Effects Of Selenium-rich Lactobacillus Plantarum On Cadmium/Lead Accumulation,Oxidative Stress And Inflammatory Reaction In The Tissues Of Luciobarbus Capito

Cadmium（Cd）and lead（Pb）are environmental pollutants that enter the water environment with the development of industry and cause great harm to human aquatic economic animals.Excessive intake of Cd and Pb will accumulate in various tissues of aquatic animals.When the accumulation reaches a certain level,it will cause oxidative stress,and then damage the body organs and lead to disease.Selenium（Se）plays an important role in supplementing the level of antioxidant enzymes.Lactobacillus plantarum（L.plantarum）is a kind of probiotics,which can regulate the intestinal flora and keep the host healthy.It is a living microorganism beneficial to the host.Both Se and L.plantarum have the effect of antagonizing heavy metals.Se can form a complex with Cd or Pb and be metabolized to the body.After L.plantarum enters the intestine,it absorbs Cd or Pb in the intestine through adsorption,and then it is discharged from the body through feces.The current research mainly focuses on the single Se or L.plantarum against the toxicity of heavy metals,however,it is not clear whether the toxicity of the combination of the two to heavy metals is synergistic or antagonistic.Therefore,this study was completed in two experimental stages.The first step is to convert inorganic Se into nano-selenium by using the biotransformation of L.plantarum to inorganic Se.The second step is to add Selenium-rich L.plantarum into the feed to explore its biological accumulation,histopathological changes,serum biochemical parameters,oxidative stress and inflammatory reaction results in fish tissues under Cd or Pb stress.The research results can provide experimental data for the mechanism of heavy metal poisoning in fish and the development of functional feed for aquatic economic animals.1.Preparation of selenium-rich L.plantarumInoculate 200μL of L.plantarum in MRS liquid medium with different concentrations of sodium selenite（Na₂Se O₃）（100,150,200,300μg/m L）,and incubate for 24 h at 37℃.The content of nano-selenium in the test tube and the number of bacterial colonies were measured,and the morphology of L.plantarum after selenium enrichment was observed by scanning electron microscope.The results show that:when the amount of sodium selenite added is 200μg/m L,the number of bacterial colonies is up to 10⁷ CFU/m L,and the content of nano-selenium is 184μg/m L.Under the electron microscope,spherical nano-selenium particles（Se NPs）can be observed attached to the surface of the cell,and the diameter of nano-selenium is about 70～140 nm.2.Effects of Se-enriched L.plantarum on Cd accumulation,oxidative stress and inflammatory reaction in the tissues of L capitoIn order to explore the effect of Selenium-rich L.plantarum on the accumulation of cadmium in tissues,serum biochemical parameters,oxidative stress and inflammatory reaction of Luciobarbus capito（L.capito）,the control group,Cd group（Cd,0.05 mg/L）and Selenium-rich L.plantarum+Cd group（SL+Cd,Cd concentration 0.2 mg/L,feeding selenium-rich feed,nano-selenium content 5 mg/kg,L.plantarum content 10⁸ CFU/kg）were set up,and the test period was 28 d.Samples of kidney,liver,gill,serum,etc.were collected on the 14th and 28th d.The bioaccumulation,antioxidant enzyme activity,histopathological changes,expression of antioxidant genes and inflammatory factors in tissues were measured.The results showed that selenium-rich L.plantarum could alleviate oxidative stress and inflammatory reaction induced by Cd through Nrf2 signal pathway and NF-k B signal pathway.Compared with the control group,Cd stress led to glomerular atrophy,large gaps,red blood cell infiltration,renal tubular epithelial cells shedding,dissolution,and nuclear disappearance could be observed;The nucleus of liver disintegrates,disappears,and pyknosis;Hepatocellular necrosis;The boundary of hepatocytes is fuzzy;Inflammatory cell infiltration;A large number of cells are vacuolated,and se-enriched L.plantarum can alleviate the above changes.Selenium-enriched L.plantarum could significantly reduce the activity of serum alanine aminotransferase（ALT）（P<0.05）,significantly reduce the accumulation of Cd in the kidney（P<0.05）,significantly increase the activities of catalase（CAT）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）in the kidney and gill（P<0.05）,and significantly reduce the content of malondialdehyde（MDA）（P<0.05）.For antioxidant genes,Cd exposure significantly decreased the m RNA expression of SOD,CAT,GSH-Px,HO-1 and Nrf2（P<0.05）,and increased the relative expression of Keap1（P<0.05）;For inflammatory factors,Cd treatment significantly up-regulated the expression level of NF-κBp65、TGF-β、IL-8、IL-1 and TNF-α（P<0.05）,significantly decreased The relative expression of I-κBα（P<0.05）.However,the above inhibition was significantly weakened after feeding selenium-rich L.plantarum.The results showed that had protective effects on oxidative stress and inflammatory reaction induced by Cd.The results showed that selenium-rich L.plantarum could alleviate Cd toxicity by enhancing tissue antioxidant capacity and reducing inflammatory reaction.3.Effects of Se-enriched L.plantarum on Pb accumulation and oxidative stress in the tissues under lead stress of L capitoIn order to explore the effect of selenium-rich L.plantarum on Pb accumulation and oxidative stress in the tissues of L.capito under Pb stress,the control group,Pb group（Pb concentration is 0.2 mg/L）and selenium-rich L.plantarum+Pb group（SL+Pb,Pb concentration is 0.2 mg/L,feeding selenium-rich feed,nano-selenium content is 5 mg/kg,L.plantarum content is 10⁸ CFU/Kg）were set up,and the test period was 14 d,and then samples were collected to determine the accumulation of Se and Pb in liver,kidney,gill,intestine and muscle,as well as the activity of antioxidant enzymes and the expression of antioxidant genes.The results showed that the accumulation of Se in the Se-enriched L.plantarum+Pb group was significantly higher than that in the control group and Pb group（P<0.05）,and the selenium content in the lead group was significantly lower than that in the control group（P<0.05）,but the intestinal tract was significantly higher than that in the control group（P<0.05）;The Pb accumulation in the Pb group and Se-enriched L.plantarum+Pb was significantly higher than that in the control group（P<0.05）.The Pb accumulation in different tissues of the fish body in the Pb group was gill>liver>kidney>intestine>muscle from high to low,and the Se-enriched L.plantarum+Pb was significantly lower than that in the Pb group（P<0.05）;Selenium-enriched L.plantarum could significantly increase the activities of catalase,superoxide dismutase and glutathione peroxidase in kidney and gill tissues under Pb stress（P<0.05）,and reduce the content of malondialdehyde（P<0.05）;The relative expression of Nrf2,HO-1 and GSH-Px in lead group and Se-enriched L.plantarum+Pb group decreased significantly（P<0.05）,and the above indexes in Se-enriched L.plantarum+Pb group increased significantly compared with lead group（P<0.05）.The results showed that the addition of selenium-rich L.plantarum in the feed could reduce the Pb accumulation in various tissues and alleviate the oxidative stress of fish body under Pb stress.4.Effect of Se-enriched L.plantarum on transcriptome of renal tissue of L capito under cadmium stressIn order to explore the effect of selenium-rich L.plantarum on the transcriptome of the kidney tissue of L capito,the control group（C）,Cd group（Cd concentration 0.05 mg/L）and selenium-rich L.plantarum+Cd group（S1L1＿Cd,Cd concentration 0.05 mg/L,nano-selenium content 5 mg/kg,L.plantarum content 10⁸ CFU/kg）were set up,and the test lasted for 28 d.Using the method of transcriptomics,the kidney of L capito in the selenium-rich L.plantarum+Cd group,Cd group and control group were analyzed by transcriptomics.Results A total of 2955 differential genes were found,of which 488 were differentially expressed in the control group and Cd group,301 were up-regulated and 187 were down-regulated.There were 1474 differentially expressed genes in the control group and the selenium-rich L.plantarum+Cd group,of which 720 genes were up-regulated and 754genes were down-regulated.A total of 989 differential genes were expressed in selenium-rich L.plantarum+Cd group and Cd group,of which 510 genes were up-regulated and 497genes were down-regulated.GO differential genes are mainly enriched in 20 pathways,including viral transcription,transcription,structural constituent of ribosome,SRP-dependent cotranslational protein targeting to membrane,r RNA processing,response to bacterium,respiratory chain complex IV,metallopeptidase activity,membrane,iron ion binding,integral component of membrane,nuclear-transcribed m RNA catabolic process,nonsense-mediated decay,immunoglobulin production,heme binding,focal adhesion,extracellular space,extracellular exosome,cytosolic small ribosomal subunit,cytosolic large ribosomal subunit and carbohydrate binding.KEGG enrichment found 11 most closely related enrichment pathways,mainly including Ribosome,Intestinal immunenetwork for Ig A production,Glutathione metabolism,Thermogenesis,Huntington disease,Parkinson disease,Non-alcoholic fatty liver disease,NAFLD,Cell adhesion molecules,CAMs,Cardiac muscle contraction,Alzheimer disease and TNF signaling pathway.