| Objective:Extracellular signal-regulated kinase (ERK) was one of the members of the family of mitogen source activated protein kinase (MAPK) signal transduction system, it could mediate a series of physiological processes include cell growth, development,differentiation, apoptosis. From the abroad literature,ERK transduction pathway had been confirmed by the mouse model was the core pathway of cellular immune regulation.From domestic study,ERK transduction pathway could induce TH2differentiation,ERK played a vital role in immune regulation mechanism.Current research on the relationship between ERK and the disease had been carried out in the areas of cancer, diabetes with kidney disease, AIDS,but in research of home and abroad, the expression of the ERK pathway in disseminated candidiasis kidney was not reported yet.IL-10was a kind of cytokines which were secreted by TH2cells,it was important in negatively regulating the innate immune response.In recent years, innate immunity and specific immunity were paid more and more attention in the pathogenesis of disseminated candidiasis.In this study, we established mouse model of disseminated Candida albicans infection and used immunohistochemical staining methods to detect the expression of ERK2and IL-10in mouse kidney at different infection time. At the same time we explored the correlation ship between ERK2and IL-10so as to find out the probable pathogenesis of disseminated candidiasis. Method:Strains:Candida albicans were saved from the Second Hospital of Hebei Medical University.Establishment of a mouse model of disseminated candidiasis:90Kunming mice, half male and half female, weighing about25g, were purchased from Hebei Medical University animal breeding center. Adapt to the environment, they were randomly divided into A, B, C, n=30. The mice of group A was the experimental group, each mouse was injected cyclophosphamide200mg/kg by ip form lth to4th day, in the5th day, each mouse was injected lml suspension of Candidaalbicans for l～5x106cfu/ml by ip. Group B was cyclophosphamide control group, each mouse was injected cyclophosphamide200mg/kg by ip form lth to4th day, in5th day, by intraperitoneal injection of saline lml; group C was blank control group: each mouse was injected saline200mg/kg by ip form lth to5th day. At experiment6th day,9th day,20th day, mice were broken neck to observe abdominal viscera in general situation.Identification of the disseminated candidiasis infection of mice kidneys:the kidneys of mice were removed under sterile conditions, sterile normal saline several times, after the grinding of the side, inoculated in cultured on Sabouraud medium.Histopathological examination and immunohistochemical:another part of the renal tissue were fixed with10% formalin solution for routine pathological HE and PAS staining to be observed pathological changes and Candida albicans infections in mice kidney tissue. We detected the expression of ERK2and IL-10by immunohistochemical method with chemical Streptomyces antibiotic protein-peroxisome connection method (SP).Result:1. Mice model of disseminated candidasis infection:During the experiment,10of mice death due to immunosuppressant cyclophosphamide caused multiple organ hemorrhage, edema;11of mice death due to disseminated infection by Candida albicans;15died of unknown causes. Finally, a total of54mice incorporated in experimental mice.The mice of group A in experiment6th day, the mice organ were edema, not formed white pus points. In experiment9th day, by the anatomy of mice, organs had white pus point generated in abdominal cavity. In20th day, all organs had a lot of white pus point generation, part of the organ were congestion, necrosis. Group B mice, in the experimental of6th day, mice abdominal internal organs were edema and congestive. In experiment9th day, after dissection, mice abdominal internal organs were part of the edema, hyperemia, necrosis. In experiment20th day, by autopsy, the mice spleen were enlarged, liver slightly congestive, renal edema. The mice of group C spirit and appetite did not change, hair gloss, activities normal. In the experimental sections6th day,9th day,20th day, the mice abdominal organs showed normal performance respectively.2. Fungal culture and identification in mice kidney tissue of disseminated Candida albicans infections:on the6th day,9th day,20th day, the kidneys tissue of group A mice were cultured in Sabouraud medium for72hours. There were white colonies growth which were identified as Candida albicans by serum culture method with chlamydospore and pseuohypha.3. Histopathological examination:the mice of group A, in the experiment6th day, microscopically there were normal renal tubules and glomeruli, tubular cytoplasm stained red degeneration. In9th day, microscopically renal tubular cell nucleus pyknosis, cytoplasm stained red, which appeared degenerative changes. Hyphae were seen by PAS stain purple. In20th day, parts of renal tissue were necrosis, microscopically renal tubular cell nucleus pyknosis, cytoplasm stained red. Hyphae were seen by PAS stain purple. Group B mice, in the6th day,9th day and20th day, microscopically there were normal renal tubules and glomeruli, tubular cytoplasm stained red degeneration. In group C mice showed normal performance.The level of ERK2protein expression:ERK is mainly expressed in the cytoplasm of positive cells which were stained brown in cytoplasm, immunohistochemical staining results showed that there were visible positive cells. In group A, OD values in6th9th20th were:1882±0.0134,0.1962±0.0098,0.1878±0.0094respectively, the value increased at first and then decreased, P>0.05, the difference was not significant. In group B, Section6d, Section9d, Section20d OD values were:1939±0.0165,0.1900±0.0250,0.1892±0.0173respectively; expression of the trend was the expression gradually decreased, P>0.05, the difference was not statistically significant.In group C, Section6d, Section9d, Section20d OD values were:0.1985±0.0129,0.1891±0.0124,0.2023±0.0156respectively. Expression of the trend was the expression decreased at first and then increased, P>0.05, the difference was not statistically significant.In the6th day of our experiment, group A, B, C, the three OD values were0.1882±0.0134,0.1939±0.0165,0.1985±.0129respectively, expression of the trend was the expression gradually increased, P>0.05, the difference was not statistically significant.In the9th day of our experiment, group A, B, C, the three OD values were:1962±0.0098,0.1900±0.0250,0.1891±.0124respectively, expression of the trend was the expression gradually decreased, P>0.05, the difference was not significant.Section20d of the experiment, group A, B, C, the three OD values were:1878±0.0094,0.1892±0.0173,0.2023±0.0156respectively, expression of the trend was the expression gradually increased, P<±0.05. The difference was statistically significant, further pairwise comparisons, group A comparison with group C, group B with group C,p<0.05. difference were both statistically significant.The level of IL-10protein expression:IL-10expression in the cytoplasm of positive cells visible cytoplasm stained brown; immunohistochemical staining results showed that A, B, and C were visible positive cells. In group A during the experimental section6d, section9d,section20d the OD values were:0.1960±0.0355,0.2084±0.0162,0.2246±0.0361respectively, expression of the trend is the expression gradually increased, P>0.05. The differences were no significant. In group B, Section6d, Section9d, Section20d the OD values were:0.1871±0.0159,0.1978±0.0149,0.2031±0.0256respectively, expression of the trend was the expression gradually increased, P>0.05, the difference was not statistically significant. In group C, Section6d, Section9d, Section20d the OD values were:0.2387±0.0430,0.2023±0.0209,0.1943±0.0167respectively, expression of the trend was the expression gradually decreased, P>0.05, the difference was not statistically significant. In the6th day of our experiment, group A, B, C, the three OD values were:0.1960±0.0355,0.1871±0.0159,0.2387±0.0430respectively, the expression of the trend first decreased and then increased, P>0.05, the difference was not statistically significant.In the9th day of our experiment, group A, B, C, the three OD values were:0.2084±0.0162,0.1978±0.0149,0.2023±0.0209respectively, the expression of the trend first decreased and then increased, P>0.05, the difference was not statistically significant. In the20th day of our experiment, group A, B, C, the three OD values were:0.2246±0.0361,0.2031±0.0256,0.1943±.0167respectively, expression of the trend was to express the gradual decline, P>0.05, the difference was not significant.ERK2overall repeated measure count:the same group at different times, F=0.092, P>0.05, the difference was not statistically significant; at the same time different groups F=0.381, P>0.05, the difference was not significant.IL-10overall repeated measure count:the same group at different times, F=0.132, P>0.05, the difference was not statistically significant; at the same time different groups, F=1.660, P>0.05, the difference was not significant.The linear correlation between ERK2and IL-10:the results of linear correlation analysis showed the expression of ERK2and IL-10were linear correlation. At6th day, r value was-0.829, P<0.05, the difference was significant. But at9th day and20th day, r values were-0.799, and-0.077respectively,P>0.05, the difference was not significant.Conclusion:1.Compared with normal control group, ERK2expression decreased in the disseminated candidiasis mouse kidney.2. The expression of IL-10in the disseminated candidiasis mouse kidney was time-dependent increasing which might cause the susceptibility to Candida, that was one of the reasons of invasive infection in mice.3. Results of our study in the6th day of the experiment, the expression of ERK2and IL-10expression was negatively correlated with that of IL-10expression might be through inhibition of the ERK2signal transduction pathway activation, ERK2might promote the apoptosis of immune cells inhibit immune response, resulting in increased susceptibility of the organism Candida albicans. |
PDF Full Text Request