Study On The Therapeutic Effect And Anti-inflammatory Mechanism Of Rebamipide On Dry Eye In Mice

Objective:Dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort,visual disturbance,and tear film instability with potential damage to the ocular surface.It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.Severe dry eye can cause obvious decline of Quality of life and serious damage to the vision of patients,affecting normal life.It has been found that despite the onset of dry eye caused by different causes,inflammatory response is the most common key factor in the development of dry eye disease.There have been lots of studies on the inflammatory response in dry eye disease and they found that proinflammatory cytokines and matrix metalloproteinases(MMPs)play an important role in the pathogenesis of dry eye.The expression of inflammatory cytokines(IL-1,IL-6,TNF-α,IL-23,IFN-γ,IL17,MMPs)increased,accompanied by lymphocytic infiltrates in lacrimal gland functional unit(LFU),which led to the gradual decline of lacrimal gland secretion,decreased tear film stability and induced dry eye clinical symptoms.As a mucosal autoimmune disease,anti-inflammatory therapy is becoming a hot spot of current research of dry eye.At present,the anti-inflammatory therapy drugs for dry eye focuses on corticosteroids,cyclosporine A,tetracycline derivative and essential fatty acids.Besides the effective controlling and alleviating the signs and symptoms of DED,they inevitably bring series of adverse reactions,and the dosage form and dosage aspects need to be further explored.Rebamipide(Reb),chemically called 2-(4-chlorobenzene formamide base)-3-[2-(1 H)quinoline ketone-4-yl]propionate,molecular formula C19H15ClN2O4,molecular weight of 370.7864,as a gastrointestinal protection drug,was used for the treatment of gastric ulcer and gastritis previously,by increasing the gastric mucosal blood flow,prostaglandin E2 synthesis and gastric mucus secretion,removal of oxygen free radicals,it promotes the improvement of the digestive ulcer healing and inflammation Recent studies have shown that Reb can be used to treat dry eye.Reb eye drops was officially used in the treatment of dry eye disease in 2012 in Japan.Reb can effectively reduce the symptoms of dry eye patients,improve barrier function and reduce the release of inflammatory cytokines,showing that as a boost drug of mucin production and secretion,it also has anti-inflammatory effects.However,there are relatively few studies on the mechanism of rebamipathy in the anti-inflammatory treatment of dry eye.Therefore,the study of the anti-inflammatory mechanism of rebamipathic in dry eye will provide a theoretical basis for further research in the future and provide a new thinking for the anti-inflammatory treatment of dry eye,which is expected to reduce the burden of drug treatment of dry eye in the future.In the pathogenesis of dry eye disease,T lymphocytes,especially T helper 17(Th17)subtypes play a vital role.It has been found that a variety of chronic inflammatory diseases can be caused by Th17 cells.Many researchers considered that Th17,which was used as a target,has great potential in the treatment of autoimmune diseases and cancer.Regulatory T cells(Treg)are a class of T cell subsets with negative regulatory function that inhibit CD4~+T cells and thereby inhibit ocular surface inflammation in dry eye.The treatment of multiple diseases is associated with the regulation of Th17/Treg cell balance.Studies have found that rebamipide can regulate differentiation balance of Th17/Treg cells in arthritis.Toll-like receptors(TLRs)play an important role in the immune response.TLR4is the first identified TOLL-like receptor,and it was confirmed as a molecule associated with autoimmune diseases.TLR4/MyD88/NF-κB pathway is highly correlated with a variety of diseases.MyD88 is a cytoplasmic adapter of TLR4 and activated TLR4could interacts with MyD88,then activate downstream NF-κB pathway and initiate downstream gene transcription,such as IL-6,TNF-α,IFN-βand other cytokines.Studies have found that the curative effect of some drugs in chronic inflammatory diseases was associated with inhibition of TLR4/MyD88/NF-κB pathway.Based on the above research results,our study will investigate the anti-inflammatory therapeutic effect of Rebamipide on dry eye from the perspective of Th17/Treg cells balance and TLR4/MyD88/NF-κB pathway.This study will use hypodermic scopolamine hydrobromide combined with dry environment simulation to establish dry eye model in mice and observe the pathological changes of dry eye and the protection of Rebamipide to dry eye tissue damage.On this basis,we will detect the influence of Rebamipide on the expression of inflammatory factor in dry eye mice,preliminarily discuss the influence of Rebamipide on Th17/Treg cells differentiation balance and its treatment mechanism of regulating TLR4/MyD88/NF-κB pathway in dry eye miceMethods:96 C57BL/6 mice at the age of 8 weeks were randomLy divided into four groups:normal control group,dry eye(DE)group,DE+balanced salt solution(DE+BSS)group and DE+2%Reb group,24 mice in each group.Mice received subcutaneous injection of NS for 2 weeks in normal control group.The dye eye model was established by subcutaneous injection of scopolamine hydrobromide for 2 weeks in the latter 3groups.BSS was administered as eye drops for 2 weeks in DE+BSS group.2%rebamipide was administered as eye drops for 2 weeks in DE+2%Reb group.The levels of tear secretion were measured after modelling and after administration by phenol red cotton method.Tear break-up time(BUT)and corneal fluorescein staining and K10immunofluorescence staining was used to detect the ocular surface damage of each group.HE staining was used to observe the pathological changes of corneal tissue and lacrimal gland tissue and conjunctival tissue in mice.The mRNA expression levels of IL-1β,IL-6,TNF-α,IL-17,IL-23,TGF-βand IL-10 in the conjunctiva and lacrimal gland were detected by Real-time PCR.The protein expression levels of IL-1β,IL-6,TNF-α,IL-17,IL-23,TGF-βand IL-10 in conjunctiva and lacrimal gland were detected by ELISA.Real-time PCR was used to detect the expression of MMP-9 in lacrimal gland of mice,and the effect of rebamipide on ocular surface and lacrimal gland inflammation was investigated.To investigate the effect of rebamipide on Th17/Treg cells balance,the expression levels of ROR-rt and Foxp3 in cervical and mandibular lymph nodes were detected by Real-time PCR,and the percentage of CD4~+IL-17~+Th17 and CD4~+CD25~+Foxp3~+Treg cells in lymph nodes was detected by flow cytometry,and the ratio of CD4~+IL-17~+and CD4~+Foxp3~+double positive cells in cornea and lacrimal gland were detected by Immunofluorescence double staining.The mRNA expression of TLR4,MyD88,IL-1βand IL-23 in mice cervical and mandibular lymph nodes was detected by Real-time PCR.Western blot was used to detect the expression of TLR4,MyD88,IκB,p-IκB and nucleus NF-κB p65 protein in lymph nodes.The effect of rebamipide on TLR4/MyD88/NF-κB pathway activation was investigated.Results:1、Tear secretion in the dry eye mice was significantly less than that in the normal mice.After treatment with rebamipide,the tear secretion of the mice in the DE+2%Reb group was slightly more than that in the DE group.Compared with normal mice,the ocular surface of the mice in the DE group was seriously injured,but ocular surface damage of rebamipide treated mice was obviously relieved,and the tear film rupture time was prolonged.The results of HE staining showed that the cornea damage was more serious than that of the normal mice.The cell arrangement was disturbed.After the treatment of rebamipide,the cells arranged in the cornea were more regular,and corneal defect was relieved.The results of lacrimal gland tissue staining showed that there was abnormal acinar,vacuolar changes and cell disorder in dry eye mice,while the treatment of rebamipide significantly improved the cell morphology of the lacrimal gland tissue.Conjunctival tissue staining results showed a decrease in the number of goblet cells in the conjunctiva of dry eye mice,whereas treatment with rebamipide significantly increased the number of goblet cells.2、The results of real-time PCR and ELISA in conjunctiva of mice showed that,the expression levels of IL-1β,IL-6,TNF-α,IL-17 and IL-23 in the conjunctiva of dry eye were increased at mRNA and protein levels,and the expression of TGF-βand IL-10 was decreased.The expression of IL-1β,IL-6,TNF-α,IL-17 and IL-23 in the conjunctiva of dry eye was down-regulated by rebamipide and the expression of TGF-βand IL-10 was up-regulated.Lacrimal gland test results was consistent with that in conjunctiva.The expression levels of IL-1β,IL-6,TNF-α,IL-17 and IL-23 in the lacrimal gland of dry eye mice were higher than those in normal mice,while TGF-β,IL-10 expression was lower than normal mice.After treatment with rebamipide,the expression of IL-1,IL-6,TNF-α,IL-17 and IL-23 was down-regulated,and the expression of TGF-βand IL-10was up-regulated.At the same time,the expression of MMP-9 was increased in the lacrimal gland of dry eye mice.After treatment with rebamipide the expression of MMP-9 was decreased compared with that in dry eye group.In dry eye mice,the expression of ROR-rt was significantly increased,the expression of Foxp3 was significantly decreased in the cervical and mandibular lymph nodes.Rebamipide can enhance the expression of Foxp3 in lymph nodes of dry eye mice and down-regulate the expression of ROR-rt.Flow cytometry showed that the proportion of Th17 cells in dry eye mice cervical and mandibular lymph nodes was significantly higher than that in normal mice,and the proportion of Treg cells did not change significantly.The results of immunofluorescence double staining showed that the number of CD4~+IL-17~+positive cells in the cornea and lacrimal gland of dry eye mice increased,and the number of CD4~+Foxp3~+positive cells decreased,while after rebamipide treatment,The number of CD4~+IL-17~+positive cells decreased and the number of CD4~+Foxp3~+positive cells increased.Rebamipide treatment can increase the proportion of Treg cells in dry eye mice and reduce the proportion of Th17 cells.Conclusions:1、Rebamipide can slightly increase tear secretion in dry eye mice and stabilize tear film,can alleviate ocular surface and lacrimal gland damage in dry eye mice.2、Rebamipide can relieve inflammation of ocular surface and lacrimal gland of dry eye mice by affecting the expression of inflammatory cytokines in the conjunctiva and lacrimal gland.Rebamipide can regulate the balance of Th17/Treg cells in dry eye mice,increasing Treg cells differentiation and reducing Th17 cells differentiation.3、Rebamipide can inhibit the expression of TLR4 and MyD88 and the activation of NF-κB,affecting the transcription of downstream inflammatory factors and reduce reduce inflammation in dry eye mice.