| Background and ObjectivesBlastocystis hominis(B.hominis)is an widely distributed opportunistic protozoan parasitizing in the host’s intestinal tract.It mainly causes diarrhea and abdominal pain-based clinical symptoms to the host.The pathogenic mechanism of the parasite is still not sure.This study aims to verify the destruction of caecum epithelial tight junction after Blastocystis hominis infection,by observing the ultrastructural damage characteristics of the infected rats’cecum ultrastructures through transmission electron microscopy,using q PCR to detect the expression of the rats’cecum epithelial tight junction protein,and using HPLC-ELSD to detect changes of the rats’intestinal permeability.At the same time,the apoptosis of cecum epithelial cells and mitochondrial damage in the infected rats were also detected.This study has preliminarily discussed the possible mechanism of the destruction of caecum epithelial tight junction in rats infected with Blastocystis hominis,providing a basis for the study of the pathogenic mechanism,clinical diagnosis and treatment of the parasite.Methods1.Establishment of the infection models and selection:ST7 B.hominis were subcultured and used for standby.Thirty male SD rats were randomly divided into five groups,with six rats each:1 week post-infection(p.i),3 weeks p.i,6weeks p.i,9 weeks p.i and uninfected control(NC).Rats in each infected group were orally infected with Blastocystis hominis trophozoites with a density of 10~8/ml,while rats in NC group were orally gavaged with PBS in equal volume.Rats in each group were sacrificed at time point of 1 week,3 weeks,6 weeks and 9weeks post-infection respectively,and their cecum tissues were taken for standby application.2.Pathological observation of the infected cecum:HE staining was conducted to observe the pathological damage of the cecum tissues of the rats at different time post-infection.Dynamic changes of cecum ultrastructure were observed by using transmission electron microscope.3.Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the RNA expression levels of tight junction cytoplasmic protein(ZO-1)and tight junction transmembrane proteins(claudin-1,occludin)in cecum tissues of the rats.4.To evaluate changes in intestinal mucosal barrier function,the ratios of lactulose(L)to mannitol(M)in urine samples of the rats before and after infection were determined by high performance liquid chromatography evaporative light scattering(HPLC-ELSD method)for intestinal permeability evaluation.5.Detection of apoptotic cells:observe the apoptosis of cecum epithelial cells of the infected rats by means of in situ terminal labeling(Tunel)method and detect the expression of B-lymphoma-leukemia-2(bcl-2)and apoptosis precursor protein(bax)in cecum tissue by q PCR.5.Detection of apoptotic cells:observe the apoptosis of cecum epithelial cells of rats by means of in situ terminal labeling(Tunel)method and detect B-lymphoma-leukemia-2(bcl-2)and apoptosis precursor protein(bax)Expression level in cecum tissue of infected rats by means of q PCR.6.Detection of mitochondrial damage:detect the m RNA expression levels of 8-hydroxyguanine DNA glycosidase(OGG1),apurinic/apyrimidine endonuclease 1(Apex1),signal transduction and transcription activator(Stat3)and protein casein tyrosine kinase(Src).Results1.Comparison of general conditions of rats:The appearance and daily activities of rats in each group were normal,and there were no statistically significant differences in the weightof the rats.The feces of the rats in control group were intact and there were no symptoms of diarrhea.Compared with the control group,the feces of rats in infected groups were softer and more watery,and the shape were incomplete.2.The result of pathological observation with HE staining:the invasion of B.hominis was found in the cecum epithelial mucosa of rats in each infected group.The injuries were most obvious at 1 week p.i,with partial microvilli shed in the intestinal epithelium and deep staining of the nuclei.3.The result of pathological observation with transmission electron microscopy:The mitochondrial morphology w ERE regularly round and the ultrastructure were normal in the rats of control group.The mitochondrial edema were obviously swollen at 1-week p.i,and the mitochondrial crests were bent or disappeared.The damages were the most serious at 1-week p.i compared to other infection groups.At 3 weeks p.i,the degree of mitochondrial swelling was slightly lighter than that at 1-week p.i.Only part mitochondria at 6 weeks p.i showed swelling and deformation,and the degree of lesions were significantly reduced.The mumber of abnormal mitochondria was the least,and only slight deformation and minimal damage at 9 week p.i.4.Detection of tight junction proteins:Compared to the control group,the m RNA expressions of ZO-1,claudin-1 and occludin all decreased significantly at the 1st and 3rd weeks p.i(P<0.05);the expression levels of Z0-1 and claudin-1 gradually increased at the 6 and 9 weeks p.i;the expression level of occludin was not significantly different at 9 weeks p.i compared to the control group(P>0.05).5.Determination of intestinal permeability of rats:the linear range of mannitol and lactulose in the urine of rats in each group is favourable.Compared to the control group,the intestinal permeability(lactulose/mannitol ratio)of rats Significantly increased at p.i.6.TUNLE cell apoptosis observation:There was no apoptosis-positive cellin the cecum epithelial of rats in the control group,while positive cells have been observed in the infection groups,and the number of positive cells was more at 1 week and 3 weeks p.i.7.Detection of apoptotic genes:Compared with the control group,the expression level of bcl-2 decreased significantly(P<0.05),and the expression level of bax increased significantly(P<0.05);at 6 weeks after infection,the expression of bcl-2 gradually increased and the expression level of bax gradually decreased(P<0.05);at the 9th week after infection,the expression levels of bcl-2 and bax approached were nealy the same with the expression of the control group(P>0.05).8.Detection of mitochondrial damage-related molecules:Compared with the control group,the m RNA expression of OGG1,Apex1 and Stat3 increased significantly at 1 and 3 weeks p.i(P<0.05),and the expression increase were more obvious at 1 week p,i.While There was no statistically significant difference in expression between the 6 and 9 week p.i;Src increased significantly at 1 week p.i(P<0.01),and then the expression level gradually decreased with the extension of the infection time.Conclusion1.B.hominis infection can cause ultrastructural damages to the cecum epithelium of rats,and the degree of infection is dynamically related to the infection time,with the most serious injury during the acute infection period.Compared with the control group,the expression of tight junction-related proteins in the infected groups decreased and the intestinal permeability increased,suggesting that the pathogenic mechanism of B.hominis may be related to the impaired intestinal barrier function caused by the destruction of cellular tight junctions.2.The pathogenic mechanism of B.hominis infection be related to the oxidative damage of mitochondria,which may induce apoptosis of intestinal epithelial cells,thereby destroys the tight junctions between intestinal epithelial cells and cause increased intestinal permeability. |
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