| Objective Candida albicans was the leading cause of fungal infection and the fourth most common cause of hospital acquired infection, with mortality rates approaching 50%. Candida albicans permanently planted within the oral cavity, low respiratory tracts, gastrointestinal and urogenital tracts of healthy humans. They could cause superficial infections, as well as leathal systemic infections through blood stream dissemination and organ invasion in immunopromised individuals. The patients suffered from candidemia, endocarditis, central nervous system infections, endophthalmitis and osteomyelitis. Interleukin-23(IL-23) involved in the process of innate immunity during Candida albicans infection. IL-23 could not only stimulate macrophages to produce more cytokines, but also activated Th17 cells, which was correlated with adaptive immune response, thus promoting Candida albicans clearance. IL-23 was essential for the survival, proliferation and reactivation of Th17 and required for the production of IL-17. Consequently, IL-23 was a major cytokine bridging the innate and adaptive immune response. Hsp90 was an evolutionarily conserved and highly abundant molecular chaperone, which was complicated in stress, inflammation and innate immune response. Additionally, Hsp90 infuenced the activity of client ptoteins and conduction of signal transduction cascades. This experiment was to explore the expression and secretion levels of IL-23 after the interaction between macrophages and Candida albicans in vitro and to investigate whether Hsp90 inhibitor will affect expression and secretion levels of IL-23.Methods 1 Mice Kunming mice, female, 4 to 5 weeks old, 15-20 g, were purchased from the experimental animal center of He Bei Medical Universit-y.Certificate number was 1404078. 2 Strains The type strain of Candida albican(ATCC 90028) was purchased from the Institute of Microbiology Chinse Academy of Science. 3 Activation and preparation of suspension The type strain of candida albican rewarmed and activated on Sabouraud Dextrose Agar for three times. RPMI 1640 medium supplemented with 0.1% Tween 80 was added to the colony and beat blended through a straw.The suspensions were counted using a hemocytometer and finally adjusted to 1-2×106/m L. 4 Isolation, culture and identification of mouse macrophages The mouse macrophages were obtained by intraperitoneal lavage with RPMI 1640 medium. After centrifuge and counting, the cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum eventually. Cellular morphorlogy was observed under inverted microscope. The macrophages were collected by centrifugation after 3 days culture and washed with phosphate-buffered saline(PBS) for three times. Then the cells were fixed with acetone for routine HE and Wright-Giemsa staining. The expression of antigen of CD68 was detected by Immunohistochemical SP method. 5 The experimental group and handling At 3th day, the macrophages were collected, centrifuged and diluted in RPMI 1640 supplemented with 10% fetal calf serum and counted with hemocytometer to adjusted the final concentration to 1-2×106/m L.Then the cells were transferred to 6 well round-bottom plates with 106 CFU/well. After incubation 24 hours, 0.5 m L candida albican suspensions were added to every well. 100mL 17-DMAG(Hsp90 inhibitor) with different concentrations of 0.1mmol/L~0.5mmol/L was added in experimental group and the same volume of RPMI 1640 supplemented with 10% fetal calf serum in control group. In 0.1mmol/L group, the cells and supernatants were collected at 2h, 4h, 6h, and 12 h. The supernatants in orther different concentrations group were collected at 4h and stored at-80℃. 3 parallel holes were seted for each well. The expression of IL-23 at 2h, 4h, 6h,12 h was determined by flow cytometric ana-lysis and sandwich enzyme-linked immunosorbent assays(ELISAs). 6 Flow Cytometric Analysis Macrophages of all groups were blowed down from six orifice, centrifugated and washed with PBS for 2 times. 0.1% Triton X- 100 was used to permeabilized, and the cells were incubated avoiding light for 20 minutes. Subsequently,IL-23 antibody was used to stain and the cells were incubated avoiding light for 15 minutes. The expression of IL- 23 levels in macrophages were detected by flow cytometry instrument, and the data was analyzed with FACSDiva Software. 7 Analysis of cytokine IL-23 Secreted by Macrophages The supernatants in all groups were rewarmed and detected according to the kit instructions.Results 1 The morphology observation of macrophage cultured. At 1th day, macrophages were round or oval, and adherent growth under inverted microscope. At 3th day, the cells volume increased slightly, as round, ellipse, polygon, and most cells had pseudopodia and bump, adherent growth. 2 The results of HE stain and Wright-Giemsa stain. The results of HE staining The pink macrophage cytoplasm was rich and the blue nuclear was big. The results of Wright-Giemsa staining The macrophage cytoplasm was light blue and the dark blue nuclei were in the side of the cells. 3 The results of immunohistochemical stain. CD68 positive reaction was observed in macrophage cell membrane and cytoplasm. 4 The results of the expression of IL-23 analyzed by FACS. At 2h, 4h, 6h and 12 h, the expression level of IL-23 in experimental group were 6.24±1.16,8.09±1.15,10.54±0.67 and 3.69±0.21 respectively, which were 8.19±1.01,10.06±0.32,12.10±1.97 and 4.76±0.86 respectively in the control group. The expression trend was increasing in early phage anddecreasing in late phage, reached a peak at 6h. The expression level of IL-23 in experimental group were lower than that in control group, F=55.643 P=0.000, the difference was statistically significant. 5 The results of the expression level of IL-23 in the supernatants. At 2h, 4h, 6h and 12 h, the expression level of IL-23(pg/ml) in experimental group were 225.17±7.95, 243.60±5.81, 257.61±12.48 and 259.19±7.92 respectively, which were 268.99±14.34, 278.62±14.54,295.96±5.65 and 309.79±4.34, respectively in the control group. The expression level increased with the prolongation of time, and the growth rate obviously slowed down at 6h. The expression level of IL-23 in experimental group were lower than that in control group, F=17.41 P=0.000 P <0.05, the difference was statistically significant. 6 Effects of different concentration of HSP90 inhibitors on the secretion level of IL-23 in supernatants. In 0.01 mmol/L,0.1 mmol/L,0.25 mmol/L and 0.5 mmol/L concentration group, the secretion level of IL-23(pg/ml) in the supernatants were 261.21±2.02, 254.09±1.92, 248.69±1.30 and 234.41±3.81, respectively. With the increasing concentration of 17-DMAG, the secretion level of IL-23 decreased correspondingly, F=64.43 P <0.05, the difference was considered statistically significant.Conclusions 1 C. albicans interacted with mice peritoneal macrophages in vitro, longer duration of action, IL- 23 expression levels rised in early stages and appeared downward trend in the late stages. 2 C. albicans interacted with mice peritoneal macrophages in vitro, IL- 23 expression and secretion level declined obviously after joining HSP90 inhibitors. 3 The influence of HSP90 inhibitors that candida albicans stimulated secretion and expression of IL – 23 in mice abdominal macrophage was concentration and time dependence. In conclusion the results showed that c. albicans interacted with mouse p-eritoneal macrophages in vitro, after joining HSP90 inhibitors, IL-23 expression level decreased obviously, time- and concentration- dependently. HSP90 inhibitors may reduce the damage to the organization by reducing the secretion and expression of IL- 23, thus had protection effect to the body. |
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