| Objective: Disseminated candidiasis was associated with the body’snatural immunity and specific immunity. In particular, the role of naturalimmunity in the pathogenesis was attracted more attention recently. Toll likereceptors(TLRs), one of the most important pattern-recognitionrecaptors(PRRs),trigger and regulate natural and adapted immunoreactionthrough the recognition of pathogen-associated molecular patterns (PAMPs).The unique glycolipid present in the cell surface of Candida albicansmannan can be recognized by TLR4, which induced the release ofproinflammatory cytokines and promoted the recruitment of neutrophil cells.Heat shock proteins (HSP) were highly conservatived and widespread inbiological evolution of proteins in prokaryotic and eukaryotic organisms. Heat,hypoxia, infection, oxidants and other stimuli can induce its expression. HSPnot only involve in oxidative stress response of the body, but also involve inthe body’s natural immune process. The association between HSP90andTLR4with the pathogenesis of disseminated candidiasis has not been reported.Studies had shown that HSP cytokine effects through TLR2and TLR4signaling pathways, leading to implementation of NF-KB factor and activationof MAPK kinase. In this study, we established the mouse model ofdisseminated Candida albicans infection and explored the relationshipbetween HSP90and TLR4expression in the kidneys of mice withdisseminated candidiasis. At the same time, we explored the immunemechanisms in the disseminated candidiasis and might provide the theoreticalbasis of the immune and biological treatment.Methods:1Experimental strains of Candida albicans from the Second Hospital ofHeBei Medical University. 2Establish disseminated candidiasis model in mice：90Kunming miceof clean grade, male and female,5to6weeks of age,20to25g weight ofeach mouse, purchased from the experimental animal center of HeBei MedicalUniversity. After adapted to environment,the mice were divided into A、 B、C group randomly by weigh（tn=30）. Mice of group A were the experimentalgroup: each mouse was injected Cyclophosphamide （200mg/kg）by ip form1thto4thday.In the5thday each mouse was injected0.2mL Suspension ofCandida albicans for (1～5×106cfu/mL) by ip. Mice of group B wereCyclophosphamide control group: each mouse was injectedCyclophosphamide （200mg/kg）by ip form1thto4thday.In5thday byintraperitoneal injection of saline0.2mL. Mice of group C were blank controlgroup: each mouse was injected saline (200mg/kg) by ip form1thto5thday.At the experiment6th,9th,20thday, mice were broken neck to observed celiacviscera in general situation.3Identification of disseminated candidiasis infection of mice kidneys：thekidneys of mice were removed under aseptic conditions, aseptic normal salineseveral times, after the grinded of the kidney tissue, inoculated in cultured onSabouraud medium.4Histopathological examination and Immunohistochemical： kidneytissue were fixed with10%Formalin for routine pathological HE and PASstaining. Pathological changes and Candida albicans infections in mice renaltissue were abserved. We detected the expression of TLR4and HSP90byImmunohistochemical methold with chemical Streptomyces Antibioticprotein-peroxisome connection method (SP).Result:1Establish disseminated candidiasis model in mice： during theexperiment, a total of11mice died, owing to multiple organedema,hemorrhage by Cyclophosphamide immunosuppressant. Because ofdisseminated infection by Candida albicans, a total of12mice died，a total of13mice died with unexplained reasons.54mice were included in theexperiment.Group A from the6thday: mice appetite and spiritual were poor、 had dull hair、weight and breathing normally. By anatomy, the mice organwere edema, not formed white pus points. In9thday：by the anatomy of mouse,organs had white pus point generated in abdominal cavity. In20thday：organshad a lot of white pus point generation, part of the organ were necrosis andcongestion. Group B from the6thday：appetite and spiritual were poor, dullhair, weight and breathing normally. By anatomy the mice abdominal internalorgans were congestive and edema. In9thday by anatomy the mice abdominalinternal organs were part of the edema, necrosis,hyperemia. In20thday byanatomy the mouse spleen were enlarged,liver congestive, renal edema. Themouse of group C appetite and spirit did not change,hair gloss and activitiesnormal. In6th,9th,20thday, the mice abdominal organs showed normalperformance.2Fungal identification and culture in mouse kidney of disseminatedCandida albicans infections: in the6th,9th,20th, the kidneys of group A micewere cultured in Sabouraud medium for72hours. There were white coloniesgrowth which were appraisal as Candida albicans by serum culture methodwith pseuohypha and chlamydospore.3Histopathological examination: the mice of group A, in6thday kidneytubule and glomerular had complete structure, no spores and hyphae were seenby PAS stain purple. In9thday：the structure of renal tubule and glomerularwere not complete,nuclear condensation,spores and hyphae were seen by PASstain. In20thday：renal tissue were necrosis,spores and hyphae were seen byPAS stain purple. The mouse of group B：in6th,9th,20thday renal tubular andglomerular structure complete,some cells degeneration,cytoplasmhomogeneous red dye, the nucleus intact. In group C mice showed normalperformance.4The level of TLR4protein expression: TLR4is mainly expressed in themembrane of positive cells which stained brown. Immunohistochemicalresults show that there were positive cells in each group. In group A：ODvalues6th,9th,20thday were:0.1935±0.2019、0.2183±0.0369、0.1991±0.3083respectively, the expression trend was rising in early and declining in late phage.P <0.05, the difference was statistically significant. In group B：ODvalues6th,9th,20thday were:0.1980±0.0290、0.2107±0.0240、0.2100±0.0288respectively, the expression trend was rising in early and declining in latephage. P>0.05, the difference was not statistically significant. In group C：ODvalues6th,9th,20thday were:0.1834±0.0182、0.2017±0.0258、0.1962±0.0232respectively, the expression trend was rising in early and declining in latephage. P <0.05, the difference was statistically significant. In6thday groupA,B, C, the three OD values were0.1935±0.2019、0.1980±0.0290、0.1834±0.0182respectively, the TLR4expression in experimental groupincreased than that of control group.P>0.05, the difference was notstatistically significant. In9thday group A,B,C, the three OD values were0.2183±0.0369、0.2107±0.0240、0.2017±0.0258respectively, the TLR4expression in experimental group increased than that of control group, P>0.05,the difference was not statistically significant. In20thday group A,B,C, thethree OD values were0.1991±0.3083、0.2100±0.0288、0.1962±0.0232respectively, the TLR4expression in experimental group increased than thatof control group. P>0.05, the difference was not statistically significant.Repeated measures design ANOVA: there was significant difference at thedifferent time points, F=8.093,P<0.05the difference was statisticallysignificant. there was not significant difference between the groups,F=3.595,P>0.05the difference was statistically significant.5The level of HSP90protein expression: HSP90is mainly expressed inthe cytoplasm of positive cells which stained brown. Immunohistochemicalresults show that there were positive cells in each group. In group A：ODvalues6th,9th,20thday were:0.1851±0.0201、0.2155±0.0345、0.1894±0.0266respectively, the expression trend was rising in early and declining in latephage, P <0.05the difference was statistically significant. In group B：ODvalues6th,9th,20thday were:0.1769±0.0183、0.1961±0.0234、0.1924±0.0310respectively, the expression trend was rising in early and declining in latephage, P>0.05the difference was not statistically significant. In group C：ODvalues6th,9th,20thday were:0.1825±0.0300、0.1945±0.0345、0.1925±0.0351 respectively, the expression trend was rising in early and declining in latephage, P>0.05the difference was not statistically significant. In6thday groupA,B,C, the three OD values were0.1851±0.0201、0.1769±0.0183、0.1825±0.0300respectively, the HSP90expression in experimental groupincreased than that of control group, P>0.05, the difference was notstatistically significant. In9thday group A,B,C, the three OD values were0.2155±0.0345、0.1961±0.0234、0.1945±0.0345respectively, the HSP90expression in experimental group increased than that of control group, P>0.05,the difference was not statistically significant.In20thday group A,B,C, thethree OD values were0.1925±0.0351、0.1924±0.0310、0.1894±0.0266respectively, the HSP90expression in experimental group increased than thatof control group, P>0.05, the difference was not statistically significant.Repeated measures design ANOVA: there was significant difference at thedifferent time points, F=8.375，P<0.05the difference was statisticallysignificant. there was not significant difference between the groups, F=0.914，P>0.05the difference was statistically significant.6The expression of TLR4and HSP90in experimental group in6th、9th、20thday had no linear correlation, r=0.002,0.000,0.081respectively. P>0.05the difference was not statistically significant.Conclusion:1Expression of TLR4in renal tissue with disseminated candidiasismice increased in early stage of infection, in the late stages appeareddeclining trend.2Expression of HSP90in renal tissue with disseminated candidiasis miceincreased in early stages of infection, in the late stages appeared decliningtrend.3Expression of TLR4and HSP90in the late stages both showed a trendof decline, which may be one of the reasons for Candida albicans invasiveinfection in kidney.4Expression of TLR4and HSP90in kidney with disseminatedcandidiasis mice elevated than that of normal mouse kidney. 5The expression of HSP90and TLR4was not linear relation indisseminated candidiasis mouse kidney. |
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