| Diquat,a non selective herbicide,is widely used in agricultural production.The large-scale application of diquat has caused inevitable risks to human and animal health and safety,and has potential harm to the ecological environment.It is reported that diquat can be detected not only in food and drinking water,but also in air,soil,surface water,groundwater and other environmental media.In this study,zebrafish（Brachydanio rerio）was used as the experimental organism to explore the toxic effect and its mechanism of diquat intoxication,so as to provide reference for the use of this compound and lay the foundation for finding effective therapeutic targets according to its toxic mechanism.1.The acute toxicity of diquat to adult zebrafish was tested by static test method.And according to the measured 96 hour half lethal concentration(LC50),1/2,1/4 and 1/10diquat concentration groups of it were set for acute exposure to zebrafish.After exposure,the acute damage effects of diquat on zebrafish gills and liver tissues were observed.The results showed that the 24,48,72,96-hour LC50of diquat to zebrafish were 27.85,21.36,17.95 and 16.92 mg·L-1.The safety concentration of diquat was 1.69 mg·L-1(SC=96 h LC50×0.1).After acute exposure to 8.46、4.23、1.69 mg·L-1of diquat for 96 hours,the results of hematoxylin and eosin（H-E）staining showed that the gill fragments were curled and shortened,and epithelial cell edema,deformation and necrosis.Hepatocytes showed obvious enlargement,and local necrosis and dissolution of hepatocytes and vacuolation of cytoplasm were observed.The pathological changes in the gills and liver of zebrafish were aggravated with the increase of diquat concentration.2.According to the results of acute toxicity test,1/5,1/10 and 1/50 of 96h LC50were used as the exposure concentration of diquat respectively.The changes of blood glucose,superoxide dismutase（SOD）,glutathione-s transferase（GST）,malondialdehyde（MDA）,cholesterol（TC）,triglyceride（TG）and low density lipoprotein（LDL）in the liver of zebrafish were determined during the exposure;The liver tissue of zebrafish was stained with hematoxylin eosin（H-E）to observe its histopathological morphology,the distribution of glycogen in cells was observed by periodate schiff reaction（PAS）staining,and the apoptosis of liver tissue was observed by terminal deoxynucleotidyl transferase mediated nick end labeling（TUNEL）staining;The expression levels of Akt and p-Akt in zebrafish liver were detected by Western blot.The results showed that compared with the blank control group,the blood sugar value of zebrafish exposed to 3.38,1.69 and 0.34 mg·L-1diquat showed an upward trend with the increase;Zebrafish were exposed to diquat for 56days,the SOD activity in the liver of zebrafish in each group showed a change process that decreased first,then increased,and then gradually decreased,and there was a certain concentration-effect relationship;the activity of GST in liver of 1.69 mg·L-1and 3.38mg·L-1diquat treatment groups increased first and then decreased,which were significantly lower than that of control group at 28th and 56th day（p<0.01）,0.34 mg·L-1diquat treatment group（p<0.05）compared with the control group,the content of MDA in the liver of the treatment group did not change significantly on the 7th and 14th day（p>0.05）,and the content of MDA in the liver of the 1.69 mg·L-1and 3.38 mg·L-1diquat treatment groups increased significantly on the 28th and 56th day（p<0.05）;the content of TG in the liver of zebrafish in the treatment group increased significantly from the 14th day to the 14th day（p<0.01）compared with the blank control group,TC in the liver of zebrafish treated with 3.38 mg·L-1diquat increased to a certain extent from 28th day,but there was no significant difference（p>0.05）;the liver LDL level of zebrafish treated with3.38 mg·L-1diquat was higher than that of the blank control group at 28th and 56th day（p<0.01）There was a significant increase on 56th day（p<0.05）in the treatment group of 1.69 mg·L-1diquat.The results of hematoxylin and eosin（H-E）staining showed that the liver cells of zebrafish were disordered,swollen,the cell boundaries were blurred,the number of cells decreased,the nucleus atrophied and shifted,the red blood cells exuded,the blood sinuses were congested,the perisinusoidal space became smaller,and the degree of damage was aggravated with the increase of exposure concentration;glycogen staining section showed that with the increase of exposure concentration of diquat the distribution of glycogen in zebrafish liver cells in the treatment group was gradually reduced and uneven,with partial aggregation;TUNEL staining showed that with the increase of exposure concentration of diquat,the number of positive cells increased;Western blot results showed that compared with the blank control group,the protein expression levels of Akt and p-Akt in liver of zebrafish in diquat treatment group were decreased,and the protein expression level of p-Akt in 3.38 mg·L-1diquat treatment group was significantly different from the control group.3.High throughput sequencing of 16S r RNA gene was used to study the structure and diversity of intestinal microbial community in zebrafish exposed to diquat.Zebrafish were exposed to 1.69 mg·L-1and 0.34 mg·L-1diquat.After 28 day,their intestines were dissected and collected.The samples were sequenced by Illumina sequencing platform to analyze the composition and diversity of intestinal microflora.LEf Se analysis was used to identify the microbial flora with statistical differences.PICRUSt2 was used to predict the characteristics of functional changes of microflora.The results showed that the number of OTUs（operational taxonomic unit）of zebrafish intestinal microflora decreased with the increase of exposure concentration;the number of OTUs shared by blank control group and 0.34 mg·L-1diquat treatment group was more than that shared by 1.69 mg·L-1diquat treatment group the results showed that the number of OTUs was small;the structure and proportion of dominant microflora in zebrafish intestinal microflora changed after exposure to diquat;principal coordinate analysis（PCo A）showed that the total microflora of zebrafish intestinal in 1.69 mg·L-1diquat treatment group and blank control group were significantly separated,although 0.34 mg·L-1diquat treatment group had no significant difference.LEf Se results showed that there were 9 kinds of intestinal microflora between0.34 mg·L-1diquat treatment group and normal control group,and 87 kinds of intestinal microflora between 1.69 mg·L-1diquat treatment group and normal control group.Picrust2function prediction of zebrafish intestinal microflora showed that the microbial populations involved in development and regeneration,neurodegenerative diseases,cardiovascular diseases,cancer,signal transduction and other processes were significantly changed in diquat exposure group.Moreover,the changes of intestinal microflora of zebrafish exposed to 1.69 mg·L-1diquat affected the metabolism of carbohydrate,lipid,amino acid and other substances.The above studies show that diquat in water has a serious acute damage effect on zebrafish.Zebrafish exposed to diquat for a long time will have oxidative stress reaction in the liver,lead to lipid peroxidation of biofilm,cell structure damage,and liver glucose disorder of glucose and lipid metabolism.The mechanism may be related to molecular changes in PI3K/Akt signaling pathway.Exposure to diquat can lead to the disorder of intestinal microflora structure and function,which leads to the disorder of zebrafish metabolism. |
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