| Purpose: At present,the number of diabetes patients in China is as high as 114 million,ranking first in the world.As a chronic metabolic disease,diabetes can cause a variety of eye complications.About 50% of patients with diabetes have dry eyes(with or without symptoms)and primary keratopathy.Diabetic dry eyes are characterized by abnormal tear secretion,decreased tear film stability,ocular surface inflammation and decreased corneal sensitivity.Diabetic keratopathy is characterized by delayed wound healing or nonunion after corneal epithelial wound and corneal nerve damage.Persistent corneal epithelial defect can be followed by complications such as corneal infection,corneal ulcer and corneal perforation,which seriously affected the patient’s vision.At present,the pathogenesis of diabetic dry eyes and diabetic keratopathy is still unclear,and there is a lack of effective therapeutic strategies in clinical practice.Oxidative stress caused by excessive production of reactive oxygen species(ROS)in mitochondria is a common pathogenesis of diabetic complications.Mitochondria-targeted antioxidants specifically target mitochondria ROS,which has shown to have a beneficial effect in a number of basic researches on diabetic nephropathy,diabetic retinopathy and other diseases related to oxidative stress.Therefore,in this study,type 1 diabetic mice induced by intraperitoneal injection of streptozotocin(STZ)were used as an animal experimental model to explore the effects and related mechanisms of mitochondria-targeted antioxidants on diabetic dry eye and diabetic keratopathy.Methods: 1.Comprehensive evaluation of dry eyes in type 1 diabetic mice induced by STZ(1)Evaluation of dry eye related manifestations and macroscopic visualization of lacrimal gland tissues in type 1 diabetic mice induced by STZ Comparing differences between the type 1 diabetic mice and age-matched control C57BL/6J mice in blood glucose,body weight,tear secretion volume,corneal Rose Bengal staining,tear ferning test,the conjunctival goblet cell density,corneal sensitivity,cytokeratin 10(K10)expression in corneal epithelial,weight of lacrimal gland and lipid content in meibomian gland,diabetic mice related dry eyes were systematically evaluated.(2)Evaluation of oxidative stress and antioxidant status status of lacrimal glands in type 1 diabetic mice induced by STZ Pathological changes of lacrimal tissue samples in diabetic mice and age-matched controls were observed by HE staining,Masson staining and immunofluorescence staining.The expressions of 8-hydroxydeoxyguanosine(8-OHg G)as a marker of mitochondrial DNA oxidative damage,antioxidant enzymes such as Mn SOD,NQO-1 and Catalase in lacrimal tissue samples of diabetic mice and age-matched controls were compared by immunohistochemistry staining and Western Blot respectively.The difference of ATP levels in lacrimal tissue samples between diabetic mice and controls was detected by chemiluminescence technology.(3)AGEs/RAGE expression and senescence-related changes in lacrimal tissue samples The advanced glycosylated end products(AGEs)and receptor of advanced glycosylated end products(RAGE)were detected by immunohistochemistry staining,immunofluorescence staining and Western Blot.The aging degree of lacrimal gland and the expression differences of senescence-related genes such as p16 and p53 at m RNA level were detected by frozen section β-gal staining and quantitative real-time RT-PCR respectively.2.Effects and mechanisms of mitochondria-targeted antioxidants on dry eyes in diabetic mice.(1)Effects of mitochondria-targeted antioxidants on dry eye related indicators in diabetic mice.With type 1 diabetes mice induced by STZ as an animal model of dry eye and 0.05% cyclosporine A(Cs A)eye drops as the positive control drug,the effects of mitochondria-targeted antioxidants SS-31(10mg/ml)and Sk Q1(0.155μg/ml)on Rose Bengal staining,tear secretion volume and corneal sensitivity were evaluated at 1,2,and 3 weeks after treatment,respectively.PAS staining was used to evaluate the effects of various drugs on the conjunctival goblet cell density in diabetic mice after 3 weeks treatment.(2)Effects of mitochondria-targeted antioxidants on pathology and ultrastructure of lacrimal glands in diabetic mice HE staining and CD45 immunofluorescence staining were used to evaluate the effect of SS-31 and Sk Q1 on the infiltration of inflammatory cells in the lacrimal gland of diabetic mice after 3 weeks treatment.The effect of SS-31 eye drops on the ultrastructure of lacrimal gland in diabetic mice was evaluated by transmission electron microscopy.3.Effects of mitochondria-targeted antioxidants on diabetic mouse keratopathy(1)Effects and mechanism of mitochondria-targeted antioxidants on the healing of corneal epithelial injury in diabetic mice STZ-induced type 1 diabetic mice and control C57BL/6J mice were used to establish corneal epithelial wounded models.The healing area of corneal epithelium at 24 h,48h and 72 h after epithelial scraped was compared by fluorescein sodium staining.Corneal tissue samples were taken at the above time points for immunofluorescence staining to detect expression of 8-OHd G.The effects of Mn TBAP and SS-31 on corneal epithelial wound healing were determined using an in vivo model of wound healing described above.After 72 h of corneal epithelium wounded,corneal tissue samples were taken and oxidative stress status was detected by measurement of ROS generation and GSH content.Immunofluorescence staining was used to detect the expression of 8-OHd G,antioxidant enzymes(Mn SOD,NQO-1 and Catalase)and the expression of signal transduction molecule such as p-AKT,Sirt1,and p-EGFR involved in corneal epithelium wound repair.The effect of Mn TBAP on the stability of mitochondrial DNA in TKE2 cells cultured in high glucose environment was evaluated by 8-OHd G immunofluorescence staining.Flow cytometry was used to detect the effect of Mn TBAP on ROS generation in TKE2 cells under high glucose environment,and JC-1 staining was used to detect the changes of mitochondrial membrane potential.(2)Effects of mitochondria-targeted antioxidants on corneal nerve repair in diabetic mice After 7 days of corneal epithelium(diameter 2.0mm)scraped in diabetic mice and controls,nerve staining of the whole cornea to evaluate the effects of Mn TBAP and SS-31 on the regeneration of corneal nerve fiber,and the changes of corneal sensitivity before and after corneal epithelium wounded were tracked.Results: 1.Comprehensive evaluation of dry eyes in type 1 diabetic mice induced by STZ(1)Dry eye related symptoms in type 1 diabetic mice induced by STZ were evident Compared with controls,diabetic mice had significantly increased blood glucose and reduced body weight(P<0.001),decreased tear secretion volume(P<0.001),decreased or disappeared tear fern,showed stronger corneal Rose Bengal staining(P<0.001),increased expression of K10 in corneal epithelium,decreased density of conjunctival goblet cell(P<0.001),attenuated corneal sensitivity(P<0.001)and less innervation.The lacrimal glands of diabetic mice were atrophic and weight decreased.The ratio of lacrimal gland weight to body weight was significantly lower than those of age-matched controls(P<0.001)and Tabby mice(P<0.01).There was no significant difference lipid content in meibomian gland between diabetic mice and controls.(2).Lacrimal glands of STZ-induced type 1 diabetic mice showed inflammatory cell infiltration,fibrotic changes and oxidative stress damage The results of HE and Masson staining showed that a large number of inflammatory cells infiltration and more collagen expression surrounding ducts and acinus in lacrimal glands of diabetic mice.The results of immunofluorescence and immunohistochemical staining showed that more CD45 positive cells,α-SMA,and 8-OHd G expression in lacrimal gland samples of diabetic mice.ATP levels decreased significantly(P < 0.01)in lacrimal glands of diabetic mice.Western Blot results showed that the protein levels of antioxidant enzymes such as Mn SOD,NQO-1 and Catalase in the lacrimal tissue samples of diabetic mice were significantly lower than those in controls(P<0.05).(3)Increased expression of AGEs/RAGE and the aging degree in lacrimal gland of diabetic mice The results of immunohistochemistry,immunofluorescence staining and Western Blot showed that AGEs accumulation and increased expession of RAGE in the lacrimal tissue samples of diabetic mice.Quantitative real-time RT-PCR results showed that increased m RNA expression of p16 and p53 in lacrimal gland samples of diabetic mice(P<0.05),which was consistent with the results of β-gal staining.2.The therapeutic effect and mechanism of mitochondria-targeted antioxidants on dry eyes in diabetic mice(1)Mitochondria-targeted antioxidants have been shown to have a beneficial effect in a diabetic mice model of dry eye SS-31-treated diabetic mice decreased corneal Rose Bengal staining similar to that of Sk Q1 group,the staining scores were significantly lower than that of vehicle-treated group after 1-3 weeks treatment respectively(P<0.05),while the staining score in the Cs A group began to decline at 3 weeks after treatment compared with controls(P<0.05).SS-31 increased tear production similar to that in the Sk Q1-treated diabetic mice after 1-3 weeks respectively(P<0.05),while in the Cs A group was improved compared with controls at 2-3 weeks(P<0.05).The corneal sensitivity of SS-31-and Sk Q1-treated mice was higher than that of the vehicle-treated group at all time points(P<0.05),while the Cs A was inactive.PAS staining showed that the density of conjunctival goblet cell increased significantly in all drug treatment groups after 3 weeks compared with that in the vehicle-treated group(P<0.05).(2)Mitochondria-targeted antioxidants can reduce the infiltration of inflammatory cells and improve mitochondrial structure of lacrimal gland in diabetic mice The results of HE and CD45 immunofluorescence staining showed that the infiltration degree of inflammatory cells was reduced in the lacrimal gland of the SS-31-,Sk Q1-and Cs A-treated mice.The results of transmission electron microscopy showed that SS-31 treatment could increase the number of secrete vesicles in lacrimal gland acinar cells of diabetic mice and improve ultrastructure and external morphology of mitochondria.3.The effects and mechanisms of mitochondria-targeted antioxidants on corneal wound healing and nerve regeneration in diabetic mice(1)Mitochondria-targeted antioxidants promote wound healing in diabetic mice Compared with normal control mice,the healing rate of corneal epithelium was significantly delayed at 48 h and 72 h in diabetic mice after corneal epithelium wounded(P<0.001).The result of Immunofluorescence staining showed that 8-OHd G consistently expressed at high levels in corneal epithelium during corneal epithelial wound healing.Mn TBAP treatment significantly inhibited the up-regulated expression of 8-OHd G in TKE2 cells under high glucose environment.Moreover,ROS generation in TKE2 cells after hyperglycemia was significantly reduced(P<0.001),and the mitochondrial membrane potential of TKE2 cells was reversed under hyperglycemia(P<0.001)after Mn TBAP treatment.Subconjunctival injection of Mn TBAP and SS-31 significantly promoted the corneal epithelium wound healing(P<0.001),decreased the corneal epithelial ROS level and 8-OHd G expression,increased the expression of GSH,Mn SOD,NQO-1,Catalase,and up-regulated the expression of p-AKT,Sirt1,p-EGFR and Ki67 in corneal epithelial cells of diabetic mice.(2)Mitochondria-targeted antioxidants promoted the regeneration of corneal nerve fbers impaired in diabetic mice After the corneal epithelial wound in diabetic mice,subconjunctival injection of Mn TBAP and SS-31 significantly promoted the regeneration of corneal nerve fibers in diabetic mice(P<0.05)and the recovery of corneal sensitivity(P<0.01).Conclusion: 1.The mice with type 1 diabetes induced by STZ intraperitoneal injection were mainly manifested as lacrimal gland atrophy,decreased tear secretion,increased corneal Rose Bengal staining,decreased conjunctival goblet cells,and decreased corneal sensitivity,which were similar to clinical manifestations and could be used as an animal model in research for diabetic dry eye.2.It was shown that increased inflammatory cell infiltration,increased expression of mitochondrial DNA oxidative damage marker 8-OHd G,and decreased expression of antioxidant-related enzymes in the lacrimal tissue samples of diabetic mice,suggesting that oxidative stress may be involved in the occurrence of diabetic dry eye.3.Mitochondria-targeted antioxidants SS-31 and Sk Q1 have certain therapeutic effects on dry eyes of diabetic mice,which can improve the production of tear secretion,increase the density of goblet cells,improve the corneal sensitivity and tear film stability,as well as reduce the infiltration of lacrimal inflammatory cells and improve ultrastructure and external morphology of mitochondria.4.Mitochondria-targeted antioxidants promote the promote corneal epithelial wound healing and regeneration of corneal nerve fibers in diabetic mice;The mechanism of action may be related to anti-oxidative stress,improvement of mitochondrial DNA stability,and up-regulation expression of p-AKT and Sirt1.Innovation and meaning: In this study,it was found for the first time that type 1 diabetes mice with dry eyes induced by STZ showed lacrimal gland atrophy with ratio of lacrimal gland weight to body weight significantly lower than the normal control mice and Tabby mice which is a relevant meibomian gland dysfunction-related dry eye model,suggesting the pathogenesis of diabetes related dry eye may differ substantially from other types of dry eyes.In addition,it was found for the first time that mitochondrial DNA oxidative damage was found in lacrimal gland and in corneal epithelium during corneal epithelial wound healing in diabetic mice induced by streptozotocin.With it as a target,mitochondria-targeted antioxidants were used for treatment of diabetic mice with dry eyes and keratopathy.It was found that Mitochondria-targeted antioxidants can alleviate the inflammatory changes of lacrimal glands in diabetic mice,improve the morphological structure of lacrimal mitochondria,and increase the amount of tear secretion.It was found that it can promote corneal epithelial wound healing and regeneration of corneal nerve fibers in diabetic mice.The multifunctional physiological effects of mitochondria-targeted antioxidants provide potential therapeutic strategies and new targets for the clinical treatment of diabetic dry eye and diabetic keratopathy. |
