The Research Of Inhibitory Effect Of Keratinocyte On Trichophyton Rubrum

BackgroundTrichophyton rubrum(T.rubrum)is one of the most important agents of fungal infetion.The characteristics of the infectious diseases caused by T.rubrum are chronic,easy to relapsed and difficult to cure.T.rubrum usually invades the superficial keratinous structures of humans,but under the conditions of immunosuppression and trauma,it can invade the deep tissue structure and causes deep fungal infection.Due to the side effect of drugs,long period of treatment,poor patient compliance and so on,the therapeutic effect of fungal infectious diseases is not good,seriously affects the quality of people s life.The study found that there are a number of immune cells in the skin,such as langerhans cells,macrophages,neutrophils,keratinocytes and so on.Keratinocytes can recognize antigens,release a variety of antimicrobial peptides,regulate the inflammatory factors and induce the occurrence of inflammatory reactions.As a result,it achieve the purpose of defense against fungal infection.Keratinocytes are the first line of defense in the skin.The surface of keratinocytes has a variety of pattern recognition receptors(PRRs),can identify the pathogen associated molecular patterns(PAMPs)which on the surface of the fungal cell wall.After combined with PRRs and PAMPs,it can induce the production of inflammatory cytokines and initiate a further immune response.However,in contrast,when keratinocytes and fungi interact with each other,what will happen to the fungi themselves and the specific mechanism within the fungi has not been studied.The abundant reactive oxygen species(ROS)and nitric oxide(NO)in the cells of T.rubrum have the ability to damage the growth and activity of the fungus,and even cause the cell death.In this study,we mainly investigated the internal change of T.rubrum when it invaded the keratinocytes.We co-cultured T.rubrum and keratinocytes to explore whether keratinocytes have the inhibitory effect on the growth of T.rubrum.And to investigate the damage effects of ROS and NO in T.rubrum.ObjectiveWe aim to investigate the inhibitory effect of HaCaT cells on T.rubrum and its mechanism.The results play an important role for further understanding of the relationship between dermatophytes and skin barrier,and the immune mechanism of fungal infections cutaneous.Methods(1)The HaCaT cells and T.rubrum(ATCC4438)were cultured in suitable condition.For the dose and time response assay,we divided the experiment into two groups:T.rubrum,T.rubrum +HaCaT,and chose the optimal co-cultured dose and time.(2)In the follwing study,we divided the experiment into three groups:T.rubrum,T.rubrum + HaCaT,T.rubrum + DPI(NADPH oxidase inhibitor)+ HaCaT.Determination of fungal activity,the levels of ROS and fungal keratinase activity.The micromorphology of fungus was observed using scanning electron microscopy(SEM)and transmission electron microscopy(TEM).(3)In the follwing study,we divided the experiment into three groups:T.rubrum,T.rubrum + HaCaT,T.rubrum + L-NMMA(NOS inhibitor)+ HaCaT.Determination of fungal activity,the contents of NO,NOS and fungal keratinase activity.The micromorphology of fungus was observed using SEM and TEM.(4)In the follwing study,we divided the experiment into three groups:T.rubrum,T.rubrum + HaCaT,and test the expression of ADMA.Results(1)A ratio of HaCaT cells to T.rubrum suspension was 4:1 and 24 h of co-cultured time was determined for the following experiment.(2)After co-cultured with HaCaT,the fungal activity and keratinase of T.rubrum was decreased,the fungal micromorphology was damaged,and the content of ROS was increased.DPI has the ability to reduce the inhibition effect of HaCaT.(3)After co-cultured with HaCaT,the fungal activity and keratinase of T.rubrum was decreased,the fungal micromorphology was damaged,and the expression of ADMA in T.rubrum was decreased,the content of NOS and NO were increased.L-NMMA has the ability to reduce the inhibition effect of HaCaT.Conclusions(1)After co-culture with HaCaT cells,T.rubrum may generate the production of ROS and NO,leading the damage to the fugus.As the result,HaCaT cells can reduce the fungal activity and invasiveness.(2)The Nox and NOS may be the targets of inhibiting the growth of T.rubrum by HaCaT cells.(3)The research offer a theoretical basis for further understanding of the relationship between dermatophytes and skin barrier,and the immune mechanism of fungal infections cutaneous.