The Association Between Dry Eye And Corneal Epithelial Cellular Energy Metabolism And The Therapeutic Effects Of Amino Acid Supplement

Part Ⅰ:The association between corneal epithelial cellular energy metabolism and dry eyePURPOSE:To investigate the association between corneal cellular energy metabolism and dry eye,and role of L001 in cellular energy metabolism.METHODS:Desiccating stress(DS)was induced in 8-10-week old female C57BL/6(C57)mice by subcutaneous injection of scopolamine hydrobromide(0.5 mg/0.2 mL)four times a day(9:00,12:00,15:00,18:00)and exposure to a low humidity environment(relative humidity<40%)for 5 days.L001(DS5+L001)or vehicle(DS5+Vehicle)eye drops were topically applied in mice subjected to DS.Human corneal epithelial cells(HCECs)were cultured in hyperosmolarity medium(69 mM sodium chloride,450 OsM),medium with(HOsM+L001)or without(HOsM)L001 was used as a group treatment.Cell viability of HCECs was assessed using a cell counting kit(CCK8).Western blot(WB)was used to detect expression of the markers of energy metabolism in HCECs and murine corneal cells,including phosphorylated adenosine monophosphate activated protein kinase(p-AMPK),AMPK,p-acetyl-CoA carboxylase(ACC),ACC,AXIN and liver kinase B1(LKB1).RESULTS:The cell viability of HOsM+L001 cells was significantly higher than that of HOsM cells.The ratio of p-AMPK/AMPK and p-ACC/ACC in HOsM group was significantly higher than that in the normal control group,and the ratio in HOsM+L001 group was significantly lower than that in HOsM group.There was no significant difference in the expression of AXIN and LKB1 among each group.The ratio of p-AMPK/AMPK and p-ACC/ACC in corneal cells of dry eye(DS5)group was significantly higher than that of normal(NS)group.The ratio in corneal cells of DS5+L001 group was significantly lower than that of DS5+Vehicle group.There was no significant difference in the expression of AXIN and LKB1 among each group of mice.CONCLUSIONS:Significant abnormality of corneal cellular energy metabolism appears in dry eye and L001 can alleviate dry eye induced cellular energy metabolism abnormality.Part Ⅱ:Therapeutic effects of L001 on murine dry eyePURPOSE:To investigate the role of L001 in ocular surface damages of dry eye.METHODS:The establishment and treatment of the murine dry eye model was the same as the previous part.The phenol red cotton test was used to measure tear production,and Oregon green dextran(OGD)staining was performed to assess corneal epithelial integrity.Periodic acid-schiff(PAS)staining was used to quantify conjunctival goblet cells.Suamous metaplasia in ocular surface was evaluated by immunofluorescent staining of Keratin(K10)and Small proline-rich protein IB(SPRR1B).Immunofluorescent staining and quantitative real time-polymerase chain reaction(Q RT-PCR)were used to assess the expression of matrix metalloproteinase(MMP)-3 and MMP-9 in corneal epithelium.Cell proliferation was assessed by immunofluorescent staining of Ki-67.Apoptosis in ocular surface was assessed by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)and immunofluorescent staining for activated(AC)caspase-3 and caspase-8.Infiltration of CD4-T cells in conjunctiva was assessed by immunochernistry staining.Levels of proinflammatory cytokines and T helper(Th)cytokines in conjunctiva were detected by Q RT-PCR and enzyme-linked immunosorbent assay(ELISA),including interleukin(IL)-1 p,tumor necrosis factor(TNF)-α,IL-13,IL-17A and interferon(IFN)-γ.RESULTS:Compared with vehicle-treated mice,L001-treated mice showed increased tear production,decreased goblet cell loss and alleviated OGD staining.Topical application of L001 suppressed corneal and conjunctival epithelial keratinization in dry eye.Topical application of L001 decreased the expression of MMP-3 and MMP-9 in corneal epithelium,promoted cell proliferation and suppressed cell apoptosis in ocular surface under DS.Topical application of L001 decreased CD4+T cells infiltration,with decreased production of IL-1β,TNF-α,IFN-γ and IL-17A and increased production of IL-13 in conjunctiva.CONCLUSIONS:Topical application of L001 effectively alleviated ocular surface damages via promoting cell proliferation,suppressing cell apoptosis and inflammation in ocular surface of dry eye.Part Ⅲ:Clinical evaluation of the efficacy of L001 eye drops in dry eyePURPOSE:To investigate the clinical efficacy of L001 eye drops in dry eyeMETHODS:The dry eye patients enrolled in this clinical trial had been treated with other medications,but the therapeutic effects had been poor,and they had been drug discontinued for more than 2 weeks before enrollment.The enrolled patients were divided into three groups(Group 1-3)according to the Ocular Surface Disease Index(OSDI)scores before treatment.Then patients were treated with L001 eye drops for 2 weeks(four times a day,one drop at a time).Symptoms and signs of dry eye were evaluated before and after treatment,including OSDI scores,tear film break-up time(TBUT)measurement,corneal fluorescein staining(CFLS)scores,bulbar conjunctival hyperemia(BCH)scores.RESULTS:After two weeks of treatment with L001 eye drops,OSDI scores of the dry eye patients decreased significantly.Among all the symptoms,ocular pain alleviated the most significantly.After treatment with L001 eye drops,the patients’ TBUT increased significantly,and CFLS scores was significantly reduced.BCH scores showed a downward trend after treatment,but the difference was not statistically significant.CONCLUSIONS:L001 eye drops can effectively alleviate the symptoms and signs of dry eye patients,and have good curative effects especially on the patients who have poor therapeutic effects with other drugs.