The Role And Mechanism Of SIRT1 In Regulating COL1A1 Expression In Human Trabecular Meshwork Cells

Purpose:Primary open-angle glaucoma(primary open Angle glaucoma, POAG) is an optic neuropathy characterized by optic disk cupping and visual field loss caused by pathological elevated intraocular pressure while anterior chamber angle become open.At present, its pathogenesis has not been fully elucidated. Among them, abnormal deposition of extracellular matrix in trabecular meshwork is the key factors leading to obstruction of aqueous humor outflow and higher intraocular pressure. Therefore, to seek solutions to figure out the science problems become the important thing. We found the deacetylation enzyme SIRT1(sirtuin1) had anti-fibrosis and antioxidant effect in other diseases. Moreover, resveratrol, the agonist of SIRT1, applied in eye experiment and had the effect of reducing intraocular pressure. We expect that SIRT1 can play a role in inhibiting the extracellular matrix abnormal deposition in the human trabecular meshwork cells. In addition, the previous research results show that there is interaction between SIRT1 and HES1(hes family bHLH transcription factor1)protein, and HES1 may directly regulate COL1A1(collagen type 1 alpha 1)transcription. In this study, we hope to confirm the presence of SIRT1-HES1-COL1A1 pathway in human trabecular meshwork cells in order to clarify the role and mechanism of SIRT1 as a glaucoma therapeutic target.Methods:Immunofluorescence technique was used to explore SIRT1 expression and localization in human trabecular meshwork cells. SIRT1 agonist resveratrol with different concentrations(0,10,25,50 ?M) was used to treat human trabecular meshwork cells respectively for 12 h. The effect of resveratrol on SIRT1 was detected by Western Blot and qPCR, and at the same time, the effect of resveratrol on COL1A1 expression was assayed by same technique. These methods were also used to determine the effects of SIRT1 on COL1A1 after knockdown or overexpression of SIRT1 through virus infectionThe expression levels of COL1A1 in HTMCs treated with HES1 shRNA was measured by Western Blot and qRT-PCR analyses. Using SIRT1 agonist resveratrol with different concentrations(0,10,25,50 ?M) to treat human trabecular meshwork cells respectively, the action time is 12 h. The effect of resveratrol on HES1 was detected by Western Blot and qPCR. These methods were also used to determine the effects of SIRT1 on HES1 after knockdown or overexpression of SIRT1 through virusinfection. Moreover, HES1 binding to the promoter of the COL1A1 gene was confirmed by DNA pull-down assay. Furthermore, the resistance of SIRT1 to COL1A1 accumulation under oxidative stress was investigated through Western Blot analysis.Results:The results of immunofluorescence technique showed that SIRT1 distributed in the nucleus of human trabecular meshwork cells, resveratrol upregulated SIRT1 protein level and inhibit the expression of extracellular matrix component COL1A1.In addition, overexpressing SIRT1 downregulated the expression of COL1A1 in human trabecular meshwork cells. COL1A1 expression was upregulated in HTMCs with SIRT1 knocked down by shRNA lentivirus.HES1 protein reduced the expression of COL1A1, which was consistent with previous results. In addition, overexpressing SIRT1 downregulated the expression of HES1 in human trabecular meshwork cells. HES1 expression was upregulated in human trabecular meshwork cells with SIRT1 knocked down by shRNA lentivirus.Furthermore, knockdown of SIRT1 enhanced the binding of HES1 to the promoter of the COL1A1 gene and led to increased level of COL1A1. Finally, SIRT1 activation attenuated HES1 and COL1A1 production under oxidative stress in HTMCs.Conclusions:1. SIRT1 negatively regulated COL1A1 synthesis and HES1 positively regulate COL1A1 synthesis.2. Knockdown of SIRT1 enhanced the binding of HES1 to the promoter of the COL1A1 gene.3. SIRT1 downregulated the expression of COL1A1 in HTMCs under oxidative stress by inhibiting HES1. Resveratrol reduced the expression of HES1 and COL1A1 by activating SIRT1, which may play a role in lowering intraocular pressure.