| Research Background and PurposeSporotrichosis is an infectious disease caused by Sporothrix schenchii var.luriei,which affects skin,subcutaneous tissue,mucosa,and localized lymphocyte system,and can lead to severe systemic disseminated infections,the global incidence of which climbs yearly.Current therapies to treat sporotrichosis are very limited,and mainly include antifungal drugs,photodynamic therapy,thermotherapy,cryotherapy.and surgery,but the efficiency of treatment is often unsatisfactory.With the research on the pathogenesis of sporotrichosis,it has been found that the adaptive immunity mediated by CD4+T cell subsets is closely correlated with host resistance to fungus,and the NOD-like receptor thermal protein domaia associated protein 3(NLRP3)inflammasome may play an important role.However,the role of NLRP3 in the pathogenesis and progression of sporotrichosis is still unclear and requires further study.This study aimed to detect NLRP3 expression in the skin lesions of patients with sporotrichosis and explore the role of NLRP3 in the pathogenesis and progression of sporotrichosis,providing new targets for the diagnosis,assessment,and treatment of sporotrichosis.Laser therapy is a promising new type of antifungal therapy that may replace traditional therapy-It has many advantages,including high efficiency,economy.safety,and non-resistance,and can be used to treat onychomycosis(mainly caused by Trichophyton rubrum),chromoblastomycosis,Candida albicans,Malassezia.and Aspergillus fumigatus.Different lasers have certain differences in their action mechanisms and methods.Among them,the Nd:YAG 1064-nm laser has strong penetrability and can reach deeper layers of tissue.In addition,the melanin in the fungal wall can selectively absorb the N:YAG 1064-nm laser and produce a photothermal effect,which can avoid damage to surrounding tissues-Our previous work showed that the Nd:YAG1064nm laser was effective in the treatment of sporotrichosis.However,the associated mechanism is still unclear.So in this study we aim to investigate the therapeutic effects and mechanism of Nd:YAG 1064-nm laser on sporotrichosis and provide a theoretical basis for its clinical application in the treatment of sporotrichosis.Antimicrobial peptides(AMPs)are small peptides commonly found in natural organisms They have the advantages of broad spectrum,small molecular weight,high efficiency,stability,selective toxicity,and drug resistance,as well as anti-pathogen function.A previous study showed that scorpion venom-derived antimicrobial peptide ToAP2(AP02759)could inhibit the growth of Candida albicans.Moreover.ToAP2 has the characteristics of low hemolytic activity,low cytotoxicity,and remarkable antifungal biofilm activity,suggesting great potential for the development of new antifungal drugs.Because the biological characteristics of Sporothrix globosa are very similar to those of Candida albicans,which are both dimorphous fungi,we used the ToAP2 structure as a template for molecular modification to study the inhibitory effects of antimicrobial peptides combined with CO2 fractional laser on sporotrichosis,in order to and provide a new method for the clinical treatment of sporotrichosis.Method1.Skin lesion samples from 60 patients with sporotrichosis and 30 cases of healthy skin were selected Based on the duration of the disease,the included patients were divided into the acute group(?3 months)and non-acute group(>3 months),with 30 cases in each group.The expression of NLRP3 in skin lesions was detected by immunohistochemical methods,and the correlation between NLRP3 levels and disease progression was analyzed.2.Investigate the therapeutic effects and mechanism of Nd:YAG 1064-nm laser on sporotrichosis through experiments in vitro and in vivo.(1)In vitro experiments:The isolated and cultured Sporothrix globosa were inoculated into brain heart solid medium.The optimal irradiation parameters of the Nd:YAG 1064-nm laser on the Sporothrix globosa were determined by the CCK-8 viability assay.Scanning electron microscopy was used to observe the influence of the Nd:YAG 1064-nm laser on the morphology of Sporothrix globosa.A confocal laser scanning microscope was used to observe apoptosis and necrosis(Hoechst/PI double staining)of Sporothrix globosa after Nd:YAG 1064/nm laser irradiation.A stress sensitivity test was used to observe H2O2 and SDS sensitivity changes of Sporothrix globosa after Nd:YAG 1064-nm laser irradiation.A spectrofluorometer was used to detect the accumulation of ROS and the changes in mitochondrial membrane potential of Sporothrix glob os a after Nd;YAG 1064-nm laser irradiation.A confocal laser scanning microscope was used to detect metacaspase activation of Sporothrix glob os a after Nd:YAG 1064-nm laser irradiation.(2)Invivo experiments:An animal model of localized cut aneous sporotrichosis in BALB:'c mice was constructed,and the successful establishment of the model was confirmed by histopathological changes Mice were divided into infection,laser,and itracnoazole groups.?Infection group:Neither Nd:YAG 1064-nm laser irradiation nor drug treatment was given:?Laser group:Nd:YAG 1064-nm laser irradiation was admibistered once every 7 days with an energy density of 100 J/cm2,emitted at 30 ms pulses of duration at a 1 Hz frequency:?Itraconazole group:The mice of the itracorazole group were gavaged daily with itracorazole at a dose of 60 mg/kg The control group consisted of same-age healthy mice.Photoshop CC 2019 software was used to calculate the foot pad area of mice in each group daily.On days 14:22:and 30:the expression of NLRP3,caspase-1,and caspase-3 in the foot pad tissue of each group were compared using immunohistochemical staining.The expression levels of serum cytokines IFN-?:IL-17 and TGF-?1 in each group of mice were compared using enzyme-linked immunoassay(ELISA).3.ToAP2 derivative peptides were designed and modified by incorporating effective fragments and amino acid substitutions,which physical and chemical parameters were screened and analyzed using bioinformatics software.ToAP2 derivative peptides ToAP2A,ToAP2C:and ToAP2D were synthesized by solid-phase synthesis The derivative peptide with the strongest antifungal activity was screened using the inhibition zone test for in vitro and in vivo experiments.(1)In vitro experiments:The isolated and cultured Sporothrix globosa were inoculated into brain heart solid medium.After 24 h,the experimental group was evenly coated with the strongest antifungal activity derivative peptide(4 mg/ml),while the control group was treated with the same amount of normal saline.The morphology of Sporothrix globosa.was observed by scanning electron microscopy.Confocal laser scanning microscopy was used to observe apoptosis and necrosis(Hoechst/PI double staining)of Sporothrix globosa.Flow cytometry was used to detect the accumulation of RUS:changes in mitochondrial membrane potential,and metacaspase activity of Sporothrix globosa.(2)In vivo experiments:An animal model of localized cutaneous sporotrichosis in BALB/c mice was constructed,and the successful establishment of the model was confirmed by histopathological changes Mice were divided into infection,lattice laser,lattice laser+derived peptide(joint),and itraconazole groups ? Infection group:Neithex CO2 lattice laser irradiation nor ToAPS2D treatment was administered;?Lattice laser group:CO2 lattice laser irradiation was administered once every 3 days with an energy density of 120 mJfmm2 and a coverage rate of 25.0%;?Lattice laser+ToAP2D group:Laser irradiation was the same as the lattice group and 4 mg/ml derived peptide solution was administered daily for external treatment every;?Itraconazole group;Mice were gavaged with itraconazole at a dose of 60 mg/kg every day.The control group consisted of healthy mice of the same age Photoshop CC 2019 software was used to calculate the foot pad area of mice in each group once every 4 days.HE and immunohistochemical staining were used to compare the infiltration of inflammatory cells and the expression of caspase-3 in the footpads of mice in each group.Result1.The expression of NLP3 in the skin lesions of patients with sporotrichosis was significaotly higher than that in normal skin tissues(P<0.001);The expression of NLRP3 in sporotrichosis tissues was not related to the patient gender or age(P>0.05)but was related to the duration of the disease The expression level of NLRP3 in skin lesions in the acute group was significantly higher than that in the non-acute group(P<0.001).2.The therapeutic effects and mechanism of Nd:YAG 1064-nm laser on sporotrichosis(1)In vitro experiments:The CCK-8 viability assay indicated that the Nd:YAG 1064-nm laser could effectively inhibit the growth of Sporothrix globosa,and the optimal laser energy against Sporothrix globosa was 400 J/cm2(actual energy density absorbed by Sporothrix globosa was 191 J/cm2).Scanning electron microscopy showed that the Nd=YAG 1064-nm laser caused membrane shrinkage,destruction,and leakage of contents.The Hoechst/PI double staining test with confocal laser scanning microscope showed that Nd:YAG 1064-nm laser can induce apoptosis and necrosis of Sporothrix globosa.The stress sensitivity test showed that the Nd:YAG 1064-nm laser can cause an increase in the sensitivity of Sporothrix globosa to H2O2 and SDS.The fluorescence ratio of the mitochondrial membrane potential detected by the spectrofluorometer in the laser group(9.90±1.4%)was significantly lower than that in the control group(100%;P<0.001);the ROS accumulation level in the laser group(43.7%±7.8%)was significantly higher than that in the control group(3.5%±1.3%;P<0.01).Confocal laser scanning microscopy detected a significant increase in metacaspase activity after laser irradiation.(2)In vivo experiments1)A mouse model of localized cutaneous sporotrichosis was successfully established using histopathology.2)Footpad area change in sporotrichosis mice.Compared with the control group,the footpad sizes in the laser,itraconazole,and infection groups increased significantly by days 14 and 22(P<0.001);on day 30,the footpad sizes of the mice in the laser and itraconazole group had returned to normal(P>0.05),while the footpad sizes in the infection group were still significantly higher than those in the control group(P<0.01).Compared with the infection group,the footpad sizes of the mice in the laser and itraconazole groupswere significantly reduced by days 14,22,and 30(P<0.01).3)Effects of Nd:YAG 1064-nm laser treatment on the NLRP3-Caspase-1 pyroptosis pathway and Caspase-3 apoptosis in a mouse model of sporotrichosis.Compared with the control group,the expression of NLRP3 and Caspase-1 proteins in the foot pad tissue of the laser group had increased significaotly by day 14(P<0.05)The expression level of Caspase-1 protein increased significantly in the infection group(P<0.01),whereas there was no significant difference in the expression of NLRP3(P>0.05).On day 22,the expression of NLRP3 and Caspase1 proteins in the footpads was significantly increased in the infection and laser groups(P<0.01).On day 30,the expression levels of NLKP3 and Caspase-1 proteins in the laser group had returned to normal levels(P>0.05)The expression levels of Caspase-1 protein in the infection group returned to normal(P>0.05),whereas the expression levels of NLRP3 protein were still higher than those in the control group(P<0.01).In addition,compared with the control group,on days 14 and 22,the expression of Caspase-3 in the footpads of the laser group was significantly increased(P<0.05),whereas the expression of Caspase-3 in the infection group was not significantly different(P>0.05).On day 30,compared with the control group,there was no significant difference for the expression of Caspase3 in the infection and laser groups(P>0.05)4)Effect of Nd:YAG 1064-nm laser treatment on serum IEN-?,IL-17 and TGF-?1 levels in a mouse model of sporotrichosis.On day 14,the expression levels of serum IL-17 and IFN-? in the laser group were significantly higher than those of the control group(P<0.01)The expression level of IFN-? in the infection group was signfficantly higher than that of the control group(P<0.001),whereas the expression level of IL-17 was not significantly different from that of the control group(P>0.05).The expression level of IL-17 in the laser group was significantly higher than that in the infection group(P<0.01).On day 22,the expression of IL17 in the laser group returned to normal(P>0.05),whereas the expression level of IFN-? was still significantly higher than that of the control group(P<0.01).The expression levels of IL-17 and IFN-? in the infection group were significantly higher than those of the control group(P<0.001)The expression levels of IL-17 and IFN-? in the laser group were significantly lower than those in the infection group(P<0.05).;On day 30,the expression levels of IL-17 and IFN-? in the laser and infection groups were not significantly different from those in the control group(P>0.05),whereas the expression levels of IL-17 in the laser group were significantly lower than those in the infection group(P<0.001).On day 14,the expression levels of TGF-?1 were not significantly different between the laser and infection groups from those in the control group(P>0.05).while there was no significant difference between the laser group and the infection group(P>0.05).On day 22,the expression levels of TGF-?1 in the laser and infection groups were significantly lower than those in the control group(P<0.05),whereas there was no difference between the laser and infection groups(P>0.05).On day 30,the expression levels of TGF-?1 in the laser and infection groups were not significantly different from those in the control group(P>0.05),but the expression level of TGF?1 in the laser group was significantly lower than that in the infection group(P<0.05).5)Correlation analysis between NLRP3/Caspase-land IL-17/IFN-? The expression levels of NLRP3 and Caspase-1 in the laser group were positively correlated(r=0.580,P=0.012).The expression levels of NLRP3/caspase-1 were positively correlated with IL-17(r=0.519,P=0.027;r=0.627,P=0.005)and IFN-?(r=0.624.P=0.006;r=0.876.P=0.000).3.After molecular modification,the secondary structure of three derived peptides,ToAP2A,ToAP2C,and ToAP2D,was dominated by ? helix;compared with ToAP2,the modified peptide had a significantly shorter length,lower molecular weight,and lower instability coefficient;the aliphatic index,hydropathic index and protein binding increased They were synthesized by solid-phase synthesis and the purity of all peptides was greater than 95%.All three derived peptides could inhibited the growth of Sporothrix globosa.Among them.ToAP2D had the strongest antifungal activity with an MIC value of 156.25 ?k/mL.(1)In vitro experiments:Scanning electron microscopy showed that the derived peptide ToAP2D caused membrane shrinkage,destruction,and leakage of contents.The Hoechst/PI double staining test with confocal laser scanning microscope showed that the derived peptide ToAP2D could induce apoptosis and necrosis of Sporathrix globosa.The ROS accumulation level detected by flow cytometry in the ToAP2D group(40.84%± 5.69%)was significantly higher than that in the control group(4.65%±1.00%.P<0.01).The fluorescence ratio of the mitochondrial membrane potential detected by flow cytometry in the ToAP2D group(4.30±1.29%)was significantly lower than that in the control group(26.73±8.58%,P<0.01).The Metacaspase level of the ToAP2D group was 62.05%±4.15%,which was significantly higher than that ofthe control group(20.73%±3.05%.P<0.001).(2)In vivo experiments1)A mouse model of localized cutaneous sporotrichosis was successfully established using histopathology.2)Footpad area change of sporotrichosis mice:Compared with the control group,the footpad sizes in the lattice laser+ToAP2D,lattice laser,itraconazole,and infection groups had increased significantly on day 14(P<0.01),while on day 26,the footpad sizes of the mice in the lattice laser and infection group were still significantly higher than those in the control group(P<0.001),but the footpad area of mice in the lattice laser+ToAP2D group and itraconazole group was not significantly different from that of the control group(P>0.05).3)Inflammatory cell infiltration:Compared with the control group,HE stained irflammatory cells in the lattice laser+ToAP2D,lattice laser,and infection groups were significantly increased on day 18(P<0.01).On day 26,the number of inflammatory cells in the lattice laser and infection group were still significantly higher than that in the control group(P<0.01),but there was no significant difference in the number of inflammatory cells between the lattice laser+ToAP2D group and the control group(P>0.05).4)Caspase-3 protein expression Immunhistochemical results showed that,on days 18 and 26,the expression of Caspase-3 in the footpads of the lattice laser+ToAP2D group was significantly increased compared with the control group(P<0.05).whereas the expression of Caspase-3 in the lattice laser and infection group was not significantly different(P>0.05).Conclnsion1.The expression of NLRP3 protein in the lesions of patients with sporotrichosis was significantly higher than that in normal skin,while the expression level of NLRP3 was related to the duration of the disease.These data indicate that the high expression of NLRP3 might play an important role in the occurrence and development of sporotrichosis,which is expected to become a new target for the diagnosis assessment,and treatment of sporotrichosis.2.The Nd:YAG 1064-nm laser had an obvious therapeutic effect on an animal model of sporotrichosis,inhibiting Sporothris globosa growth,destroying morphological structure,and inducing apoptosis and necrosis.The laser decreased the mitochondrial membrane potential and led to the accumulation of ROS,which activated the caspase-dependent apoptosis pathway,activated the NLP3-Caspase-1 pyroptosis pathway,and regulated the immunity of Thl and Th17 cells and promoteCaspase-3 apoptosis.These findings provide a theoretical basis for the clinical application of Nd:YAG 1064-nm laser in the treatment of sporotfichosis.3.Three successfully synthesized ToAP2 derived peptides,ToAP2A,ToAP2C,and ToAP2D,all had obvious inhibitory effects on sporotrichosis.Among them,ToAP2D had the strongest antifungal activity,good serum stability,and no acute toadcity.In vitro experiments showed that the derived peptide ToAP2D could induce apoptosis and necrosis in Sporothrix globosa.The derived peptide ToAP2D decreased the mitochondrial membrane potential and led to the accumulation of ROS,which activated the caspase-dependent apoptosis pathway.In in vivo experiments,we found that the derived peptide ToAP2D combined with the CO2 lattice laser had a significant therapeutic effect on the Sporothrix globosa animal model,the mechanism of which was related to reducing footpad inflammation and promoting caspase-3 apoptosis.This provides a basis for the clinical application of the derived peptide ToAP2D and also provide a new idea for the clinical treatment of sporotrichosis. |
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