| Objective (1) To inhibit the expression of alkaline serine protease (ALP) and phospholipase B (PLB) gene in aspergillus fumigatus (A.fumigatus) by RNA interference using constructed plasmid vectors. (2) To detect the expression and enzymic activities of ALP, PLB gene and identifiy the gene-silenced A.fumigatus strains. (3) To establish a rat model of keratomycosis that permits evaluation the effect of ALP, PLB gene silence on invasion of A.fumigatus and corneal inflammation. (4) To investigate in vivo thereaputic effect of RNA interference on keratomycosis by inhibiting the expression of ALP, PLB gene of invasive Afumigatus with a lithium acetate-based transformation protocol. Methods (1) Double stranded RNA corresponding to the ALP and PLB gene of Afumigatus were designed and inserted into vector pBC-hygro to construct pALP and pPLB used in RNA interference. The reduction of mRNA and protein of ALP, PLB in A.fumigatus were detected by RT-PCR and Western-blotting. Enzymic activities were analysized using special culture medium and gene-silenced strainsΔALP andΔPLB were thus screened out. (2) Corneas of triamcinolone-treated adult Wistar rat were topically inoculated with 106 spores ofΔALP2,ΔPLB1 to establish a rat model of gene-silenced Afumigatus keratitis. Standard A.fumigatus strain was also inoculated as a control. The decreased level of MMP 2,9 mRNA in cornea were analyzed by Real-time quantitative PCR, and proteins were detected by immunohistochemistry stain and Western-blotting. The severity of keratomycosis and the invasiveness of aspergillus were evaluated by a 12-point clinical scoring with the aid of slit lamp and histopathological examination. (3) In vivo experimental therapy towards rat model of aspergillus keratomycosis was carried out with RNA interference targetting the ALP and PLB gene of invasive Afumigatus, in which separated or combined subconjunctival injection of pALP2, pPLB1 and lithium acetate eyedrops as a transform media were given simultaneously. Enzyme linked immunosorbent assay (ELISA) was used to analyze the levels of TNF-a and IL-1βin corneas. Western-blotting and immunohistochemistrical stain were used to detect the expression ratio of MMP-2,9 and tissue inhibitor of metalloproteinase (TIMP) 1,2. Clinical scoring and histopathological examination were performed to evaluate the therapeutic effect of RNA interference. Results (1) The constructs of plasmid vectors were certificated by enzyme digestion and gene sequencing. The expression of mRNA of ALP, PLB in the gene-silenced A.fumigatus strain were significantly inhibited and so did the interest protein (F=23.77-176.48, p<0.01). Among the gene-silenced A.fumigatus strain,ΔALP2,ΔPLB1 achieved better inhibitory effect to enzymatic activities than other strains (q=16.082,33.507, p<0.01). (2) Elevated expression of mRNA and protein of MMP-9 in corneas were detected in all groups 1 to 8 days after inoculation, but increasing of MMP-2 were only detected in late stage of keratomycosis. Expression of MMP-9 in corneas were significantly inhibited inΔALP group andΔPLB group (P<0.01). Expression of MMP-2 mRNA and protein inΔPLB group were much less than that of control group (P<0.01). However, expression of MMP-2 mRNA inΔALP group was higher than in control group (P<0.01). Both of the invasiveness of aspergillus and corneal inflammation were obviously inhibited inΔALP group andΔPLB group. (3) In control group and pBC-hygro group (injection with null plasmid), upregulation of TNF-a, IL-1βand MMP-9/TIMP-1were found from 6 hours to 7 days after injection (P<0.01) and MMP-2/TIMP-2 only in late stage, but no significance was found between the two groups (P>0.05). Expression of TNF-a, IL-1βand MMP-9/TIMP-1 were significantly inhibited in pALP, pPLB group and particularly in combined group (P<0.01). Expression of MMP-2/TIMP-2 decreased in pPLB group and combined group but slightly upregulated in pALP group in late stage. Both of the invasiveness of aspergillus and corneal inflammation were obviously inhibited in pALP, pPLB group and particularly in combined group. Conclusions (1) pALP and pPLB vectors are successfully constructed and consistent with the design for RNA interference. (2) RNA interference can significantly downregulate the expression of ALP and PLB gene in A.fumigatus. The incomplete inhibitation to ALP and PLB may related to the efficiency of RNA interference. (3) ALP and PLB play an active role in the mechanism of asperggillus.fumigatus keratitis through up-regulating the expression of MMP-2 and MMP-9 in host cornea. (4) Through RNA interference mediated by plasmid vector and lithium acetate transformation, inhibition to the expression of ALP and/or PLB gene of A.fumigatus can efficiently downregulate the expression of proinflammatory cytokine and re-balanced the ratio of MMPs/TIMPs in host cornea. The results suggest that this method can inhibit the corneal destruction during fungal keratitis and may be a novel therapeutic strategy for fungal keratitis.
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