Effect And Mechanism Of LRG1 In Promoting Corneal Fibrosis After Alkali Burn In Mice

Objective:Leucine-rich α-2-glycoprotein-1(LRG1)is a novel pro-fibrotic factor that modulates TGF-β signaling.However,its role in the corneal fibrotic response remains unknown.This paper intends to establish a mouse alkali burn model and administer exogenous LRG1 treatment,to explore the fibrosis promoting effect and mechanism of LRG1 in mice after alkali burn.Methods:1.Changes of corneal LRG1 in mice after alkali burn and its effect on fibrosisThe expression level of LRG1 in mouse cornea after alkali burn was detected by immunofluorescence and Western Bolt(WB).To explore the influence of high and low levels of LRG1 on corneal fibrosis,alkali burned mice were treated with LRG1 solution to increase LRG1,and siLRG1 was injected subconjunctival to inhibit LRG1.The expression of alpha-smooth muscle actin(α-SMA)was detected by immunofluorescence.WB was used to detect the expression of α-SMA,Collagen type Ⅰ(Collagen Ⅰ)and Connective tissue growth factor(CTGF).2.The effect of LRG1 on neutrophil infiltration and its relationship with fibrosisThe effect of LRG1 on neutrophils(PE-CD11 b,APC-Ly6 G double positive cells)infiltration in mouse cornea was detected by flow cytometry.The effects of LRG1 on the chemotaxis of neutrophils were detected by Transwell assay.Immunofluorescence co-reference with α-SMA and Ly6 G were used to detect the relationship between corneal fibrosis and neutrophils when LRG1 levels is high or low after burn.In order to investigate the influence of neutrophils on the regulation of fibrosis by LRG1,LRG1-treated alkali burn mice were injected with LY6 G neutralizing antibody subconjunctival to clear neutrophils,and the expression of α-SMA was detected by immunofluorescence.WB was used to detect the expression of α-SMA and CTGF.To evaluate the influence of LRG1 and neutrophils on TKE2 fibrosis.Mouse corneal epithelial stem/progenitor cell line(TKE2)was cultured in vitro.TKE2 was co-cultured with LRG1,neutrophils and both of them,respectively,and the expression level ofα-SMA was detected by immunofluorescence.WB was used to detect the expression ofα-SMA,Collagen Ⅰ and CTGF.3.The effect of LRG1 on IL-6/ Stat3 signaling pathway and its relationship with fibrosis and neutrophil infiltrationMice after alkali burn and neutrophils were treated with LRG1,and their levels of Stat3 phosphorylation and IL-6 were detected by WB.To explore the role of neutrophils in LRG1 regulation of IL-6/ Stat3 signaling pathway,the neutrophils were removed from the cornea of mice with alkali burn treated by LRG1,TKE2 was co-cultured with LRG1,neutrophils and both of them,their phosphorylation level of Stat3 and the level of IL-6were detected by WB.The mice treated with LRG1 were injected with Stat3 inhibitor subconjunctival to inhibit the activation of Stat3.Flow cytometry was used to detect the effect of inhibiting IL-6/ Stat3 pathway on promoting neutrophils infiltration of LRG1.To evaluate the relationship between the fibrosis promoting effect of LRG1 and the IL-6/ Stat3 pathway,WB was used to detect the expression of α-SMA,Collagen Ⅰ and CTGF.Results:1.LRG1 was increased after alkali burn and LRG1 can promote corneal fibrosis.(1)The expression level of LRG1 protein increased in mouse cornea 72 h after alkali burn.It suggested that LRG1 was involved in corneal repair after alkali burn.(2)With LRG1 treatment,the protein expressions of α-SMA,CTGF and Collagen Ⅰ in the cornea were up-regulated,the level of fibrosis was enhanced.Silencing of LRG1 with specific si ILRG1 resulted in reduced levels of corneal fibrosis.These suggested that LRG1 is involved in and promotes corneal fibrosis after alkali burn.2.LRG1 enhances corneal fibrosis after alkali burn through neutrophil infiltration(1)The infiltration of neutrophils was significantly increased by LRG1.At the same time,LRG1 promotes neutrophils migration.These suggested that LRG1 has chemotactic effect on neutrophils.LRG1 administration can promote fibrosis and increase neutrophil infiltration.Silencing LRG1 resulted in reduced fibrosis and neutrophil infiltration.These suggested that the fibrosis promoting effect of LRG1 is related to neutrophils.(2)When neutrophils were removed from the cornea,the promoting effect of LRG1 on corneal fibrosis was inhibited,suggesting that LRG1 may mediate the promoting effect of corneal fibrosis through neutrophils.(3)Both LRG1 and neutrophils can promote the fibrosis of TKE2 cells,and the addition of neutrophils can enhance the fibrosis promoting effect of LRG1 on TKE2 cells,suggesting that neutrophils infiltration can enhance the pro-fibrosis effect of LRG1.3.LRG1 regulates neutrophil infiltration and fibrosis through the IL-6/ Stat3 signaling pathway.(1)LRG1 can increase the expression of IL-6 and the phosphorylation of Stat3 in corneal epithelium and neutrophils in vitro;Clearance of neutrophils inhibited the promotion of p-Stat3 and IL-6 by LRG1,moreover neutrophils significantly enhanced the production of IL-6 and p-Stat3 in TKE2 cells which promoted by LRG1.These suggested that LRG1 can activate the IL-6/ Stat3 signaling pathway of cornea and TKE2 after alkali burn,and neutrophils play an important mediating role in this activation process.(2)Inhibition of Stat3 signaling pathway by S3I-201 can reduce neutrophil infiltration and reduce the corneal fibrosis promoting effect of LRG1 in alkali burn cornea.These suggested that LRG1 promotes fibrosis and neutrophil infiltration by activating the IL-6/ Stat3 pathway.Conclusion:LRG1 promotes corneal fibrosis by regulating the IL-6/ Stat3 signaling pathway,which may be mediated by neutrophils.LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.