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. General Significance: While at low concentrations, TGF-β1 protects cells from antioxidation, whereas at high concentrations, it accumulates in the extracellular matrix, causing further HTMC dysfunction.

中文翻译：

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高血糖对小梁网细胞TGF-β通路的影响

 背景

高血糖可导致细胞凋亡、肥大、纤维化，并因氧化应激而诱发糖尿病血管并发症的过度炎症。为了阐明 TGF-β1 和 TGF-β2 在人小梁网细胞 (HTMC) 中的潜在双重作用和调节信号转导，我们使用 5.5、25、50 和 100 mM d-葡萄糖在 HTMC 中建立了氧化细胞模型-补充培养基并表征TGF-β相关的氧化应激途径。

 方法

进行了进一步的分析以调查信号转导引起的 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，例如细胞活力/凋亡分析、细胞内活性氧（ROS）检测、钙释放浓度分析、免疫印迹分析以检测相关蛋白表达变化以及细胞纤维化分析研究不同严重程度的高血糖的影响。此外，我们还阐述了 TGF-β1/2 在 shRNA 介导的敲低或重组人 TGF-β1 蛋白 (rhTGF-β1) 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，高血糖下 HTMC 中 p-JNK、p-p38、p-AKT 和相关 SMAD 家族成员表达上调。在细胞功能测定中，在高血糖条件下用 rhTGFβ-1 (1 ng/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

总之，HTMC 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。一般意义：在低浓度时，TGF-β1 可以保护细胞免受抗氧化，而在高浓度时，它会在细胞外基质中积聚，导致进一步的 HTMC 功能障碍。