| Objective: To investigate the anti-inflammatory effect and mechanism of thymoquinone(TQ)in Candida albicans(C.albicans)infected keratitis,and its anti-inflammatory effect.Methods: 1.The minimum inhibitory concentration(MIC)of TQ against C.albicans was detected by the microdilution method,its toxicity to immortalized human corneal epithelial cells(HCECs)was detected by the cell counting kit(CCK-8),and its toxicity to mouse corneas was detected by the in vivo Draize test to screen for safe and effective anti-inflammatory and antifungal concentrations.2.C.albicans spores infected C57BL/6mice,then given corneal 50μg/ml TQ or 0.1% DMSO subconjunctival injection to observe the clinical scores after corneal infection;the fungal load of infected mice corneal was detected by colony counting method and periodic acid Schiff(PAS)staining;the effect of TQ on mouse corneal neutrophil recruitment was observed by immunofluorescence method,MPO method and hematoxylin-eosin(HE)staining.The effect of TQ on neutrophil recruitment in mouse corneas was observed by immunofluorescence,MPO and hematoxylin-eosin(HE)staining;the expression of COX-2,IL-1β and IL-6 m RNA and protein in corneal tissues of C.albicans-infected mice was detected by PCR and ELISA;the expression of m RNA and protein of Heme oxygenase-1(HO-1)and NF-E2-related factor-2(Nrf2)in the corneal tissues of C.albicans-infected mice was investigated by PCR and Western blot to determine the in vivo anti-inflammatory effects of TQ.3.3.75μg/ml TQ or 0.1% DMSO pretreatment of HCECs for 2 hours,and cells were stimulated with C.albicans spores for 8 or 24 hours.The expression of COX-2,IL-1β and IL-6 m RNA and protein in each group of cells was measured by PCR and ELISA to study the anti-inflammatory effect of TQ in vitro.4.HCECs were pretreated with Nrf2 inhibitor Brusatol(BT)or HO-1 inhibitor Zinc protoporphyrin(Znpp)for 2 hours,and then the cells were pretreated with 3.75μg/ml TQ or 0.1% DMSO for 2 h.Cells were stimulated with Candida albicans spores for 8 or 24 hours.The expression of COX-2,IL-1β and IL-6 m RNA and protein were measured by PCR and ELISA;the expression of HO-1 was measured by Western blot to investigate the anti-inflammatory mechanism of TQ.Results: 1.The microdilution method showed that the MIC90 of TQ on C.albicans spores was 50μg/ml,and the corneas of mice were not affected by TQ when the drug concentration was 50μg/ml,as demonstrated by the in vivo Draize test in mice;CCK-8 experiments showed that the cellular activity of HCECs was not affected by TQ when the TQ concentration was ≤ 3.75μg/ml.2.In the animal model of C.albicans keratitis,compared with the DMSO group,the inflammatory reaction was significantly reduced and the clinical score was significantly lower in the TQ group on days 3 and 5;on day 3 after infection,plate counting experiments showed that the fungal residues in the corneal tissues of the TQ group were significantly reduced,and PAS staining showed that the mycelial growth in the corneal tissues of the TQ group and its invasion into the deeper layers of corneal tissues were significantly inhibited;the immunofluorescence method,the MPO and HE staining showed that TQ inhibited the recruitment of neutrophils in the corneas of infected mice.3.In the animal model of Candida albicans keratitis,compared with the DMSO group,the m RNA and protein expression of COX-2,IL-1β and IL-6 in the corneal tissue of mice at 3 and 5 days in the TQ group were significantly decreased.4.In the in vitro experiments,compared with the DMSO group,the m RNA and protein expression of COX-2,IL-1β and IL-6 in the human corneas of the TQ group was significantly decreased.5.In the animal model of C.albicans keratitis,the m RNA and protein expression of Nrf2 and HO-1in the corneal tissue of mice in the TQ group was significantly increased compared with the DMSO group.After stimulation of HCECs by C.albicans spores,the m RNA and protein expression of Nrf2 and HO-1 were further increased in HCECs in the TQ group compared with the DMSO group.6.After stimulation of HCECs by C.albicans spores,the protein expression of HO-1 in infected HCECs was significantly reduced by giving BT pretreatment before TQ treatment compared with pretreatment of cells with TQ alone;The m RNA and protein expression of COX-2,IL-1β and IL-6 in HCECs was increased when BT or Znpp pretreatment was given to HCECs followed by TQ treatment compared with cells treated with TQ alone.CONCLUSION: TQ plays a protective role in murine C.albicans keratitis by activating the Nrf2/HO-1 signaling pathway,inhibiting the growth of C.albicans,reducing the recruitment of neutrophils,and suppressing the expression of inflammatory factors. |
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