| Sporotrichosis is a common deep mycosis with diverse lesions.Most cases of sporotrichosis are localized to the skin and subcutaneou tissues and dissemination to ostearticular structures and viscera are uncommon.The course of disease is prolonged.Sporotrichosis is distributed worldwide and the pathogen is the Sprothrix schenckii complex,which contains 7 genotypes,of which S.schenckii sensu stricto,Sporothrix globosa,and Sprothrix brasiliensis are the main pathogenic types.In China the epidemic strain is S.globosa.However,the pathogenisis of sporotrichosis is not completely clear,and the treatment is limited,which contains potassium iodide and the antifungal agents like itraconazole,terbinafine and amphotericin B.The course of treatment is usually 3-6 months.Potassium iodide is cheap and effective,but it may lead to adverse reactions such as rashes,gastrointestinal reactions.Long-time application of antifungal drugs may cause liver and kidney dysfunction,gastrointestinal reactions and other adverse reactions,and even the emergence of drug-resistant strains,leading to treatment failure.Other treatments include thermotherapy,liquid nitrogen cryotherapy,physiotherapy,etc.Melanin is an important virulence factor of S.globosa,and protects strains from the antifungal effects of antifungal agents and environments such as extreme temperature,acid and alkali,ultraviolet rays and ionizing radiation,affecting the therapeutic effect.Recently cases of photodynamic treatment on sporotrichosis have been reported,but the investigations are few.Thus,in order to initially clarify the effects ofphotodynamic therapyand physiotherapy such as WIRA,red light and red light-emmitting diodes(LED)on S.globosa,the effect of melanin,the mechanism of ALA-PDT and the enhancement of ALA-PDT by potassium iodide(KI),the wild-type S.globosa Mel+,melanin-inhibited by tricyclazole in medium S.globosa TCZ-Mel+,UV-induced albino mutant S.globosa Mel-and 7 S.globosa isolated from the leisions of 7sporotrichosis patients were employed,and 5-aminolevulinic acid(ALA)as photosensitiser,red LED as a light source were used to conduct the following experiment,aming to explore an alternative therapy for sporotrichosis to achieve a shorter treatment course and a better effect.In the first part,the Mel+conidia were exposed to WIRA,red light and LED with dose of 54,108 and 162 J/cm~2 and calculate the survival rate,then TCZ-Mel+and Mel-conidia were exposed to investigate whether S.globosa was protected by melanin during exposure.The results showed that the survival rate of Mel+irradiated by WIRA was 78.9%,significantly lower than that of the control group,indicating that in vitro WIRA irradiation had antifungal activity against Mel+and red light and LED had no antifungal effect on Mel+.The TCZ-Mel+and Mel-conidia were killed partly by irradiation,but the survival rates were lower significantly than that of Mel+after exposure to WIRA,red light and 162 J/cm~2LED,which showed that melanin protected Mel+conidia during exposure.The second part was aimed to explore the antifungal activity of ALA-PDT against S.globosa in vitro and the role of melanin.Mel+conidia and yeast cells were incubated spectively with 0,0.3M,0.6M,1.19M ALA at 28?in dark and then exposed to 0,54,108,162J/cm~2 LED to determin the antifungal effect.Then TCZ-Mel+and Mel-conidia were treated in the same way to investigate the protect of melanin.The conidia and yeast cells of 7 S.globosa were treated with 1.19M ALA and 162 J/cm~2 LED to demonstrate the effect of ALA-PDT on other S.globosa.CCK-8 assay was used to detect the viability of HaCaT treated with ALA-PDT to evaluate the safety of ALA-PDT.The result revealed that following incubation with1.19M ALA for 30min at 28?in dark and 162 J/cm~2 irradiation in vitro,the antifungal activity of ALA-PDT was highest and the killing rate of Mel+conidia and yeast cells were 97.38%and 100%.The yeast cells were more sensitive to ALA-PDT.Compared with Mel+conidia,the survival rates of TCZ-Mel+and Mel-were significantly lower than that of Mel+,indicating that melanin protected the strain against ALA-PDT.The viability of 7 S.globosa strains and 2 reference strains were also reduced significantly after the treatment of 1.19M ALA and 162J/cm~2 LED.There was no difference between the viability of HaCaT treated with ALA-PDT and untreated,revealing that ALA-PDT had no killing effect on HaCaT.The mechanism of ALA-PDT against S.globsa was explored in the third part.Mel+conidia were incubated with 1.19M ALA and irradiated by 162J/cm~2 LED.Then DCFH-DA probe,scanning electron microscopy(SEM),transmission electron microscopy(TEM)and comet assay were employed to detect ROS generation,were conducted with DCFH-DA probe to detect ROS,observe the morphology and ultrastructure of Mel+conidia and explore the DNA damage.We found that Mel+conidia treated with ALA-PDT produced more ROS,and the conidial surface appeared wrinkled with burrs or protrusions obversed with SEM.The melanin in the outer layer disappeared,and damaged organelle and porous cellular stucture were obversed by TEM.The comet rate of Mel+conidia after ALA-PDT increased significantly,indicating more DNA damage.Then we invesigated the effect of KI on ALA-PDT against Mel+in the fourth part.0.6M ALA and 108J/cm~2 LED were employed in this part.Adding KI before or after ALA-PDT and KI concentration were determined firstly.The conidia of Mel+and 7 S.globosa were treated with ALA-PDT adding 100m M KI to evaluate the effect of KI on the antifungal activity of ALA-PDT.Then the ROS of Mel+conidia treated by ALA-PDT added KI was detected with DCFH-DA probe.The results showed that the antifungal activity of ALA-PDT against Mel+was enhenced more when adding KI before ALA-PDT with 100m M KI.With 0.6M ALA as the photosensitiser and108J/cm~2 LED as the light source,the killing rate of Mel+conidia was only 12.06%.However,when 100m M KI added,it was increased to 50.5%.The enhancement of the kiling effect was also observed in the experiments of 7 other S.globosa.And the ROS of Mel+conidia treated by ALA-PDT added KI was significantly reduced compared with conidia treated with ALA-PDT only.The study demonstrated that WIRA and ALA-PDT had antifungal activity against S.globosa in vitro,and melanin could protect the strain from ALA-PDT and exposure of WIRA,red light and 162J/cm~2 LED.And we also found that antifungal activity was resulted from more ROS generation induced by ALA-PDT,leading to ultrastructure changes and DNA damage and KI enhanced the antifungal activity of ALA-PDT.The study provides a new idea for the treatment of sporotrichosis. |
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