The Role Of GSDMD In The Induction Of Early Alkali Burn-induced Corneal Inflammation And The Effect Of Glibenclamide On Inhibiting Inflammation

Alkali Burn is one of the ophthalmic emergencies.Because alkaline chemicals can dissolve fat and protein,lye easily penetrates into the eye through the cornea and spreads.The damaged tissue further secretes protease to exacerbate tissue cells.Dissolution,necrosis,repeated and difficult to control inflammation and tissue adhesion,neovascular growth,and eventually develop into eye deformity/blindness.Although a variety of treatments are used clinically,including:immediate lavage,removal of harmful substances;drug treatment;early promotion of epithelial formation,control of inflammation,reduction of scar formation in the later stage;surgical treatment.However,for moderate to severe corneal alkali burns,the therapeutic effect is not satisfactory and the prognosis is poor.Therefore,exploring the mechanism of inflammation of corneal alkaline chemical burns and finding possible therapeutic methods are the research direction that ophthalmologists have been working on.Pyroptosis,one of the ways of cell death,has recently attracted researchers’attention.Unlike cell necrosis and apoptosis,pyroptosis is considered to be a programmed lytic cell death with inflammation.Innate immunity recognizes signs of injury or inflammation,then assembles and activates inflammasomes,which in turn produce activated caspase-1/4/5/11.The activated caspase in turn cleaves the gasdermin（GSDMs）family proteins and pro-inflammatory factor precursors.The N-terminal domain of the GSDMs is perforated on the cell membrane,and mature inflammatory factors such as IL-1β and IL-18 are released.They produce pro-inflammatory signals to neighboring cells and rapidly initiate inflammatory responses through cascade amplification.GSDMs family proteins are key performers of pyroptosis,including GSDMA,GSDMB,GSDMC,GSDMD,GSDME,etc.,which differ in the expression levels of different tissues and organs.Most of them can be cleaved by inflammatory caspase,the fragments that are cleaved play an important role of punching and destroying the integrity of cell membrane.Among GSDMs family,GSDMD is one of the most widely studied key menbers in the world,and it has been proven to play an important role in various diseases such as diabetes,kidney disease and infectious diseases.Glibenclamide is a commonly used sulfonylurea oral hypoglycemic agent.In addition to being a hypoglycemic agent,studies have found that intraperitoneal injection of glibenclamide in an animal model of bladder inflammation,sinusitis with nasal polyps,etc.,can reduce IL-1β,TGF-β1,TNF-α by inhibiting the activation of NLRP3 inflammatory bodies.However,its application in ophthalmic anti-inflammatory treatment has not yet been seen.Objective 1.To investigate whether GSDMD-mediated pyroptosis is involved in the early inflammatory response of corneal alkali burn;2.To explore the effect and mechanism of topical application of glibenclamide on corneal alkali burn.Methods:Part Ⅰ:Establish a mouse corneal alkali burn model,observe and collect corneal tissue on the 3rd,5th,7th,and 14th day respectively.Real-time quantitative PCR was used to detect the mRNA levels of NLRP3,caspase-1,GSDMD and IL-β.The protein expression of NLRP3,caspase-1/caspase-1p20,GSDMD/GSDMD-N,IL-1β/IL-1βcleave was detected by Western Blot..Part Ⅱ:Comparison of effects of intraperitoneal injection and topical application of glibenclamide on blood glucose in mice.A mouse corneal alkali burn model was established,which consisted of a solvent eye drop control group（DP）,a 100 uM eye drop group（G1）,a 200 uM eye drop group（G2）,and a 500 uM eye drop group（G5）.The healing of corneal epithelium in each group was compared by fluorescein sodium staining and tissue HE staining.Real-time quantitative PCR was used to detect the mRNA levels of NLRP3,caspase-1,GSDMD,IL-1β and MMP-9/13 in the corneal tissues of each group after burn.The protein level of NLRP3,caspase-1/caspase-1p20 and GSDMD were detected by Western Blot.Results:1.After alkali burn,the mRNA and protein levels of NLRP3,caspase-1,GSDMD,IL-1β in corneal tissue increased（P<0.05）,and the increasing trend was roughly consistent.2.Intraperitoneal injection of glibenclamide caused a transient decrease in blood glucose in mice,and recovered 24 hours after administration.（P<0.001）Glyburide eye drops had no significant effect on blood glucose in mice.3.Sodium fluorescein staining score and HE staining after corneal alkali burn:The sodium fluorescein staining score of the solvent control group was higher than that of the glibenclamide concentration group（P<0.001）.The results of HE showed that the corneal epithelial defects in the solvent control group were more severe than the other groups,and the infiltration of inflammatory cell was obvious.4.Topical application of glibenclamide in the treatment of corneal alkali burn significantly reduced the mRNA and protein levels of NLRP3,caspase-1,GSDMD,IL-1β as well as the mRNA expression of matrix metalloproteinase MMP-9/13（P<0.05）.Conclusion:It is preliminarily confirmed that GSDMD-mediated pyroptosis may be involved in early inflammatory response after corneal alkali burn.Topical application of glibenclamide may inhibit the activation of NLRP3 inflammasome,thereby preventing pyroptosis and reducing the early inflammation of corneal alkali burns in mice.