| Objectives:To establish a stable rat model of B.hominis by modifying the way of infection of rats with B.hominis.To compare the immunopathological changes of rats in different weeks of infection,observe the pathological changes and ultrastructural changes of intestinal tissues of infected rats.The expression of IL-33/ST2 and related factors in B.hominis-infected rats and possible pathways were detected.To provide new ideas for the diagnosis and treatment of Blastocystis protozoosis.Methods:Part One A modified method to infect rats with B.hominisLock’s-Egg-Serum(LES)medium was used for culture and breeding.Thirty male Sprague-Dawley rats were randomly divided into five groups,one control group and four infection groups,with six rats in each group.The rats in infected group were orally infused with trophozoites of B.hominis by the numbers of 105,106,107 and 108,respectively,while the control group was infused with an equal volume of PBS.Rats’ Fecal examination was started on the third day after infection.After being cultured in LES medium for 72 hours,B.hominis were observed,if there were any.The feces were collected every 3days for in vitro culture and continuously cultured for 5 times,and the number of infected rats was calculated.On the 21 st day after infection,all the rats were sacrificed by cervical dislocation.The duodenum,jejunum,ileum,cecum and colon tissues were taken,and the intestinal contents of each intestinal segment were cultured and counted in vitro.The infection of rats were finally confirmed by PCR.The genotypes of the strains before and after infection were determined by sequencing,and the pathological damage of the intestinal tissue were observed by HE staining.Part Two Expression of IL-33/ST2 and related factors in B.hominis infection1.Establishment of infection model: Thirty male SD rats were randomly divided into five groups,one control group and four infection groups,with six rats in each group.The infected group was infected for 1 week,3 weeks,9weeks and 24 weeks respectively.The rats in infected group rats were orally infused with trophozoites of B.hominis by the numbers of 108,and the control group was infused with an equal volume of PBS.Rats were euthanized at the 1st week,3 week,9 week and 24 week after infection.Blood was collected and the rats were sacrificed by cervical dislocation.The ileum,cecum and colon tissues were taken for use.2.Immunopathological observations of infected rats: The pathological damage of ileum,cecum and colon tissues were observed by HE staining.Ultrastructural damage of cecum was observed by transmission electron microscopy.The expression levels of IL-33,ST2 L,s ST2,IL-2,TGF-β,Foxp3,IL-10,IL-4 and IL-6 m RNA in the cecum were detected by RT-q PCR.The expression levels of Ig G in serum were detected by ELISA.Results:Part One A modified method to infect rats with B.hominisThe number of infected rats were 2,3,5 and 6 in the 105,106,107 and 108 infected groups,respectively.In the 105 infected group,2 positive mice appeared on the 6th day and continued until the 21 st day.The number of positive mice in the 106 infection group fluctuated,and the number of positive mice detected at each time point ranged from 1-4.In the 107 infected group,6 positive mice were found on the 6th day,and 5 positive mice were found at the other time,which was stable.In the 108 infected group,5 positive mice were found on the 3rd day,and 6 positive mice were found at the other time.The infection status was the most stable.At each time point,no rats got infected in the control group.Using18 s r DNA analysis and sequence alignment,we confirmed that the subtype of B.hominis were ST7,no matter before or after infections.The examination of intestinal contents after in vitro culture showed that B.hominis were found in the ileum,cecum and colon,and the number of B.hominis in cecums was positively correlated with that in the colons(p<0.05).B.hominis and mucin lay found in the section of cecums with H.E staining,while no parasitic protozoa were found in the rest setion of intestines.Part Two Expression of IL-33/ST2 and related factors in B.hominis infection1.Intestinal pathological changes: The results of HE staining showed that the structure of the ileum was intact,the intestinal villi were arranged regularly,and no obvious pathological changes occurred.In the cecum and colon,B.hominis invaded the epithelial tissue,and even the B.hominis invaded the lamina propria in the colon.Transmission electron microscopy observation of cecal tissue showed that the microvilli of B.hominis rats were severely detached and sparsely arranged,the tight junctions between cells were fused,the boundary was blurred,and the nucleus was shrank.2.RT-q PCR was used to detect the expression levels of IL-33,ST2 L,s ST2,IL-2,TGF-β,Foxp3,IL-10,IL-4 and IL-6 m RNA in the cecal of B.hominis-infected rats.At the 1st week and the 9th week of infection,the expression levels of each cytokine were significantly changed compared with the control group(p<0.01),the expression levels of IL-33 and ST2 L were significantly up-regulated,and the signal pathway-related factor IL-2 was significantly increased.The expression level was significantly higher than that of the control group(p<0.01).The soluble receptor s ST2 of IL-33 has a negative feedback regulation on the IL33/ST2 signaling pathway.Our results show that the m RNA expression level of s ST2 was significantly higher than that of the control group at the 3rd week and the 9th week of infection(p<0.05).Activation of the IL-33/ST2 signaling pathway promotes the activation of Treg cells.We found that the expression of TGF-β in the first week after B.hominis infection was significantly up-regulated compared with the control group(p<0.01),then gradually decreased,and the expression level was significantly lower at the 9th week than the control group(p<0.05),while the expression levels of Treg cell markers Foxp3(p<0.01),anti-inflammatory factors IL-10 and IL-4(p<0.05)were significantly increased at the first week of infection,and the expression of pro-inflammatory factor IL-6 was also observed.The level was also significantly higher than the control group(p<0.01).At the 24 th week of infection,the expression levels of each factor tended to be normal,and there was no difference between the two groups(p>0.05).Activation of the IL-33/ST2 signaling pathway promotes the activation of Treg cells and secretes IL-10 and other factors to exert an anti-inflammatory effect.3.Immunoglobulin changes: ELISA was used to detect Ig G antibodies in serum.The results showed that the expression levels of total Ig G antibody were different at different time points(p<0.001).LSD test for pairwise comparison,the infection group of 3th week and 24 th week were all significantly up-regulated compared with the control group(p<0.05).Conclusions:1.The rat infection model can be successfully established by using the trophozoites.105 B.hominis trophozoites were able to successfully infect rats,and the infection effects of 108 trophozoites were stable.Invasive B.hominis can be found in the cecum and colon after infection with rats.The cecum and colon may be the sites where the B.hominis are settled and pathogenic.The results of electron microscopy showed that B.hominis infected rats could cause damage to the ultrastructure of rat cecal epithelial cells.2.The expression of IL-33/ST2 and related factors were up-regulated after B.hominis-infected rats,suggesting that the body may stimulate IL-2 production by activating IL-33/ST2 signaling pathway,co-stimulation with TGF-β,causing up-regulation of Foxp3 expression.Treg cells are activated to increase the secretion of IL-10 to participate in immune regulation.3.The expression of IL-33/ST2 and IL-4,IL-6 and Ig G antibodies were up-regulated in B.hominis-infected rats,suggesting that the body may also promote the expression of Th2 cytokines by activating IL-33/ST2 signaling,thereby mediating humoral immunity. |
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