| PurposeThe cornea is a crucial component in the ocular refraction imaging system,but as the outermost layer of the eye,it is directly exposed to the environment and is vulnerable to various damage factors（eg.abrasions,infections,and burns）.When the process of corneal repair is abnormal,it will lead to corneal edema,neovascularization,and scar formation,resulting in poor vision.The global blindness and visual impairment caused by corneal opacity are also one of the priorities of national prevention and treatment of blindness.It is urgent to find a safe and effective drug to promote the proliferation of corneal epithelial cells and reduce the formation of corneal opacity and neovascularization.At present,it has been proved that when the corneal epithelial cells are damaged,the necrotic cells will release various endogenous factors--alarmin,which mediates inflammatory cells migration towards the damaged tissue,and then generates an excessive inflammatory response,leading to corneal stromal fibrosis,corneal ulcer and so on.Among them,high mobility group box 1（HMGB1）,as one of the classical alarmins,has been demonstrated to act as an inflammatory factor aggravating the development of the disease in keratitis,uveitis,glaucoma,diabetic retinopathy,and retinal degeneration,while inhibiting the expression and function of HMGB1 is expected to become a new therapeutic approach for these diseases.In this study,we investigated whether HMGB1participates in the process of repair and the expression of extracellular HMGB1 at different times after corneal injury through a mechanical injury model of corneal epithelium in mice,and the related regulatory mechanism of HMGB1 on inflammatory response and Haze formation during wound repair.Therefore,we examined the corneal epithelial defect rate,corneal histopathological changes,the expression of extracellular HMGB1 at different times,cell proliferation,the number of neutrophils,the expression of inflammation-related factors,Haze score and the expression of Haze formation-related factors in each group to investigate the expression of extracellular HMGB1 and whether using the inhibitor glycyrrhizin（GL）to inactivate extracellular HMGB1 function could affect the outcome of inflammation response and Haze formation in the process of corneal wound healing in mice.Methods1.105 8-week-old healthy C57BL/6J mice were divided into three groups using a random number table,which were healthy group（A）,corneal epithelial injury+PBS eye drop group（B）,and corneal epithelial injury+GL eye drop group（C）,with35 mice in each group.One day before modeling,groups A and B were intraperitoneally injected with 100μL of PBS solution,respectively,and group C was intraperitoneally injected with 100μL of 2 g·L-1 GL.When modeling,the model of corneal epithelial injury was not established in group A.The model of corneal epithelial injury was established in the right eyes of group B and C（left eyes without the process）.After establishing the model,the right eyes of mice in groups B and C were topically instilled with 5μL GL or PBS every six hours for three consecutive days and then twice a day for another eleven days.2.After the model of corneal epithelial injury was established,the corneal epithelial defect was observed by staining with 100 g·L-1 fluorescein sodium solution at 24h,48h,and 72h,and filmed for the record.Haze score was performed on corneal transparency at 1 and 2 weeks after modeling,and data were recorded.3.72 hours after t the model of corneal epithelial injury was established,the mice were killed by cervical dislocation.The whole eyeballs were harvested,sliced in paraffin,and stained with HE.The histopathological changes of the cornea were observed under a light microscope.4.At 24 h,48 h,72 h,1 week,and 2 weeks after the model of corneal epithelial injury were established,the mice were sacrificed by cervical dislocation,the complete eyeballs were removed,and the paraffin sections were subjected to immunohistochemical staining（IHC）to detect the expression of extracellular HMGB1,myeloperoxidase（MPO）,nuclear-associated antigen Ki-67（Ki-67）,nuclear factor-κB（NF-κB）,interleukin-1β（IL-1β）,transforming growth factor-β1（TGF-β1）,α-smooth muscle actin（α-smooth muscle actin,α-SMA）,fibronectin（FN）,collagen type III（collagen type III）proteins in each group.5.HMGB1,IL-1β,NF-κB,nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome（NLRP3）,α-SMA,and FN protein were detected in corneal tissues at different time points in each group of mice by Western blot.6.The relative m RNA expressions of HMGB1,NF-κB,IL-1β,chemokine 2（CCL2）and C-X-C motif chemokine ligand 2（CXCL2）were detected by Quantitative Real-time PCR（RT-q PCR）in corneal tissues at different time points in each group of mice.Results1.HMGB1 expression in corneal tissue after corneal epithelial injury.At 24 h,48 h and 72 h after modeling,the results of IHC staining showed that HMGB1protein was not expressed in the normal corneal epithelial extracellular and stromal layer in group A.In group B and C,HMGB1 protein was translocated from the nucleus of corneal epithelial cells to cytoplasm and extracellular space,and positive expression of HMGB1 protein was observed in the stromal layer.However,with an application of GL,the positive expression of HMGB1 protein in the corneal epithelial extracellular and stroma in group C was significantly weakened compared with group B.Similarly,RT-q PCR and Western blot results of HMGB1 also showed that the m RNA and protein expression levels of HMGB1 in corneal tissues of groups B and C were significantly increased at each time point compared with group A,while the increased level in group C was significantly lower than that in group B.2.The healing of corneal injury at different time points,and Haze scores.At 24 h,48 h and 72 h after modeling,the corneal epithelial defect rates in group B were（87.74±3.57）%,（32.21±4.02）%and（3.80±1.86）%,respectively,which were significantly higher than those in group C（78.75±5.81）%,（21.58±4.53）%and（0.83±0.72）%,and the differences were statistically significant（all P<0.01）,which can be seen that the proliferation ability of corneal epithelial cells in group C is stronger than that in group B.At 1 and 2 weeks after modeling,Haze scores of the cornea in group B were（2.50±0.54）points and（1.50±0.54）points,respectively,which were significantly higher than those in group C（1.67±0.51）points and（0.92±0.58）points,and the differences were statistically significant（all P<0.05）,which can be seen that the corneal Haze degree of group C is lower than that of group B.3.Corneal tissue structure repair and epithelial cell proliferation.At 72 h after modeling,HE staining results showed that:in group A,the structure of normal corneal tissue was complete,the epithelial layer was composed of 3 to 5 layers of epithelial cells,and the collagen fibers in the stromal layer were arranged regularly and tightly;in group B,the corneal epithelial cells grew 0 to 2 layers,and the epithelial cells were disorganized;while in group C,the corneal epithelial cells grew2 to 3 layers,and the epithelial cells were arranged less regularly.At the same time,72 hours after modeling,the staining results of Ki-67 protein in epithelial cells showed that the positive expression rate of Ki-67 protein in corneal epithelial cells of group C was（82±3.57）%,which was significantly higher than that of group B（78±4.02）%,and the differences between two groups were considered statistically significant（P<0.05）.These results indicated that the proliferation ability of corneal epithelial cells in group C was stronger than that in group B.4.Extracellular HMGB1 regulates IL-1βexpression after corneal epithelial injury.Western blot results showed that the mature IL-1βprotein was not expressed in the normal corneal tissues of group A,but only the pro-IL-1βprotein;at 24 h,48 h,and 72 h after modeling,the mature IL-1βprotein was highly expressed in the damaged corneal tissues of both groups B and C,and with an application of GL to inhibit the expression and function of extracellular HMGB1,the expression level of mature IL-1βprotein in the corneal tissues of group C was significantly lower than that of group B at each time point.Similarly,IHC staining of IL-1βprotein also showed that in the normal corneal tissues of group A,IL-1βprotein was only expressed in the corneal epithelial layer but not in the stroma;IL-1βprotein was highly expressed in the corneal epithelial layer,and stromal layer in both groups B and C.The mean optical density（MOD）of positive IL-1βprotein expression in the corneal stroma of group C was lower than that of group B at each time point.Besides,the results of IL-1βRT-q PCR also showed that the m RNA level of IL-1βof B and C group in corneal tissue was significantly increased after corneal injury,the m RNA level of IL-1βin group C was significantly decreased compared with B group.In addition,we further investigated extracellular HMGB1 how to regulate the expression of IL-1βexpression by Western blot,RT-q PCR,IHC,and other methods,and found that extracellular HMGB1 could regulate the protein expression of IL-1βthrough the NF-κB-P65/NLRP3 signaling pathway.5.After the corneal epithelial injury,extracellular HMGB1 induces neutrophils migration into the corneal stroma and exacerbates inflammatory injury to the damaged tissue.At 24 h,48 h,and 72 h after modeling,IHC staining results of MPO protein showed that there was no neutrophil infiltration in the stromal layer of normal corneas in group A.At 24 h after modeling,a large number of neutrophils migrated from the limbus to the corneal stroma in both groups B and C;at 48 h after modeling,the infiltration degree of neutrophils in the corneal stroma of both groups B and C reached the peak,and it was（93.33±3.18）cells per 200 fold field of view in the C group and the number was considerably lower than that of the B group[（142.66±4.50）cells per 200 fold field of view],the difference was statistically significantand（P﹤0.01）.At 72 h after modeling,it was（10.66±5.13）cells per 200 fold field of view in the C group and the number was considerably lower than that of the B group[（40.00±6.08）cells per 200 fold field of view],the difference was statistically significantand（P﹤0.01）.From these results,we found that the number of neutrophilic infiltrates in the corneal stromal layer was significantly lower in group C than in group B by using GL to inactivate extracellular HMGB1 function.Similarly,the results of RT-q PCR also showed that:at 24 h,48 h,and 72 h after modeling,the m RNA expression levels of CCL2 and CXCL2,which are effective molecules for chemotactic neutrophil migration,in the corneal tissues of group C were significantly lower than those of group B.6.After the corneal epithelial injury,extracellular HMGB1 induces fibroblast differentiation and Haze formation by regulating the expression of TGF-β1.At 72h,1week and 2 weeks after modeling,IHC staining results showed that the positive expression of TGF-β1 protein was observed in the corneal stroma of group B at 72h compared with the normal corneal tissues of group A.After 1 week,the positive expression of TGF-β1 protein continued to increase,while the positive expression of TGF-β1 protein of group C in the stromal layer was significantly decreased at each time point compared with B group through applying GL to inactivate extracellular HMGB1 function.Meanwhile,at 1 and 2 weeks after modeling,mature myofibroblasts with positive expression ofα-SMA protein appeared in the corneal stromal layer of group B,and a large amount of collagen III and FN protein accumulated in the stromal layer.However,the application of GL in group C reduced the formation of myofibroblasts,as well as the abnormal accumulation of collagen III,FN collagen in the stromal layer.ConclusionsWhen corneal epithelial injury,it can induce the abnormal expression of HMGB1 in corneal tissue.Extracellular HMGB1 is an important regulatory molecule that mediates the migration,cytokine production of neutrophils,and the mature of myofibroblasts,which induce an excessive inflammatory response to damaged tissues,delay healing and increase Haze degree.However,GL can effectively antagonize the function of extracellular HMGB1,reduce the overexpression of cytokines and neutrophil infiltration in injured tissues,thereby reducing the inflammatory response,promoting the healing of corneal epithelial layer as well as reducing corneal Haze. |
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