| Objective:Dry eye disease(DED)is a common ocular surface disease and one of the common ocular surface diseases that causes blindness,which seriously affects the quality of life of patients.At present,the treatment methods for dry eye mainly include artificial tears replacement,immunosuppressive eye instillation and punctum closure,etc.,but the treatment effect is often poor.Therefore,it is urgent to develop more targeted treatments for dry eye.In order to find targeted drugs for dry eye treatment,research in recent years has mainly focused on the etiology and pathogenesis of dry eye.The pathogenesis of dry eye is related to the abnormal quantity and quality of tear secretion and the dysfunction of the corneal barrier caused by various reasons.Although the etiology of dry eye is varied,it is currently believed that the core mechanism of dry eye pathogenesis is inflammation,and the chronicity of dry eye is related to the persistent imbalance of inflammatory regulation.In addition,the increase of apoptosis and the changes of sex hormone levels in vivo are also involved in the pathogenesis of dry eye.As a multifunctional cytokine,IL-6 has a wide range of immunoregulatory functions and plays an important role in the proliferation and differentiation of T and B lymphocytes.Intervention of its activation process is of great significance to solve various inflammatory diseases.In recent years,studies have found that in addition to the elevated levels of IL-6 in the serum of dry eye patients,the levels of IL-6 in tears are also elevated.IL-6 can be used as a biomarker in the early stage of dry eye and a key point for targeted therapy.Tocilizumab(TCZ),a recombinant humanized,anti-human immunoglobulin G1k subclass monoclonal antibody against soluble and membranebound IL-6R,was first introduced as an orphan drug in 2005 Approved in Japan for the treatment of Castleman disease.Whether TCZ plays a role in the treatment of dry eye has not been reported.Tocilizumab works by inhibiting inflammatory activation,and studies have shown that IL-6 inhibition reduces NLRP3 inflammasome activation.NLRP3,ASC and caspase-1 constitute the inflammasome,the formation of this protein complex promotes ASC oligomerization,ASC binds to pro-caspase-1,promotes caspase dimerization and activation,leads to caspase-1 activation,and promotes the participation of proinflammatory cells The factors IL-1β and IL-18 mature into biologically active forms and cleave GSDMD.Studies have shown that the mRNA and protein expressions of the NLRP3 inflammasome are up-regulated in human dry eye,and the downstream inflammatory factors caspase-1,IL-1β and IL-18 are also elevated in patients with dry eye,indicating that the NLRP3 inflammasome is involved in the dry eye.Occurrence and development of ocular inflammation.In a mouse dry eye model,prolonged dryinginduced ROS activates the NLRP3 inflammasome.In addition,calcitriol can effectively alleviate the corneal epithelial cell damage caused by hyperosmotic stress by inhibiting the NLRP3-ASC-caspase-1-GSDMD signaling pathway.The downstream inflammatory factor IL-1β of IL-6 and NLRP3 inflammasome can induce the activation of JAK/STAT signaling pathway.Whether tocilizumab acts by regulating the NLRP3 inflammasomemediated JAK/STAT signaling pathway remains to be further investigated.In conclusion,this study established a mouse model of dry eye to explore the therapeutic effect of tocilizumab on dry eye mice and its effect on the activation of NLRP3 inflammasome on the ocular surface of dry eye mice.In addition,this study also explored whether tocilizumab regulates the injury of lacrimal epithelial cells by inhibiting the activation of the NLRP3 inflammasome-mediated JAK/STAT3 signaling pathway through primary culture of lacrimal gland epithelial cells.To provide a theoretical basis for the development of new drugs for the treatment of dry eye.Methods:The NOD mouse model of dry eye was established by low humidity and injection of scopolamine hydrobromide;the tear secretion and tear film breakup time of the mice were detected,and the corneal damage of the mice was observed by tiger red staining;the pathology of the cornea and lacrimal gland of the mice was observed by HE staining Change;TUNEL assay to detect the apoptosis rate of mouse corneal epithelial cells;qRT-PCR to detect the expression of inflammatory factors in mouse cornea and conjunctiva;qRT-PCR and immunofluorescence to detect mouse corneal matrix metalloproteinases(MMP)-3 and MMP-9 expression;ROS fluorescent probe was used to detect the level of ROS in mouse corneal tissue;Western blot was used to detect the expression of GSDMD-N protein and NLRP3 inflammasome-related protein in mouse cornea and conjunctiva;the formation of ASC spots in mouse corneal tissue was detected by immunofluorescence ELISA was used to detect the levels of IL-1β and IL-18 in the tears of mice in each group;Western blot was used to detect the expression of JAK/STAT3 signaling pathway-related proteins in the cornea and lacrimal gland tissues of mice in each group.The inflammatory response of lacrimal gland epithelial cells was induced by IL-6 to establish an in vitro model;ROS fluorescent probe was used to detect the level of ROS in lacrimal gland epithelial cells;Western blot was used to detect the expressions of GSDMD-N,NLRP3 inflammasome-related proteins and JAK/STAT3 signaling pathwayrelated proteins in lacrimal gland epithelial cells;Immunofluorescence was used to detect the formation of ASC spots in lacrimal gland epithelial cells;ELISA to detect the levels of IL-1β and IL-18 in the supernatant of lacrimal gland epithelial cells;CCK-8 to detect the viability of lacrimal gland epithelial cells.The NLRP3 overexpression plasmid was transfected into the lacrimal gland epithelial cells treated with tocilizumab,and the expressions of GSDMD-N,NLRP3 inflammasome-related protein and JAK/STAT3 signaling pathway-related proteins in the lacrimal gland epithelial cells were detected by Western blot;the lacrimal gland epithelial cells were detected by immunofluorescence.The formation of ASC spots;the levels of IL-1β and IL-18 in the supernatant of lacrimal epithelial cells were detected by ELISA;the viability of lacrimal epithelial cells was detected by CCK-8.STAT3 was silenced in cells treated with tocilizumab overexpressing NLRP3,the expression of STAT3 protein in lacrimal gland epithelial cells was detected by Western blot,and the viability of lacrimal gland epithelial cells was detected by CCK-8.Results:Tocilizumab treatment increased tear secretion and tear film breakup time in mice with dry eye,decreased corneal tiger red staining,improved corneal and lacrimal gland pathological damage,inhibited corneal epithelial cell apoptosis,and reduced mouse corneal epithelial cell apoptosis.Corneal IL-1β,IL-6,IL-17A and IFN-γexpression,reduce the expression of MMP-3 and MMP-9 in mouse corneal epithelium,reduce mouse corneal ROS level,GSDMD-N expression and ASC spot formation;inhibit NLRP3 Inflammasome and JAK/STAT3 signaling pathway activation and tear IL-1β and IL-18 secretion.Similarly,tocilizumab decreased ROS levels,GSDMD-N expression,ASC speck formation,and IL-1β and IL-18 levels in cell culture supernatants in IL-6-treated lacrimal gland epithelial cells;inhibited NLRP3 inflammasome and JAK/STAT3 signaling pathway activation;increases cell viability.Overexpression of NLRP3 increased tocilizumab-treated lacrimal gland epithelial cells GSDMD-N expression,ASC speckle formation,and IL-1β and IL-18 levels in cell culture supernatants;activated NLRP3 inflammasome and JAK/STAT3 signaling pathway,decreased cellularity vitality.Silencing of STAT3 promotes the viability of NLRP3-overexpressing lacrimal gland epithelial cells.Conclusion:Tocilizumab inhibits the JAK/STAT3 signaling pathway by inhibiting the activation of the NLRP3 inflammasome,thereby promoting the viability of lacrimal gland epithelial cells,and plays a role in the treatment of dry eye disease. |
