甜菊糖苷-白杨素滴眼液通过调控TLR4/MyD88/NF-κB信号通路改善糖尿病干眼小鼠眼表炎症的机制研究OA

Objective To investigate the mechanism of Stevioside(Ste)-Chrysin(Chr)(Ste-Chr)ophthalmic solu-tion improving ocular surface inflammation in diabetic dry eye(DDE)mice by regulating the TLR4/MyD88/NF-κB signaling pathway.Methods Forty 8-week-old SPF-grade healthy male C57BL/6J mice were selected.After one week of adaptive feeding,mice were intraperitoneally injected with streptozotocin solution at a dose of 55 mg·kg-1 for 5 consecutive days to establish DDE models.Eventually,35 mice were successfully induced into the DDE model,3 failed to be induced,and 2 died.Thirty-two mice were randomly selected from the 35 successfully modeled mice and divided into 4 groups(8 mice in each group)using a random number table method:model group,cyclosporine(CsA)group,Chr group,and Ste-Chr group.Additionally,8 healthy male C57BL/6J mice were set as a control group.Neither the DDE model group nor the control group was given any drug intervention;mice in the CsA group were administered 0.5 g·L-1 CsA eye drops;mice in the Chr group were administered 5 g·L-1 Chr suspension;and mice in the Ste-Chr group were administered Ste-Chr ophthalmic solution.The therapeutic drugs in each group were precisely aspirated using a micropipette,administered to both eyes,with 5 μL per eye,and dripped into the inferior conjunctival fornix,three times a day(at 8:00,14:00,and 20:00)for 2 consecutive weeks.Before modeling,after modeling,and after intervention,blood glucose levels of mice in each group were measured,and dry eye-related indicators such as corneal fluorescein staining(FL)score,tear film break-up time(BUT),and tear secretion volume were evaluated.Hematoxylin-eosin staining was performed to observe the morphological structure of corneal tissue in mice.Quantitative real-time polymerase chain reaction was employed to detect the mRNA expression levels of TLR4,NF-κB,tumor necrosis factor(TNF)-α,interleukin(IL)-6,and IL-1β in corneal tissue.Western blot was used to analyze the changes in protein expressions of TLR4,MyD88,and NF-κB in corneal tissue.Results After modeling and drug interven-tion,compared with the control group,blood glucose levels of mice in the model group and each drug intervention group increased significantly(all P＜0.01).After intervention,compared with the model group,the FL scores of mice in the CsA group and Ste-Chr group significantly decreased,while the BUT and tear secretion volume significantly increased(all P＜0.01).Compared with the CsA group,the FL scores of mice in the Chr group increased,and the BUT and tear secretion volume decreased(all P＜0.05),whereas the FL scores of mice in the Ste-Chr group further decreased,and the BUT and tear secretion volume further increased(all P＜0.05).After intervention,the corneas of mice in the model group and Chr group showed opacity,epithelial thickening,and disordered stromal arrangement;while the corneas of mice in the CsA group and Ste-Chr group exhibited good transparency,and the epithelial and stromal structures were regular and dense.After intervention,compared with the control group,the mRNA expression levels of TLR4,NF-κB,TNF-α,IL-6,and IL-1β in the corneal tissue of mice in the model group and each drug intervention group significantly increased(all P＜0.01).Compared with the model group,the expression of the above inflammatory factors in the corneal tissue of mice in the CsA group and Ste-Chr group significantly decreased(all P＜0.01).Compared with the CsA group,the expression of various in-flammatory factors in the corneal tissue of mice in the Chr group increased(all P＜0.05),while those in the Ste-Chr group further decreased(all P＜0.05).After intervention,compared with the control group,the expression of TLR4,MyD88,and NF-κB proteins in the corneal tissue of mice in the model group and each drug intervention group significantly increased(all P＜0.01).Compared with the model group,the expression of the above proteins in the corneal tissue of mice in the CsA group and Ste-Chr group significantly decreased(all P＜0.01).Compared with the CsA group,the expression of each protein in the corneal tissue of mice in the Chr group increased(all P＜0.05),while those in the Ste-Chr group further decreased(all P＜0.05).Conclusion Ste-Chr ophthalmic solution can effectively alleviate ocular surface inflammatory response in DDE model mice by inhibiting the TLR4/MyD88/NF-κB signaling pathway,down-regulating the gene and protein expression of TLR4,MyD88,and NF-κB in corneal tissue,and reducing the levels of pro-inflammatory factors such as IL-1β,TNF-α,and IL-6.