Study On The Role And Mechanism Of NLRP3 Inflammasome In The Formation Of Myopia In Mic

Part Ⅰ.Correlation of NLRP3 inflammasomes in the sclera with the process of myopia formation of micePurpose:To investigate whether the expression levels of NLRP3 and its signaling pathway cytokines in sclera are associated with the formation of myopia in FDM mice.Methods:Fifty-four healthy male 3-week-old wild-type C57BL/6j mice were randomly divided into three experimental groups(n=12)and three control groups(n=6).A form-deprivation myopia(FDM)mouse model was used.Via monocular form deprivation with 2-,4-week covering,and 4-week covering followed by 1-week uncovering(named as FDM2,FDM4,and FDM5 group,respectively).All mice had the right eye as the masked eye and the left eye as the control eye.Axial length(AL)and refractive power were measured to assess the specific degree of myopic shift.The protein levels of NLRP3 and related cytokines in the sclera were evaluated by Western blotting and immunohistochemistry.The survival age of the mice in the three control groups corresponded to the experimental groups,and the treatment was identical to that of the same-week-old experimental group except that no eye masking was performed.Results:Refractive levels and factor expression levels were not significantly different between the blank control eyes of the same age group and the control eyes of the experimental group(p>0.05).Compared with control eyes,the expression levels of NLRP3,caspase-1,IL-1β and MMP-2 were upregulated in the sclera of experimental eyes of FDM2 and FDM4 group(p<0.05),similar results were found in terms of axial length changes and myopic shift(p<0.05).In contrast,the expression level of Collagen-1 decreased in the experimental eyes.The myopic shift was reversed,and the up-regulated cytokines decreased in the FDM5 group compared to the FDM4 group.Conclusions:NLRP3 inflammasomes and downstream factors of its signaling pathway were detected in the sclera of FDM mice,and their expression levels were correlated with the progression of myopia in mice.Part Ⅱ.Investigation of the effects and mechanisms of NLRP3 knockdown on refractive development in micePurpose:This study aimed to investigate the impact of NLRP3 knockdown on refractive development in mice by examining myopic progression and the expression levels of NLRP3 and its signaling pathway cytokines in the posterior sclera of NLRP3 knockdown FDM mice compared to wild-type FDM mice.Methods:Fifty-four healthy male 3-week-old NLRP3-/-type pure C57BL/6j mice were selected and subjected to the same grouping and handling procedures as in Part I.To ensure comparability with Part I results,western-blot images were converted to relative gray-scale values using Image-J,and immunohistochemical images were quantitatively scored using IRS.Results:The FDM4 group of NLRP3-/-mice showed the most significant myopic shift,with significantly up-regulated protein levels of caspase-1,IL-1β,and IL-18 compared to the other groups.Similar findings were observed in NLRP3-/-mice and wild-type mice,although the myopic shift was less pronounced and the cytokine changes were less obvious in treatment group than in wild-type mice.No significant differences were observed in refraction and axial length between wild-type mice and NLRP3-/-mice of the same age group in the blank group.Conclusions:Knockdown of NLRP3 affects the progression of myopia in FDM mice.The NLRP3 may play a role in myopia progression in FDM mice by upregulating MMP-2 expression,which affects collagen I and leads to scleral extracellular matrix remodeling and myopic shift.Part Ⅲ.Effects of NLRP3 inhibitor MCC950 on refractive development in micePurpose:To investigate the effect of NLRP3 inhibitor MCC950 on refractive development in wild-type FDM mice.Methods:Sixty healthy male 3-week-old wild-type C57BL/6j mice were selected and randomly divided into four experimental groups(n=10)and four control groups(n=5).The mice were subjected to monocular form deprivation for 2 or 4 weeks and received either water or MCC950 injection.The groups were named FDM2W,FDM4W,FDM2M,and FDM4M according to the treatment they received.The four control groups corresponded to the experimental groups separately.They received the same treatment as the experimental groups,except that no eye masking was performed.The collection indexes and treatments were the same as in the first two parts.In addition,the data from the 2 and 4-week blank control groups of wild-type mice from the first part of the study were included in this study,and the data from the two mice with the highest and lowest refractive error were removed at the time of inclusion to ensure that the number of enrolled mice was equal.Results:Intraperitoneal injection of MCC950,saline,or no injection had no significant effect on refraction and AL in wild-type mice without FDM.In experimental eyes of same-week-old FDM mice,the expression levels of NLRP3,IL-1 β,IL-18,Caspase-1,and MMP-2 showed progressive increases in control eyes of MCC950 group,experimental eyes of MCC950 group,and experimental eyes of water group,and the expression in 4-week group was higher than that in 2-week group;similar changes in myopic drift and eye axis were also observed in each group.Conclusions:MCC950 can inhibit NLRP3 expression and thus affect myopia progression in FDM mice.