Regulatory Mechanism Of Aspirin Triggered-Resolvin D1 In Pyroptosis In Rats With Acute Keratitis

Purpose To investigate the clinical manifestation and the expression level of pyrolysis by lipopolysaccharide-induced keratitis in Wistar rats.And studying the role of Aspirin Triggered-Resolvin D1 in pyrolysis of the cornea,and to explore the molecular mechanism of pyroptosis in which AT-Rv Dl may be involved in.Methods This experiment is consisted of three parts.In the first part,through the establishment by LPS induced keratitis in vivo model of Wistar rats,analyzing and comparing the clinical manifestations and the characteristics of inflammation(1,3,5 days)by means of pathology method.Besides,appling q RT-PCR,immunohistochemical staining and Western-blot to detect the expression of GSDMD and Caspase-11 of corneal tissue.In the second part,in vitro,except control group,human corneal epithelial cells(HCECs)were stimulated with LPS(1000 ng/ml)and AT-Rv D1(10-8 mol/l)respectively.Cell proliferation was assayed using a cell counting kit-8 test.Besides,cell apoptosis was evaluated by flow cytometry.Furthermore,the effects of AT-Rv D1 on the protein level of GSDMD and Caspase-4 were examined using Western blotting and immunofluorescence,in order to further to explore the signal pathway that ATRv D1 may be involved in pyroptosis.In the third part,preprocessing with DHA and adding with different concentrations of AT-Rv D1(10 ng/d and 100 ng/d)on corneal tissue by local intervention respectively.Through pathological and ocular surface analysis,the different effects of different intervention methods in 3 days after stimulated by LPS were analyzed.In addition,the m RNA and protein expression of caspase-11 and GSDMD was detected by q RT-PCR,Western-blot and immunohistochemical staining.Results 1.The corneal haze was detected by LPS stimulation.The HE indicated that more neutrophils were present in corneas,which was reached peak level in the 3 day after LPS stimulation.2.q RT-PCR,immunohistochemical staining and Western-blot showed the level of Pro-Caspase-11,activated-Caspase-11,Pro-GSDMD and p30 were gradually increased after intervention by LPS and reached the peak level in the 3 day after LPS stimulation(P < 0.01).3.LPS markedly increased the number of apoptotic HCECs and decreased cell viability(P < 0.01).4.Western-blot and immunofluorescence revealed that AT-Rv D1 diminished the production of Pro-GSDMD,p30 and Caspase-4 in cells(P < 0.01).5.Topical administration of AT-Rv D1 and preprocessed with DHA resulted in a significant reduction in the severity and incidence of corneal haze compared to the disease counterparts in the 3 day after LPS administration.Infiltration of fewer neutrophils into the cornea were observed by HE staining with AT-Rv D1 and DHA treatment.6.q RT-PCR,immunohistochemical staining and Western-blot indicated that ProCaspase-11,activated-Caspase-11,Pro-GSDMD and p30 were significantly decreased after added with AT-Rv D1 or DHA,and the role of higher concentration of AT-Rv D1 was more remarkable(P < 0.01).Conclusions The acute keratitis induced by LPS in Wistar rats was self-limited.At the same time,LPS stimulation induced corneal inflammation by triggering the non-classical pathway of pyroptosis.In addition,AT-Rv D1 and DHA,could inhibit cell death by blocking the non-classical pathway of pyroptosis,meanwhile,to promote cell proliferation and tissue repair,which providing a new theoretical basis and treatment idea for clinical treatment of acute keratitis.