背景:高血糖,这可能导致细胞凋亡,肥大,纤维化,并由于氧化应激而在糖尿病血管并发症中诱导过度炎症。为了阐明TGF-β1和TGF-β2在人小梁细胞(HTMCs)中的潜在双重作用和调节信号转导,我们使用5.5、25、50和100mMd-葡萄糖补充培养基在HTMC中建立了氧化细胞模型,并表征了TGF-β相关的氧化应激途径。
方法:进行进一步分析以研究由信号转导引起的HTMC中的氧化损伤和蛋白质改变。这是通过一系列定性细胞功能研究完成的,如细胞活力/凋亡分析,细胞内活性氧(ROS)检测,钙释放浓度分析,免疫印迹分析检测相关蛋白表达改变,并对细胞纤维化进行分析,研究不同严重程度的高血糖对其影响。此外,我们阐明了TGF-β1/2在重组人TGF-β1蛋白(rhTGF-β1)通过shRNA介导的敲减或刺激引起的氧化应激损伤中的作用。
结果:蛋白表达分析结果显示p-JNK,p-p38,p-AKT,和相关的SMAD家族成员在高血糖状态下的HTMC中上调。在细胞功能测定中,在高血糖条件下用rhTGFβ-1(1ng/mL)处理的HTMC显示出更高的增殖率和更低的ROS和钙水平。
结论:总结一下,HTMC中的机制分析表明,高血糖诱导的氧化应激激活了TGF-β1及其相关途径.
结论:在低浓度下,TGF-β1保护细胞免受抗氧化,而在高浓度下,它积聚在细胞外基质中,导致进一步的HTMC功能障碍。
BACKGROUND: Hyperglycemia, which can lead to apoptosis, hypertrophy, fibrosis, and induces hyperinflammation in diabetic vascular complications due to oxidative stress. In order to elucidate the potential dual roles and regulatory signal transduction of TGF-β1 and TGF-β2 in human trabecular meshwork cells (HTMCs), we established an oxidative cell model in HTMCs using 5.5, 25, 50, and 100 mM d-glucose-supplemented media and characterized the TGF-β-related oxidative stress pathway.
METHODS: Further analysis was conducted to investigate oxidative damage and protein alterations in the HTMC caused by the signal transduction. This was done through a series of qualitative cell function studies, such as cell viability/apoptosis analysis, intracellular reactive oxygen species (ROS) detection, analysis of calcium release concentration, immunoblot analysis to detect the related protein expression alteration, and analysis of cell fibrosis to study the effect of different severities of hyperglycemia. Also, we illustrated the role of TGF-β1/2 in oxidative stress-induced injury by shRNA-mediated knockdown or stimulation with recombinant human TGF-β1 protein (rhTGF-β1).
RESULTS: Results from the protein expression analysis showed that p-JNK, p-p38, p-AKT, and related SMAD family members were upregulated in HTMCs under hyperglycemia. In the cell functional assays, HTMCs treated with rhTGFβ-1 (1 ng/mL) under hyperglycemic conditions showed higher proliferation rates and lower ROS and calcium levels.
CONCLUSIONS: To summarize, mechanistic analyses in HTMCs showed that hyperglycemia-induced oxidative stress activated TGF-β1 along with its associated pathway.
CONCLUSIONS: While at low concentrations, TGF-β1 protects cells from antioxidation, whereas at high concentrations, it accumulates in the extracellular matrix, causing further HTMC dysfunction.