| Background: With the widespread use of immunosuppressive agents,the incidence of skin fungal infections has gradually increased,but what is the specific mechanism? We speculate that natural immunity is related to the suppression of the body's natural immune function.As the first line of defense against external pathogens,natural immunity plays an important role in the anti-fungal infection,but the related research is rare at present.In this study,we constructed a model of fungal infection in vitro,and studied the reasons and specific mechanisms of the increased incidence of fungal infection caused by long-term use of immunosuppressants from the aspect of natural immunity.Objective: In this study,zymosan A was used to induce Ha Ca T cells to construct cell model of fungal infection in vitro.Observe the effect of four kinds of common dermatology immunosuppressants,such as Cyclosporin A,cyclophosphamide,mycophenolate mofetil,to Ha Ca T cells induced by zymosan A,then detecting the expression of TLR2 /Dectin-1and TNF-a /IL-6,from cellular level to explore different immune inhibitors,skin fungal infections associated with pattern recognition receptors.Methods: Part 1:Cultured Ha Ca T cells with different concentrations(0,1,10,100 ?g/ml)of zymosan A in Ha Ca T cells 24 h,using fluorescence quantitative reverse transcription polymerase chain reaction,flow cytometry to test TLR2 /Dectin-1 m RNA and protein expression level of the change,the method of enzyme-linked immunosorbent assay to detect the TNF-a,IL-6.The expression sites of Dectin-1 and TLR2 proteins in Ha Ca T cells were observed by immunofluorescence staining.Part 2:Cultured Ha Ca T cells,use the method of CCK-8 to detect the differentconcentrations(100n M,1?M,10?M,100?M,500 u M)of cyclophosphamide and Cyclosporin A,mycophenolate mofetil(10n M,100 n M,1?M,10?M,50?M))in Ha Ca T cells growth inhibition rate after24 h,to select the optimum concentration for subsequent experiments,with corresponding concentrations of drugs to stimulate Ha Ca T cells for 24 h,The expressions of dectin-1,TLR2 m RNA and protein levels in cells were detected by fluorescence quantitative PCR and flow cytometry.The concentrations of TNF-a and IL-6 in the supernatant were detected by ELISA.Part 3: Selected 100 ug / ml concentration zymosan A harmony-focused immunosuppressive impairment Ha Ca T cell co-cultivation 24 h,RT-q PCR,flow cytometric method for Dectin-1,TLR2 m RNA and protein,TNF-?,IL-6concentration in the cleansing solution in the method of ELISA.Results: Part 1: The expression levels of Dectin-1,TLR2,TNF-a,and IL-6were increased after zymosan A in Ha Ca T cells(P<0.05),which was concentration dependent and positively correlated with the increase in concentration,and the expressions of dectin-1 and TLR2 proteins were transferred to the nucleus.Part 2: Ha Ca T cells treated with cyclophosphamide,Cyclosporin A and mycophenolate mofetil at different concentrations showed certain toxic effects on cells after 24h(P < 0.05),and the toxicity increased with the increase of drug concentration.The expression levels of TLR2,Dectin-1m RNA and protein,TNF-a and IL-6 in Ha Ca T cells were higher than those in the control group,but the difference was not statistically significant.The expression levels of TLR2,Dectin-1m RNA and protein in Ha Ca T cells treated with cyclophosphamide,Cyclosporin A and mycophenolate mofetil were lower than those in the normal control group(P<0.05),and the concentrations of TNF-a and IL-6 were also lower,with statistically significant differences.Part 3: The expression levels of TLR2 and Dectin-1m RNA and protein in Ha Ca T cells induced by zymosan A were lower than those in normal control group after treated with cyclophosphamide(P>0.05),but there was no significant difference between them.The expression levels of TLR2 and Dectin-1m RNA and protein in Ha Ca T cells induced by zymosan A were lower than those in normal control group after treatment with Cyclosporin A and mycophenolate mofetil,and the difference was statistically significant(P<0.05);The concentrations of TNF-aand IL-6 in the cyclophosphamide groups were higher than those of control group,but there was no statistical significance(P>0.05).When mycophenolate mofetil and cyclosporine A acted on zymosan A-induced Ha Ca T cells,the expression levels of TNF-a and IL-6 in supernatant were significantly lower than those in normal control group t(P<0.05).Conclusion:1.Zymosan A can activate the signaling pathway of TLR2/ Dectin-1 and induce the inflammatory response of cells;2.Cyclosporin A and mycophenolate mofetil have inhibitory effects on the growth of Ha Ca T cells and are positively correlated with the concentration;3.Cyclosporin A and mycophenolate mofetil can inhibit the expression of TLR2,Dectin-1,TNF-a and IL-6 in normal and yeast polysaccharide-induced Ha Ca T cells.We speculate that Cyclosporin A and mycophenolate mofetil may inhibit the natural immunity of the body through TLR2/Dectin-1 signaling pathway.4.Mycophenolate has no significant effect on the expression of TLR2,Dectin-1,TNF-a and IL-6 in normal and yeast polysaccharide-induced Ha Ca T cells.We speculate that Cyclophosphamide may inhibit the natural immune function of the body through other signaling pathways. |
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