Relationship Of Matrix Metalloproteinase Expression And Activation Of The FAK Signaling Pathway In Herpes Stromal Keratitis

Part One:Expression of Matrix Metalloproteinase and Focal adhesionkinase in Experimental Herpes Simplex Virus KeratitisObjective: To investigate the expression of the matrix metalloproteinase (MMPs) and the focal adhesion kinase (FAK) in herpes stromal keratitis (HSK) and to investigate the relationship between the expression of MMP-2, FAK with the course of keratitis.Methods:Forty female BALB/c mice were averagely divided into4groups stochastically. Take the right eyes as the experiment eye, and dropping HSV-1on the corneas after scratched epidermis of the corneas. Keratitis was induced in BALB/c mice by inoculating the cornea with HSV-1(KOS strain). Observed changes of the corneas through the slit lamp everyday. Corneas were harvested at daysO,2,7and14.5mice in each group were infected eye paraffin-embedded, and the application of anti-MMP-2, anti-FAK polyclonal antibody stained sections of the cornea, Immunohistochemical staining was used to detect FAK expression. Extracted the total RNA from the tissue of cornea form another collection of five mice in each groups, then reverse transcripted the RNA into cDNA. Amplified the cDNA and the products were electrophoresis in the agarose gelatum. The expression of MMP-2, FAK mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR). To analyzed the data half-quantu using analysis of variance SNK-q test.Results:The typical manifestation of HSK were seen on each mice after infection2days. The epidermis of the corneas were Coloboma, then the inflammation in the superficial layer stroma occurred with the inflammation cells infiltrating. At the7days post-infection the corneal stroma were edema, the ulcer and blood vessel formate. The situation become more Severe at14days post-infection. Immunohistochemistry showed that the first two days after infection, the expression of MMP-2and FAK than uninfected eye increases and the expression in the superficial stroma and inflammatory cells in the skin. The first seven days, the expression of MMP-2and FAK weakened compared with the first two days after infection. The first14days after infection and corneal stroma seen infiltrating inflammatory cells, especially ulcers visible again significantly increased expression of MMP-2and FAK. Overall, in the HSK model, the corneal epithelial cells were deranged and the number of neutrophils with the MMP-2positive cells and the FAK-positive cells were increased; all were significantly different from negative controls (P<0.05). RT-PCR result shows MMP-2, expression FAKmRNA substantially increase the number of days with infection showed an increasing trend. Wherein expression of MMP-2mRNA at day0and the first two days the difference was not statistically significant (P>0.05) after infection, the expression of mRNA7and14days after infection gradually increased the difference was significant (P<0.01). Expression FAKmRNA in0days after infection, the first two days, the first seven days gradually increased, the first14days to express the highest value, but increased the difference on day7compared with the first two days of no significant (P>0.05), the remaining each group differences were statistically significant (P<0.01).Conclusions:After HSV-1infection of the cornea, and corneal neutrophil cells to release large amounts of proteolytic enzymes MMP-2. The expression of the FAK can be detected in HSK cornea. The expression was increase with the inflammation reaction intensify. Speculated that both play an important role in the process of dermatitis on the cornea, corneal ulcer, neovascularization, etc. Part Two:Relationship of matrix metalloproteinase expression and activation of the FAK signaling pathway in HSV-1infected human corneal epithelial cellsObjective:To investigate the relationship between matrix metalloproteinase-2(MMP-2) and activation of the focal adhesion kinase (FAK) signaling pathway in herpes stromal keratitis (HSK).Methods:Human corneal epithelial (HCE) cells cultured in vitro were infected with HSV-1, and expression of MMP-2, FAK and phosphorylated FAK (P-FAK) in HCE cells were detected using reverse transcription polymerase chain reaction (RT-PCR), Western blotting assays, and immunohistochemistry at2,20and40h.Results:Repeated measures analysis of variance of RT-PCR showed no significant difference in MMP-2and FAK mRNA expression in infected cells at different times, and no significant differences between infected cells and the negative control group. There was no interaction between groups and time points. Pairwise comparisons showed that MMP-2and FAK mRNA expression were significantly increased in virus-infected cells compared to controls. Over time, MMP-2and FAK mRNA expression did not differ significantly in virus-infected cells or in control cells. Western blotting assays showed no significant difference in P-FAK, FAK or MMP-2expression between infected cells and control cells at2h (P>0.05). Infected cells differed significantly from the control cells at20and40h (P<0.05). P-FAK, FAK and MMP-2expression in virus-infected cells at2h differed significantly from those at20and40h (P<0.05). Immunohistochemical staining results showed that longer infection time was associated with an increased number of cells staining positive for MMP-2, FAK and P-FAK.Conclusion:After HSV-1infection of the corneal epithelium, the FAK signaling pathway is activated, leading to increased secretion of MMP-2in the corneal tissue and accelerated formation of corneal ulcers and necrotic lesions.