Inhibitory effect of rapamycin-deep eutectic solvent eye drops on corneal allograft rejection in mice and its mechanism
Objective To investigate the inhibitory effect of rapamycin(RAPA)-deep eutectic solvent(DES)eye drops on corneal allograft rejection in mice and its mechanism.Methods CCK-8 assay was used to measure the influence of 10 types of DES on the viability of human corneal epithelial cells(HCECs),and the DES with a significantly higher cell viability than the con-trol group was selected for characterization to obtain the most suitable DES.BALB/c mice and New Zealand white rabbits were used to evaluate the biocompatibility of the most suitable DES.The mice were randomly divided into normal group,phosphate solu-tion(PBS)group,DES group,and RAP A-DES group.The mice in normal group were fed normally,and those in the PBS group,the DES group,and the RAP A-DES group were administrated PBS,DES,and RAP A-DES solution,respectively,to the right eye after corneal transplantation.On the next day,a slit lamp was used to observe the survival of corneal grafts,and the survival per-centage of corneal grafts was analyzed to plot the survival curve of corneal grafts.On days 7 and 16 after transplantation,the eye-balls of the mice were collected,and HE staining was used to observe corneal edema and inflammatory cell infiltration under a mi-croscope.On day 16 after transplantation,RT-qPCR was used to measure the mRNA expression levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10),interleukin-12α(IL-12α),and interleukin-17α(IL-17α)in corneal tissue,and Western blot was used to measure the protein expression levels of IL-6 and IL-17α in corneal tissue.Results CCK-8 assay showed that compared with the control group,the DES-1 group and the DES-2 group had a significant increase in cell viability(P＜0.05),and the DES-1 group had a sig-nificantly higher cell viability than the DES-2 group(P＜0.05);in addition,DES-1 had a lower viscosity than DES-2 and a pH va-lue closer to 7.4,and therefore,DES-1 was the most suitable DES and was used for subsequent experiments.The results of bio-compatibility evaluation showed that DES-1 did not cause any irritation to the eyes and cornea of mice and New Zealand white rab-bits.Compared with the PBS group,the RAPA-DES group had more transparent corneal grafts on days 7 and 16 after surgery,without significant edema or neovascularization.The RAPA-DES group had a significant increase in the survival percentage of cor-neal grafts compared with the PBS group and the DES group(P＜0.05).The median survival time of corneal grafts in the PBS,DES,and RAPA-DES groups was 16 d,16 d,and 22 d,respectively.HE staining showed significant edema and massive inflamma-tory cell infiltration in the corneal grafts of mice in the PBS group and the DES group,while there was no significant edema with a small number of inflammatory cells in the RAPA-DES group.RT-qPCR showed that there was no significant difference in the mR-NA expression level of TNF-α in corneal tissue between the RAPA-DES group and the PBS group(P＞0.05);compared with the PBS group and the DES group,the RAPA-DES group had significantly lower mRNA expression levels of IL-1β,IL-6,and IL-10(P＜0.05),and compared with the PBS group,the RAPA-DES group had significantly lower mRNA expression levels of IL-12α and IL-17α in corneal tissue(P＜0.05).Western blot showed that the RAPA-DES group had significantly lower protein expression levels of IL-6 and IL-17α than the PBS group(P＜0.05).Conclusion RAPA-DES eye drops can significantly prolong the survi-val time of corneal grafts and inhibit immune rejection reaction by reducing the levels of proinflammatory factors in corneal tissue and inhibiting the inflammatory response of corneal grafts after surgery.
国家科技期刊平台
NSTL
万方数据
维普
