| Objective: Inflammatory reaction is an essential composition of defense to pathogen, however, when it overacts or runs inadequately, harmful or even fatal results may come along. Numerous studies have confirmed that the imbalanced cytokines mediating the interaction of natural and acquired immunity contribute to human sepsis and immune dysfunctions. The inflammatory response and disease progression of candidemia presents a similar clinical picture as seen for bacterial sepsis. The clinical symptoms mainly include fever(moderate fever or high fever), hypotension, tachycardia,metabolic acidosis in the early stage, and some patients with advanced stage often developed septic shock and multiple organ dysfunction. The MAPK family is an important part of the signal transduction of inflammation and stress, and regulate a series of cellular processes. The MAPK family includes p38, JNK, and ERK, and is activated by phosphorylation. In the bacterial sepsis,the expression of p-p38, p-ERK, and p-JNK were found to increase significantly, and MAPK phosphorylation can lead to organ damage. In addition, p38 can regulate the activation of macrophages and T cells on traumatic injury reaction. Heat shock proteins, including HSP90, constitute a group of “chaperone” proteins that help to maintain the stability of client proteins and/or target the degradation of unfolded proteins when cells are exposed to heat shock or other kinds of stress.HSP90, an important molecular chaperone, is responsible for tertiary folding of client proteins such as IKK, IRAK-1 and MAP kinases and inhibition of HSP90 diminishes innate immune responses via TLR signaling. HSP90 inhibitors could attenuate inflammation and prolong survival in a murine model of sepsis. In vitro studies with macrophage stimulated by LPS, HSP90 inhibitors may exert anti-inflammatory effects by inhibiting p38 phosphorylation and enhancing apoptosis. However, it had not been reported that the influence of HSP90 inhibitor on inflammatory responses triggered by Candida albicans. In order to dissect the molecular mechanisms underlying the role of anti-infliammtory by 17-DMAG, we performed the studies with mice abdominal macrophage stimulated by Candida albicans in vitro. By deteting expression of phosphoo-p38 and proliferation of macrophages, to investigate the effect of HSP90 inhibitor on native immunity.Methods:1 MiceKunming mice, female, 4 to 5 weeks old, 18-23 g, were purchased from the experimental animal center of HeBei Medical University. Certificate number was 1508052.2 StrainsThe type strain of Candida albican(ATCC 90028) was purchased from the Institute of Microbiology Chinse Academy of Science.3 Activation and preparation of suspensionThe type strain of Candida albican were 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/mL. The suspensions were put in 65℃water for 30 min., and then Candida albicans were inactivated.4 Isolation, culture and identification of mouse macrophagesThe 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 staining. The expression of antigen of CD68 was detected by Immunohistochemical SP method.5 The experimental group and handlingAt 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 5-6×106/mL. Then the cells were transferred to 6 well round-bottom plates with 2×106 CFU/well. After incubation 8-10 hours, 0.5 mL Candida albican suspensions were added to every well. 100μL 17-DMAG(HSP90 inhibitor) with different concentrations of 0.01μmol/L~1μmol/L was added in experimental group and the same volume of RPMI 1640 supplemented with 10% fetal calf serum in control group. In the every group, the cells were collected at 12 h. 4 parallel holes were seted for each group.6 Western blot analysis were used to detect expression of phosphoo-p38Macrophages of all groups were blowed down from six orifice. Then cell were rinsed with cold PBS and lysed in RIPA buffer containing phosphatase inhibitors. Firstly, all sample total protein extracted were run on SDS-Tris-HCl Ready Gels, and then transferred onto PVDF membrane. Secondly, membranes were blocked with milk blocking solution, and subsequently incubated with primary antibodies(phosphoo-p38,1:300; β-actin,1:1000).Thirdly, membranes were incubated with appropriate secondary antibodies(1:3000). Finally, membranes were scanned by double color infrared laser scanner. The results were integrated using gel analyst software(ImageJ).7 The effect of HSP90 inhibitor on proliferation of macrophages stimulated by Candida albicansThe macrophages were counted with hemocytometer to adjusted the final concentration to 5-6×105/mL. Then cells were transferred to 96 well round-bottom plates with 100μL. After incubation 8 hours, 90μL Candida albican suspensions with 5.5-6.5×105CFU/mL were added to every hole. At the same time, join the final concentration of 0.01μmol/L, 0.1μmol/L, 0.5μmol/L, 1μmol/L HSP90 inhibitor with 10μL. By this way, macrophages, Candida albican and different concentration of HSP90 inhibitor were mixed and co-culture for 12 hours. In addition,control group(group just not drugs) and the blank group(group with medium and Candida albicans) were set up. After 12 hours, CCK-8 reagent(20μL/hole) were added to each hole. Finally, the A values of each hole were determinated in the wavelength of 450 nm after 3 hours. 3 parallel holes were seted for each group.8 Correlation between inhibition rate on the proliferation and expression level of p-p38 was analyzed.9 Statistical analysesStatistics were calculated using SPSS 13.0.Results are expressed as the mean ± standard deviation. Differences between groups were tested for statistical significances using One-Way ANOVA. Comparisons between means were assessed using S-N-K. For all test performed, P value of<0.05 was considered statistically significant.Results:1 The morphology observation of macrophage culturedAt 1th day, macrophages were round or oval, and adherent growth under inverted microscope. At 3th day, the cells volume increased, as round, ellipse, spindle-shaped, polygon, and most cells had pseudopodia and bump, adherent growth.2 The identification of macrophagesThe results of HE staining:The pink macrophage cytoplasm was rich and the blue nuclear was big.The results of immunohistochemical stain:CD68 positive reaction was observed in macrophage cell membrane and cytoplasm.3 The results of the expression of p-p38 detected by Western blot assaysIn 0.01μmol/L,0.1μmol/L, 0.5μmol/L and 1μmol/L concentration group, the expression level of p-p38 in the whole cells were 1.108±0.122, 0.699±0.054, 0.621±0.095 and 0.397±0.043, respectively. And in the control group, the expression level of p-p38 was 1.383±0.042. The results showed that the levels of p-p38 in HSP90 inhibitor treating macrophages were downregulated as compare with control group.(P<0.05) In addition, under certain concentration, the influence of HSP90 inhibitors that Candida albicans stimulated expression of p-p38 in mice abdominal macrophage was concentration dependence.(P>0.05)However, between 0.1μmol/L and 0.5μmol/L concentration group, the expression level of p-p38 had no difference.(P>0.05)4 The results of HSP90 inhibitor on the proliferation of macrophages by cell counting kit-8(CCK-8)With the HSP90 inhibior(0.01μmol/L, 0.1μmol/L, 0.5μmol/L and 1μmol/L) groups for 12 h, the inhibition rate was(43.33±10.06)%,(55.60±10.09)%,(56.33±7.14)%,(77.33±6.65)%,respectively. At the concentration of 1μmol/L, the inhibitory effect on the proliferation of macrophages was stronger than the other three groups.(P<0.05). But no significant statistic difference was found out among other three groups(P>0.05). So under a certain concentration, the effect of HSP90 inhibior on macrophages stimulated by Candida albicans can inhibit the proliferation in a dose-dependent relationship.5 Correlation between inhibition rate on the proliferation and expression level of p-p38 was analyzed.In the experimental group, there was negative correlation between inhibition rate on the proliferation and expression level of p-p38 at each concentration. r=-0.719(P<0.01)Conclusions:1 The results showed that the levels of p-p38 in HSP90 inhibitor treating macrophages were downregulated. Under a certain concentration, the influence of HSP90 inhibitors that Candida albicans stimulated expression of p-p38 in mice abdominal macrophage was concentration dependence. So with the increase of the concentration of inhibitor, the inhibitory effect of HSP90 inhibitor may enchance gradually.2 Under a certain concentration, the effect of HSP90 inhibior on macrophages stimulated by Candida albicans can inhibit the proliferation in a dose-dependent relationship.3 In the experimental group, there was negative correlation between inhibition rate on the proliferation and expression level of p-p38 at each concentration.In conclusion, the results showed that in vitro studies with macrophage stimulated by Candida albicans, HSP90 inhibitors could decrese the level of p-p38 protein expression through inhibiting cell proliferation, which may exert anti-inflammatory effects, thus protecting the organization from inflammation damage. |
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