Pathological Mechanism Of Inflammation Induced By ROS Accumulation Caused Via SVCV

The main pathological symptoms of fish infected with spring viremia of carp virus?SVCV?are inflammatory injuries including peritonitis,enteritis and others.Previous studies have shown that SVCV infection in Epithelioma Papulosum Cyprini?EPC?destroys mitochondrial structure and impairs the function of mitochondrial complex III,resulting the accumulation of H₂O₂.Reactive oxygen species?ROS?,as a product of microbial infection,induces inflammation.Therefore,this study aimed to explore the link between ROS and SVCV-induced inflammation.The main findings are as follows:1. The relationship between ROS and SVCV-induced inflammationEPC cells were treated with 0.1 MOI SVCV,500?M H₂O₂,or 1?g/m L LPS for1,3,6,12,and 24 h.The results shew that SVCV induced the expression of TNF-?and COX-2,which consistent with H₂O₂and LPS treatment;SVCV induced the expression of IL-8 at 3 h and 6 h,H₂O₂induced the expression of IL-8 at 1 h and 3 h,the expression of IL-8 was up-regulated at 1 h during LPS treatment;the expression of IL-10 was up-regulated in the early stages of SVCV infection,and then down-regulated,while H₂O₂treatment up-regulated IL-10 expression during infection,and the expression of IL-10 was down-regulated in the early stages of LPS treatment,then up-regulated.The expression of COX-2 was further verified at the protein level and shows the similar trend between SVCV infection and H₂O₂treatment in EPC cells.These results demonstrated that that SVCV can induce inflammatory response.The antioxidant N-acetyl-L-cysteine?NAC?dampened the expression of TNF-?and COX-2 which were induced by SVCV and H₂O₂,suggesting a relationship between ROS accumulation and inflammation during SVCV infection..2. The molecular mechanism of SVCV-induced inflammatory responseIn order to explore the molecular mechanism of SVCV-induced inflammatory response,we verified from NF-?B,MAPK/AP-1 and PI3K signaling pathways.?1?NF-?B pathway:the NF-?B promoter activity was verified by double luciferase reporting assays.The results shew that NF-?B promoter activity was not activated by SVCV,and that treatment with PDTC,an inhibitor of NF-?B,did not inhibit the expression of inflammatory factors.?2?MAPK pathways:The results showed that inhibition of the JNK pathway significantly inhibited the expression of COX-2 and IL-8?74%and 47.3%,respectively?,and up-regulated the expression of IL-10?1.4-fold?;inhibition of the ERK pathway significantly inhibited the expression of COX-2 and TNF-??29.2%and 51.9%,respectively?,and up-regulated the expression of IL-10?3.2-fold?;and inhibition of the p38MAPK pathway could significantly inhibit the expression of TNF-??72.9%?.?3?PI3K pathway:The cells were treated with the PI3K inhibitor LY294002 to verify the PI3K pathway.The results indicate that SVCV can induce the chemokine IL-8.Additional,inhibiting the pathways of MAPK or PI3K can promote SVCV replication.