Investigation Of Fluoride Level In Some Areas Of Songnen Plain And Its Toxic Effect On Poultry

Fluoride（F）is one of the essential trace elements for the human body.It is very common in daily life and outdoor environment.It mainly causes damage to the body through breathing,eating,drinking and other ways.In the wild environment,fluorine exceeds the standard in many areas,causing great harm to people in surrounding areas,animals and plants.Excessive fluorine will cause serious pathological damage and ultrastructural changes in poultry liver,brain and other organs,such as inflammatory infiltration,a large number of bleeding points,mitochondrial damage,etc.Ferroptosis is a new cell death method proposed in recent years,and its main manifestations are iron accumulation,lipid peroxidation,mitochondrial membrane thickening,and production of reactive oxygen species.Neutrophil external traps（NETs）are a neutrophil activation mechanism whose excessive accumulation leads to enhanced inflammation.However,until now,whether fluorine-induced tissue damage is related to ferroptosis and the mechanism of fluorine-induced NETs formation is still unclear.In order to clarify the above problems,this experiment firstly tested the fluorine content in the water bodies and sediments of the nature reserves in the Songnen Plain:Longfeng Wetland Nature Reserve,Chagan Lake Nature Reserve and Xianghai Nature Reserve.In this study,120 1-day-old Ross 308 broilers were randomly divided into 4 groups,30 birds in each group.Based on the F ion concentration and toxicological concentration detected in environmental samples,the concentration of NaF in the diets of the four groups was determined.Add different concentrations of NaF to the diets of the control group（Group C）,low-fluorine group（LF group）,medium-fluorine group（MF group）,and high-fluorine group（HF group）,so that the concentration of F ions in the diet 0,500,1000,2000mg/kg,used to replicate the field fluorosis poultry model.H&E staining,transmission electron microscope observation,combined analysis of transcriptomics and metabolomics,oil red O staining,immunohistochemistry,real-time fluorescent quantitative PCR,western blot,flow cytometry and other methods to determine the specific mechanism of fluoride-induced liver injury in poultry.Then it was verified in vitro using chicken embryo primary hepatocytes and LMH cells.Fluorescent staining,cell viability detection,real-time fluorescent quantitative PCR,western blotting,addition of oxidative stress inhibitor N-acetyl-L-cysteine（NAC）,ferroptosis inhibitor（Fer-1）,nifedipine（Nifedipine）and other means to explore the specific mechanism of fluoride-induced chicken peripheral blood NETs.By detecting the expression of chemokines in brain tissue,the generation of NETs,and establishing a co-culture system of neutrophils and neurons,we hope to clarify the role of fluorine-induced NETs in brain inflammation.The research results are as follows:（1）The fluorine content of 7 sampling sites in the Longfeng Wetland Nature Reserve was tested and found that the F^-concentration in the water was 0.9-1.47mg/L,with an average of1.16mg/L,slightly higher than the national standard of 1mg/L.The F^-content in the bottom mud is 287-759mg/kg,with an average value of 502mg/kg,which is slightly higher than the background value of 480mg/kg in Chinese soil.In the detection of Chagan Lake Nature Reserve,it was found that the F^-concentration of 12 water body sampling points was 0.91-1.47mg/L,with an average of 1.24mg/L,and the F^-concentration detected by 8 sediment sampling points was408-945mg/kg.The F^-content in the 8 water body sampling points in Xianghai Wetland was the highest among the three regions,ranging from 2.17-3.26mg/L,with an average value of2.47mg/L.The results showed that the fluorine in the environment exceeded the standard in some areas of the Songnen Plain.（2）The body weight of F poisoned poultry decreased,the coefficient of liver organs increased,and the liver function decreased.Oil red O staining showed that lipid accumulation in the liver decreased with increasing concentration.Histopathological observation showed that hepatic hemorrhage points increased and inflammatory cell infiltration appeared with the increase of F concentration.Ultrastructural observation showed abnormal mitochondrial structure and vacuolation in both MF and HF groups.Combined analysis of transcriptomics and metabolomics revealed significant changes in FOXO signaling pathway,abnormal glutathione metabolism,sphingolipid metabolism,and neuroactive ligand-receptor interactions.Afterwards,iron accumulation in liver tissue and levels of oxidative stress-related markers（T-AOC,CAT,MDA,GSH）were detected.qRT-PCR and WB were used to detect the FOXO signaling pathway,oxidative stress-related pathways,and ferroptosis-related factors from the levels of transcription and translation.It was found that F can increase the iron content in the liver,cause oxidative stress,activate the FOXO signaling pathway,and oxidative stress signaling pathway lead to ferroptosis.In vitro experiments using primary chicken hepatocytes and LMH cells proved the above point by means of CCK-8,qRT-PCR,WB,iron ion and ROS fluorescent staining,and flow cytometry.In addition,fluorine can also cause cell cycle arrest in S phase,hinder DNA synthesis,and lead to cell death.（3）Firstly,different doses of NaF（0m M,0.25m M,0.5m M,1m M）were used to induce the formation of NETs,and then combined with phorbol-12-myristate-13-acetate（Phorbol-12-myristate-13-acetate,PMA）were compared to screen out the optimal dose for inducing NETs in vitro.Afterwards,qRT-PCR was used to detect the transcriptional levels of genes related to NETs formation（MPO,NE,H3,NOX-2,PKCα,PKCβ,PKCζ）.The protein levels of MPO and NE were detected by WB and immunofluorescence,and it was found that the optimal concentration of NaF to induce chicken peripheral blood NETs was 1m M.Then Hoechst staining was used to observe the formation of NETs,the Fe Rh Nox-1 probe was used to observe the accumulation of Fe²⁺during the formation of NETs,and the changes of glutathione（GSH）and malondialdehyde（MDA）were observed by Fer-1 and NAC treatment.The results showed that Fer-1 could significantly reduce the generation of NETs induced by NaF,but could not reduce the generation of NETs induced by PMA,and NAC had an inhibitory effect on both PMA and NaF.In addition,the accumulation of Fe²⁺was observed during the formation of NaF-induced NETs,which could be relieved by Fer-1,but this phenomenon was not observed under the action of PMA.The detection results of GSH and MDA showed that Fer-1 and NAC could alleviate the decrease of GSH and the increase of MDA caused by NaF.（4）Fer-1 pretreatment can inhibit the expression of genes and proteins related to NETs production（MPO,NE）and ferroptosis（GPX4,SLC7A11,FTH1,NCOA4,ALOX12）.However,Fer-1 does not alleviate the mitochondrial depolarization induced by NaF.Next,the Fluo-4AM calcium ion fluorescent probe was used to detect the accumulation of Ca²⁺in neutrophils under the action of NaF.It was found that NaF would increase the Ca²⁺content in neutrophils,but Fer-1 could not rescue this phenomenon.Neutrophils were pretreated with L-type calcium channel（LTCC）inhibitor nifedipine（Nifedipine）.It was found that Nifedipine can reduce the occurrence of NaF-induced Ca²⁺increase and ferroptosis by inhibiting the expression of LTCC-related proteins（p-CAMKII,CAMKII,CACNA1D）.The mitochondrial membrane potential was detected by the mitochondrial membrane potential detection kit JC-1,and the generation of ferroptosis-related factors and NETs were detected by WB and Hoechst staining.It was found that Nifedipine can alleviate the phenomenon of mitochondrial depolarization.And reduce the expression of NETs production-related proteins by reducing ferroptosis.（5）H&E staining and transmission electron microscope observations were carried out on the brain tissue induced by different concentrations of NaF,and it was found that there were inflammatory infiltration and mitochondrial damage.Next,chemokines（CCL1,CCL4,CCL17,CSCL12,CXCL13,CXCL14）were detected,and the results showed that neutrophils were recruited in brain tissue under the action of NaF.Afterwards,the detection of ferroptosis,NETs and LTCC-related indicators in brain tissue found that NaF can cause ferroptosis in brain tissue,open LTCC in brain tissue,and increase the generation of NETs.Finally,a co-culture model of neutrophils and primary neurons was constructed,and it was found that NaF could aggravate the inflammatory response of neurons by causing NETs.In summary,fluorine exceeds the standard in some areas of the Songnen Plain,and high-fluorine diets can cause ferroptosis in the liver of poultry,resulting in liver damage.The combined analysis of transcriptomics and metabolomics found that NaF induced by SIRT1/FOXO3 pathway Hepatic ferroptosis was verified by using primary hepatocytes and LMH cells in vitro.In addition,excessive NaF can induce the generation of NETs by causing ferroptosis of neutrophils,and strengthen the inflammatory response in the brain.The innovation of this study is to combine field fluorine content surveys with laboratory models for the first time to carry out research related to environmental toxicology,and secondly,through multi-omics joint analysis to correlate fluorosis with ferroptosis and the regulation of the liver-brain axis detection.And it is the first time to discuss the damage mechanism of fluorine to the body from the perspective of ferroptosis,and discuss the reasons for the formation of NETs caused by NaF,which provides an important theoretical basis and data support for fluorine-induced body damage,and enriches the biological content related to fluorine.