Study On The Mechanism Of Pyroptosis Induced By Environmental Pollutant 4-Octylphenol In Common Carp Spleens

4-octylphenol（4-OP）,a toxic pollutant mainly present in the aqueous environment,is hazardous to human and animal health.Common carp（Cyprinus carpio L.）is considered as one of the common economic freshwater fish in China.The spleen as an important immune organ in fish is also a target organ attacked by toxic substances.However,the toxic effect of 4-OP on common carp spleen is not yet reported.Hence,a 4-OP exposure model of common carp was established in this experiment.81 fish were randomized into three groups:the control group,the solvent control group,and the 4-OP group（the concentration of 4-OP was 17μg/L）.After initial weight and length of fish were measured,a 45-day attack test was conducted.During the test.On the 45th day of the experiment,final body weight and length of carps were recorded,and whole blood and spleen tissues were collected.Hematological parameters of common carp were examined with a fully automated animal blood cell analyzer.In order to study the damage caused by 4-OP exposure in common carp spleens,spleen tissues were accurately weighed to calculated the spleen coefficient and were subjected to microstructural observation,and ultrastructural observation.In this experiment,the kit method was used to detect oxidative stress indicators,inflammation-related indicators and LDH activity;q RT-PCR was adopted to detect m RNA expression of immune-related,inflammation-related,and pyroptosis-related genes,as well as miRNA expression;Western blot was employed to determine pyroptosis-related protein levels;immunofluorescence staining（IFS）was performed to detect GSDME expression in common carp spleens.The present study was designed to investigate the toxic effect of 4-OP on common carp spleens and the molecular mechanism of spleen damage caused by 4-OP exposure in common carps.The main results are as follows:（1）4-OP exposure decreased WGR,LGR,and SGR（P<0.05）.4-OP exposure reduced RBC,WBC,Hb,HCT,MCH,and MCHC（P<0.05）,and increased MCV（P<0.05）.The above findings indicating that 4-OP exposure inhibited carp growth,caused changes in blood parameters and affected carp health.（2）4-OP exposure led to an increase in spleen coefficient（P<0.01）in carp.4-OP exposure caused splenic pathological changes such as swollen cells in red pulp and white pulp,mitochondrial swelling and even vacuolization,some vacuoles in the cell membrane,and cell membrane rupture.These results demonstrated that 4-OP exposure caused inflammatory injury and pyroptosis in common carp spleens.（3）Exposure to 4-OP increased H₂O₂and MDA contents（P<0.01）,and decreased the activity of CAT,GSH-Px,GST,SOD,and T-AOC as well as GSH content（P<0.01）in common carp spleens,suggesting that 4-OP exposure caused oxidative stress in common carp spleens through triggering the imbalance of oxidation and anti-oxidation.（4）4-OP exposure down-regulated mRNA expression（P<0.01）of Th1-related factors（T-bet,TNF-β,and IFN-γ）and Treg-related factors（TGF-βand Fox P3）,up-regulated mRNA expression （P<0.01）of Th2-related factors（IL-4 and GATA3）and Th17-related factors（IL-17,IL-21,and IL-22）,indicating that 4-OP exposure caused immunosuppression via Th1/Th2 and Treg/Th17imbalances.（5）4-OP exposure elevated NO content and iNOS activity（P<0.01）,up-egulated m RNA expression of TLR2a（P<0.05）,myd88（P<0.01）,IKKα（P<0.01）,IKKβ（P<0.01）,NF-κB（P<0.01）,ICAM-1（P<0.01）,COX-2（P<0.01）,and PTGES（P<0.01）,and down-regulated m RNA expression of IκBα（P<0.01）.The results suggested that 4-OP exposure induced inflammatory injury in comon carp spleens via TLR2a/IκBα/NF-κB pathway.（6）4-OP exposure resulted in elevated LDH activity（P<0.01）and percentage of GSDME-positive cells（P<0.01）,elevated m RNA expression of NLRP3（P<0.01）,ASC（P<0.05）,NEK7（P<0.01）,Caspase-1（P<0.01）,Caspase-3（P<0.01）,gsdmea（P<0.01）,and IL-1β（P<0.01）,as well as elevated protein levels（P<0.01）of Caspase-1,Caspase-3,GSDME,and IL-1βin common carp spleens.The results indicated that 4-OP exposure caused pyroptosis in common carp spleens via NEK7-NLRP3-Caspase-1-Caspase-3-GSDME pathway.（7）NEK7 and gsdmea were predicted to be the target genes of miR-200a by a bioinformatic prediction website.4-OP exposure down-regulated miR-200a expression（P<0.01）.There was a highly significant negative correlation between miR-200a and NEK7 and gsdmea,respectively.The results demonstrated that miR-200a mediated in 4-OP-induced splenic pyroptosis by targeting NEK7 and gsdmea.（8）Oxidative stress can mediate immunosuppression,immunosuppression can induce inflammation,and inflammation can mediate pyroptosis.The results suggested that oxidative stress,immunosuppression,and inflammatory response participated the complex mechanism of4-OP-induced pyroptosis in common carp spleens.In summary,4-OP exposure affected carp health and caused common carp spleen damage.miR-200a,oxidative stress,immunosuppression,and inflammation took part in the complex mechanism of 4-OP-induced pyroptosis in common carp spleens.The present study revealed the toxic effect of 4-OP on common carp spleens for the first time.It provided a new idea for the study of the toxic effects of 4-OP and provided a reference for the risk assessment of environmental endocrine disruptors.