCR and immunofluorescence(IF)analysis.The results showed that after Giardia stimulation,the PGE2 content in the culture medium and COX-2 expression in J774 A.1 cells were both increased significantly(p value of density < 0.01).The above results indicated that Giardia could induce COX-2 up-expression in infection model in vitro and in vivo,suggesting that COX-2 may play a role in Giardia infection.RT-q PCR and WB were used to detect the expression of inflammatory factors such as tumor necrosis factor-?(TNF-?),interleukin-6(IL-6),and interleukin-1?(IL-1?).The results showed that Giardia stimulated J774 A.1 cells to over-express inflammatory factors TNF-?,IL-6 and IL-1?,while the pretreatment with COX-2 inhibitor NS398 reduced(p value of density < 0.01)the up-expression of these pro-inflammatory cytokines induced by Giardia.In addition,COX-2 inhibition significantly decreased the production of nitric oxide(NO)from Giardia-treated J774 A.1 cells(p value of density < 0.01).Similarly,the nitric oxide synthase(i NOS)m RNA and protein expression also increased(p value of density < 0.01)after Giardia stimulation,but COX-2 inhibition led to a decrease(p value of density < 0.01)in i NOS over-expression.It was shown that COX-2 up-regulation may promote NO production through affecting i NOS expression in Giardia-treated J774 A.1 cells.Subsequently,RT-q PCR,WB and IF were used to detect the phosphorylation levels of nuclear transcription factor ?B(NF-?B),p38 and ERK1/2.Then we further explored the effects of SB203580(inhibitor of p38),SCH772984(inhibitor of ERK1/2)or JSH-23(inhibitor of NF-?B)pretreatment on COX-2 expression in Giardia-infected cells and non-infected cells.Our results show that the expression of COX-2 was mediated by the phosphorylation of p38,ERK1/2 MAPK and NF-?B.Flow cytometry and IF detections revealed ROS accumulation in Giardia-treated macrophages.In addition,we observed that ROS inhibitor NAC efficiently reduced p38,ERK1/2 MAPK and NF-?B activation in J774 A.1cells and blocked the nuclear translocation of NF-?B induced by Giardia.Overall,our results provide evidence that COX-2 is capable of inducing production of pro-inflammatory cytokines through ROS mediated MAPK or NF-?B pathway and serves as a key regulator in inflammation induced by Giardia.This study revealed that COX-2 regulated the expression of pro-inflammatory mediators through ROS mediated MAPK or NF-?B pathways in Giardia-infected J774 A.1 cells.These results deepened our understanding of the mechanism of inflammation in macrophages caused by Giardia stimulation.In conclusion,our study suggested that COX-2 inhibitor may represent an effective therapeutic intervention for giardiasis.It is necessary to determine the key molecular mechanisms of COX-2 for treating Giardia-induced inflammatory diseases in future studies.