| Section one:Study on the pharmacokinetics of mouse cornea withimpact of diacerein eye dropsObject:To investigate the pharmacokinetics differences of diacetin eye drops for multiple administrations and single.Methods:Male Kunming mice were randomly divided into the multiple administration group and the single administration group.The multiple administration group were given diacerein eye drop every 2 minutes(total of 3 times).The concentrations of the metabolites of diacerein in the cornea were measured by high performance liquid chromatography after given eye drop 5,15,30,60,120,and 180 min.the pharmacokinetic parameters were calculated by pharmacokinetic software(DAS2.1.1).Results:The metabolites of diacerein was rhein in the cornea at each time point.The concentration of the metabolite of diacerein in the cornea was 318.678±40.88,210.02±25.66,188.83±31.74,112.24±11.70,90.28±22.01 and 57.67±13.71?g/g after given eye drop 5,15,30,60,120,and 180 min in the multiple administration group.The concentration in the single administration group was145.17±19.29,97.95±10.49,71.18±18.70,39.11±2.44,18.10±2.34 and 9.08±2.04?g/g respectively.The concentration of rhein in the cornea was the highest at5min after the administration in the two groups.The concentration of the multiple administration group was higher than that in the single administration group at 5,15,30,60,120,and 180 min(P<0.01 or 0.05).The half-life of multiple administration groups was(1.48±0.33)h,and the half-life of single administration group was(0.89±0.31)h.Conclusion:Conjunctival sac multiple administration has the advantages of high effective drug concentration and long maintenance time compared with single administration.Section two:Effect and mechanism of diacerein in reducing inflammatory keratitis inflammation and tissue destructionObject:To investigate the effect and mechanism of diacerein eye drops in reducing inflammatory keratitis inflammation and tissue destruction,in order to provide a new basis for the treatment of fungal keratitis.Methods:45 male C57BL/6J mice of SPF grade were selected.The experiments were divided into simple model group,natamycin group and natamycin+diacerein group,30 eyes in each group(n=30).The ocular surface of the mice was observed and photographed at 18h,36h,48h,72h,96h,120h,144h and 168h after the model was established and the degree of corneal lesions was clinically scored.After the mice were sacrificed,the cornea was harvested and subjected to immunofluorescence staining of the whole cornea.FITC anti-mouse Ly-6GandCalcofluor White(CFW)were used to label neutrophils and fungal hyphaerespectively.The two-photon laser scanning microscope was used to scan the cornea,paramedian and central cornea on the Z-axis,and the content of neutrophils and the volume of fungi in each cornea were measured.The expression of IL-1,IL-6,ICAM-1,MIP-2,MMP-8,MMP-9,and MMP-13 in the cornea of mice was measured by enzyme-linked immunosorbent assay(ELISA).The activity of MMPs was measured.Results:1 The clinical corneal scores of the natamycin group at 36h,72h,96h,120h,144h,and 168h were significantly lower than that of the model group(P<0.05);The clinical scores of the natamycin+diacerein group at 36h,48h,72h,96h,120h,144h and 168h were significantly lower than that of the model group(P<0.05).The clinical corneal scores at 72h,96h,120h,144h,and 168h were significantly lower than that of the natamycin group(P<0.05).The perforation rates of the model group,natamycin group and natamycin+diacerein group were 60%,40%,and 20%,respectively.The perforation rate of natamycin+diacerein group was significantly lower than the model group(?~2=8.403 P=0.004).2 The fungal load in the natamycin group at 36 h was significantly lower than that in the model group(P<0.05);the fungal load in the natamycin+diacerein group at 48 h was significantly lower than that in the model group(P<0.05).The neutrophil volume at 48h and 72h in natamycin group was lower than that in model group(P<0.05);the neutrophils volumeinnatamycin+diacerein group at 48h,72h and 120h was lower than in the model group(P<0.05);the neutrophils volume of natamycin+diacerein at 72h were lower than that in the natamycin group(P<0.05).3 The expression of IL-1 in natamycin group and natamycin+diacerein groupat 48 hand 72h was significantly lower than that in the model group(P<0.05);The expression of IL-1 in natamycin group and natamycin+diacereinat 72 h was significantly lower than that in the natamycin group(P<0.05);the expression of IL-1 in the diacerein group at 120hwas significantly lower than that in the model group(P<0.05).After natamycin and diacerein were used,the expression of IL-6 in fungal keratitis was decreased at 60 h,the expression of IL-6 in thenatamycin+diacerein group was lower than in the natamycin group(P<0.05).4 The expression of ICAM-1 in natamycin group at 72h was significantly lower than that in the model group(P<0.05);and the expression of ICAM-1 in natamycin+diacerein group was significantly lower than that in model group and natamycin group(P<0.05).The expression of MIP-2 in natamycin group and natamycin+diacerein group at 72h was significantly lower than that in the model group(P<0.05);the expression of MIP-2 in natamycin+diacerein group at 120h was significantly lower than that in the model group(P<0.05).5 The expression of MMP-8 in natamycin group and natamycin+diacerein group was significantly lower than that in the model group at 48h(P<0.05);the expression of MMP-8 in natamycin+diacerein group was significantly lower than that in the natamycin group at 48h(P<0.05);the expression of MMP-8 in the natamycin group and the natamycin+diacerein group was significantly lower than that in the model group at 72h(P<0.05).The expression of MMP-9 in natamycin+diacerein group was significantly lower than that in the model group at 48h and 96h(P<0.05).The expression of MMP-13 in natamycin+diacerein group was significantly lower than that in the model group at120h(P<0.05).The activity of MMPs in the natamycin group and natamycin+diacerein group was significantly lower than that in the model group at 48h,72h,and120h(P<0.05);the activity of MMPs in the natamycin+diacerein group was significantly lower than that in the natamycin group at the 48h and 72h(P<0.05).Conclusion:Diacerein eye drops can reduce the secretion of IL-1 and IL-6 in fungal keratitis,and inhibit the infiltration of neutrophils by inhibiting the expression of MIP-2 and ICAM-1,thereby inhibiting the reduction of collagen by MMPs Degrades and reduces damage to the cornea caused by inflammation of fungal keratitis. |
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