| Carbon black is a major carbonaceous nanomaterial,which is widely used as industrial raw materials in rubber,tire and pigment industries.Due to its increasing market demand,carbon black has been widespread in the environment.Carbon black nanoparticles can produce toxic effects on the direct contact organs such as lungs and intestines,and can also produce toxic effects on the indirect contact organs such as knee joint,heart and liver.Intestinal mucosal barrier is the host's first line of defense resist the invasion of commensal bacteria and intestinal pathogens.Once the intestinal defense mechanism is damaged,bacterial translocation to extraintestinal tissues will cause abnormal activation of the immune system.A healthy gut microbiota ecosystem is a necessary condition for maintaining the orderly functions of the body.Major changes in the composition,activity and function(imbalance)of normal intestinal microorganisms caused by environmental or host related destructive events can damage metabolism,inflammation and nervous system,leading to disease attack.The gut-liver axis is a two-way relationship between the gut and its microbiota and the liver.The gut is connected to the systemic circulation,brain and liver through mesenteric lymph nodes,nerves and portal vein,which can affect the gut immune response,intestinal barrier function,liver and systemic inflammation.At present,there are many studies on carbon black nanoparticles as air pollutants,but there are few reports on whether carbon black nanoparticles as water environmental pollutants will damage the direct contact target organs of aquatic organisms,such as intestines,and affect the function of indirect contact target organs,such as liver.Therefore,this experiment took zebrafish as the research object,and randomly divided 360 3-month-old zebrafish into four groups: carbon black concentration 50 mg/L group(CBNPs.L),the 100 mg/L group(CBNPs.M),200 mg/L group(CBNPs.H)and the control group(NC).The zebrafish were continuously exposed to water containing different concentrations of carbon black nanoparticles for30 days,and the intestinal,liver and intestinal contents were collected as the main research samples.Using RT-PCR,Western blotting,16 S bioinformatics analysis technology,oil red O staining,H&E staining,scanning electron microscopy and transmission electron microscopy and other methods,observe the growth of zebrafish,intestinal and liver tissue damage,and detect intestinal microbial homeostasis,intestinal permeability,and expression levels of genes related to liver glycolipid metabolism.The main results are as follows:(1)The observation results of the structure characteristics of carbon black nanoparticles CBNPs show that the carbon black Printex 90 nanoparticles appear in the form of spherical clusters,and the average diameter of the nanospheres is about 89 nm,which is a typical carbon black nanoparticle structure.Observing the growth status of zebrafish found that CBNPs exposure had no significant effect on the length,weight and body mass index of zebrafish.(2)Observation of intestinal histopathology showed that CBNPs exposure caused obvious intestinal damage,thinning of the intestinal tissue wall,coarse intestinal villi,enlarged gaps,different cell sizes,and disordered arrangement.In CBNPs.H group,the loss of intestinal villi was also observed.Transmission electron microscopy results showed that with the exposure of CBNPs the number of pinosome in intestinal epithelial cells increased and merged and expanded,and the most significant change was in CBNPs.L group.With the increase of the exposure concentration of CBNPs,it was observed in CBNPs.M and CBNPs.H group,the tight junction structure was damaged,and gaps appeared between adjacent epithelial cells.The detection of tight junction related genes showed that the expressions of Occludin,ZO-1,and Rap2 were down-regulated,and the expressions of Rho A,ROCK,and MLC2 were up-regulated(P<0.05).The above results indicate that CBNPs exposure can cause damage to the intestinal microvilli of zebrafish and cause changes in the permeability of the intestinal mucosal barrier.(3)The 16 S bioinformatics analysis results showed that there were 1745,666,977 and 548 OUTs in the NC,CBNPs.L,CBNPs.M and CBNPs.H groups,respectively.Alpha diversity analysis showed that the exposure of CBNPs affected the intestinal microbial diversity,with less impact on the CBNPs.M group,and greater impact on the CBNPs.L and CBNPs.H groups.The results of the Beta diversity index heat map suggest that the species diversity of the intestinal flora increases with the increase of CBNPs exposure concentration.The above results indicate that exposure of CBNPs to low concentration(50 mg/L)and high concentration(200 mg/L)can significantly affect the microbial diversity in the zebrafish intestine,and the medium concentration of 100 mg/L CBNPs has little effect on microbial diversity.(4)The cluster analysis results showed that the CBNPs.L and CBNPs.M groups were more similar in microbial composition,followed by the CBNPs.H group,and finally the control NC group.The relative abundance results of species showed that CBNPs exposure caused an increase in the proportion of proteobacteria in the intestines of each group.Among them,the CBNPs.M group changed little,and the CBNPs.L and CBNPs.H groups increased significantly.CBNPs exposure also caused the proportion of Firmicutes and Bacteroides to decrease with the increase of CBNPs exposure concentration.The results showed that the ratio of Firmicutes and Bacteroides in the CBNPs exposure group increased significantly(P<0.05),and the increase in the CBNPs.M group was the most significant(P<0.01).The results of LEf Se analysis showed that CBNPs exposure caused significant differences in 37 species from phylum to genus level,and CBNPs.M group could cause the largest number of significant changes in taxa.Bacteroidota and Limnochordia played an important role in NC group,Enterobacteraies played an important role in CBNPs.L group,Actinobacteria and Alphaproteobacteria played an important role in CBNPs.M group,and Gammaproteobacteria played an important role in CBNPs.H group.These results indicate that CBNPs exposure can affect the composition of intestinal microbes in zebrafish,and the composition of microbes varies according to different exposure concentrations.(5)CBNPs exposure caused typical watery degeneration of zebrafish liver,with significant enlargement of cell body,blank cytoplasm,and nucleus being squeezed to one side.Transmission electron microscope results further showed that intracellular organelles were significantly reduced and disappeared,and only a small amount of mitochondria and other organelles remained in the cell membrane and near the nuclear membrane.With the increase of CBNP exposure concentration,the loss of hepatocyte organelles became more serious,indicating that CBNPs exposure caused liver damage.Oil red O staining results showed that CBNPs exposure caused the lipid droplet content in the liver to increase with the exposure concentration.The detection of glycolipid metabolism related genes in liver tissue showed that CBNPs exposure could up-regulate glycolysis-related genes PFKFB3,LDHA and LEPr(P<0.05),down-regulate the expression of GSK-3(P<0.05),upregulate the expression of lipid metabolism-related genes LIPC,PPAR-?,PPAR-?,apoa4,Fabp2 and Fabp11(P<0.05).The above results indicate that CBNPs exposure can cause disorders of glucose and lipid metabolism in the liver of zebrafish and lead to lipid accumulation.In conclusion,CBNPs exposure can change the diversity of intestinal flora,cause zebrafish intestinal tissue damage,increase the permeability of the intestinal mucosal barrier,and cause liver tissue damage through the intestinal liver axis,causing liver glucose and lipid metabolism disorders to form lipid accumulation.This result helps to provide the main information about the potential risk of carbon black to zebrafish,as well as the basis for studying the toxic effects of carbon black nanoparticles on aquatic ecosystems,and also provides a reference for comparative medicine. |
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