Study On Caenorhabditis Elegans-based Screening Technology Of Intestinal Damage Caused By Toxins

Recently,intestinal injury assessment is mostly limited to rat and cell models.There have been few reports of Caenorhabditis elegans（C.elegans）used as model for this field.C.elegans has the biological characteristics as a model of intestinal injury assessment:small size,rapid life cycle,easy cultivation,simple operation,high homology with human genes,non-toxic and harmless,and widely used in toxicity test and toxicity assessment.In addition,it has unique advantages in studying intestinal injury:transparent body,easy to observe after intestinal staining.The intestinal epithelial cells of C.elegans are similar to human intestinal epithelial cells.Therefore,this study developed the detection technology for C.elegans intestinal injury caused by model substances,and established a technology that is appropriate for screening the potential intestinal toxicity of exposure to environmental toxins.Part I Study on dose-response relationships between exposures of intestinal injury model substances and mortality outcomes in C.elegansObjective:To determine the dose-response relationships between exposures of intestinal injury model substances and mortality outcomes in C.elegans.Methods:At stage L4,C.elegans were exposed to different concentrations of model compounds[Dextran sodium sulfate（DSS）,ethanol,sodium dodecyl sulfate（SDS）,lipopolysaccharide（LPS）,glucose)]in solution for 24 h,then count the death number of C.elegans under an optical microscope.Three parallel wells were set for each test of each compound per concentration,and the number of C.elegans per well was controlled at 60-80;The standard of C.elegans death is loss of exercise capacity,stiffness or rod-like shape,and no response to platinum wire touching.The experiment was repeated 3 times.Probit regression analysis was used to calculate the 24 h-LC₁（one percent of lethal concentration）,24 h-LC₁₀（ten percent of lethal concentration）and 24h-LC₅₀（median lethal concentration）of C.elegans.Results:1.DSS,ethanol,SDS,LPS,glucose made dose-response curve of C.elegans’ death showed S type.2.Model compound-induced C.elegans 24 h-LC₁were DSS group（3.4%±0.9%;g/m L）,ethanol group（3.1%±0.6%;v/v）,SDS group（6.2±1.1;mg/L）,LPS group（0.07±0.025;mg/m L）,glucose group（133.6±17.3;mmol）,24 h-LC₁₀were DSS group（15.7%±1.8%;g/m L）,ethanol group（4.2%±0.3%;v/v）,SDS group（45.7±9.8;mg/L）,LPS group（0.3±0.05;mg/m L）,glucose group（375.1±12.8;mmol）,24 h-LC₅₀DSS group（32.7%±2.4%;g/m L）,ethanol group（5.9%±1.1%;v/v）,SDS group（146.9±22.3;mg/L）,LPS group（3.2±0.3;mg/m L）,glucose group（671.3±21.4;mmol）.Conclusions:The dose-response relationship of C.elegans death caused by intestinal injury model substances provided a foundation for the subsequent dose setting in followed research about intestinal permeability detection technology.PartⅡEstablishment of C.elegans-based intestinal injury detection technologyObjective:To develop C.elegans-based intestinal injury detection technology.Methods:In this part,we used different concentrations of DSS（2%,4%,8%,16%,32%）to induce C.elegans intestinal injury,After the exposure,using K solution to clean C.elegans,and stained with bright blue dye and low-molecular Fluorescein isothiocyanate-dextran（FD-4）.During the test,concentrated E.coli OP50 was used as nematode food.When the process of staining is over,C.elegans recovered on the Nematode Growth Medium（MGM）plate for a period of time.The percentage of stained C.elegans and the fluorescence intensity of a single C.elegans FD-4 were used as indicators to evaluate the degree of C.elegans intestinal injury.On the basis of this test procedure,the test conditions（the concentration of OP50,the concentration and staining time of bright blue and FD-4,recovery time of C.elegans after staining）were optimized.Results:1.When the stain volume is 200μL,the fluorescence intensity of the single nematode in the control group was set to 1,and the fluorescent intensity（3.4±0.3）of the single nematode added with 10μL OD=1 bacterial solution is stronger than that of the single nematode added with 5μL group（2.5±0.4）.2.The fluorescence intensity of single nematode with dye concentration of 0.05%,FD-4 had statistical difference compared with the control group（P<0.05）.3.When recovering on the NGM plate for 30 min,the average fluorescence intensity of C.elegans in the 8%DSS group was statistically different from that of the control group（P<0.05）.Conclusions:The test conditions of intestinal permeability detection technology based on C.elegans are determined as follows:OD=1 OP50 concentration is 5%;the final concentration of Brilliant blue and FD-4 staining are 5%and 0.5%;the staining time is 3 h;The recovery time on the NGM board is 45 minutes.Part III Study on intestinal injury induced by model compounds in C.elegansObjective:Verification of reliability of C.elegans-based intestinal injury detection technology.Methods:The intestinal permeability detection technology established in PartⅡof this study,which was used to evaluate the intestinal injury ability of C.elegans caused by DSS,ethanol,SDS,LPS and glucose.For the glucose test group,in order to avoid the effect,which the hypertonic effect of glucose on the physiological state,an additional concentration of mannitol was added as the test control group,and the remaining test chemicals were controlled by K solution.SPSS 21.0 software package was used for data analysis,and the inspection level was P<0.05.Results:1.With the exposure dose increases,the Brilliant blue dye percentage of C.elegans intestinal induced by DSS,ethanol,SDS,LPS,and glucose were increased,and the average fluorescence intensity of a single C.elegans FD-4 were increased,too.2.Collecting the percentages of brilliant blue dye stained outside of C.elegans intestine.In the low-dose group of the above five substances were:2%DSS group（5.0%）,2%ethanol group（17.2%）,1 mg/L SDS group（9.5%）,0.025 mg/m L LPS group（13.9%）,when glucose dose lower than 12.5mmol,the nematode gut was not externally stained;the percentage of bright blue staining in the middle-dose group were:8%DSS group（70.3%）,4%ethanol group（57.1%）,25 mg/L SDS group（54.2%）,0.1 mg/m L LPS group（73.7%）,200 mmol glucose group（17.5%）;the percentage of bright blue staining in the high-dose group were:32%DSS group（94.5%）,6%ethanol group（84.4%）,125 mg/L SDS group（77.9%）,3 mg/m L LPS group（100.0%）,400 mmol glucose group（30.9%）.Conclusion:C.elegans can detect the intestinal injury of model substances with different intestinal toxicity mechanisms,which suggested that the technology based on C.elegans established in this study can be used to evaluate the intestinal injury caused by environmental substances.