Dataset Information

Protective effects of triptolide against oxidative stress in retinal pigment epithelium cells via the PI3K/AKT/Nrf2 pathway: a network pharmacological method and experimental validation.

ABSTRACT:

Purpose

Among aging adults, age-related macular degeneration (AMD), is a prevalent cause of blindness. Nevertheless, its progression may be halted by antioxidation in retinal pigment epithelium (RPE). The primary effective constituent of Tripterygium wilfordii Hook. F., triptolide (TP), has demonstrated anti-inflammatory, antiproliferative, and antioxidant properties. The mechanics of the protective effect of triptolide against the oxidative damage in retinal pigment epithelial (RPE) were assessed in this study.

Methods

ARPE-19 cells were pretreated with TP, and then exposed to sodium iodate (SI). First, cell viability was assessed using CCK-8. Subsequently, we measured indicators for cell oxidation including reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). Then, we used network pharmacological analysis and molecular docking to explore the signaling pathway of TP. Last, we used western blot, ELISA, and immunofluorescence assays to clarify the potential mechanistic pathways.

Results

The network pharmacology data suggested that TP may inhibit AMD by regulating the PI3K/Akt signaling pathway. Experimental results showed that the potential mechanism is that it regulates the PI3K/Akt pathway and promotes Nrf2 phosphorylation and activation, thereby raising the level of antioxidant factors (HO-1, NQO1) and reducing the generation of ROS, which inhibit oxidative damage.

Conclusion

Our findings suggested that the effect of TP on SI-exposed RPE cells principally relies on the regulation of oxidative stress through the PI3K/Akt/Nrf2 signaling pathway.

SUBMITTER: Pan F

PROVIDER: S-EPMC10929812 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Publications

Protective effects of triptolide against oxidative stress in retinal pigment epithelium cells via the PI3K/AKT/Nrf2 pathway: a network pharmacological method and experimental validation.

Pan Fuying F Shu Qinxin Q Xie Hao H Zhao Long L Wu Ping P Du Yong Y Lu Jing J He Yuxia Y Wang Xing X Peng Hui H

Aging 20240221 4

<h4>Purpose</h4>Among aging adults, age-related macular degeneration (AMD), is a prevalent cause of blindness. Nevertheless, its progression may be halted by antioxidation in retinal pigment epithelium (RPE). The primary effective constituent of Tripterygium wilfordii Hook. F., triptolide (TP), has demonstrated anti-inflammatory, antiproliferative, and antioxidant properties. The mechanics of the protective effect of triptolide against the oxidative damage in retinal pigment epithelial (RPE) wer ...[more]

PMID: 38393691

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Project description:Proliferative vitreoretinopathy (PVR) is a disease leading to the formation of contractile preretinal membranes (PRMs) and is one of the leading causes of blindness. Connective tissue growth factor (CTGF) has been identified as a possible key determinant of progressive tissue fibrosis and excessive scarring. Therefore, the present study investigated the role and mechanism of action of CTGF in PVR. Immunohistochemical staining was performed to detect the expression of CTGF, fibronectin and collagen type III in PRMs from patients with PVR. The effects and mechanisms of recombinant human CTGF and its upstream regulator, TGF‑β1, on epithelial‑mesenchymal transition (EMT) and the synthesis of extracellular matrix (ECM) by retinal pigment epithelium (RPE) cells were investigated using reverse transcription‑quantitative PCR, western blotting and a [3H]proline incorporation assay. The data indicated that CTGF, fibronectin and collagen type III were highly expressed in PRMs. In vitro, CTGF significantly decreased the expression of the epithelial markers ZO‑1 and E‑cadherin and increased that of the mesenchymal markers fibronectin, N‑cadherin and α‑smooth muscle actin in a concentration‑dependent manner. Furthermore, the expression of the ECM protein collagen type III was upregulated by CTGF. However, the trends in expression for the above‑mentioned markers were reversed after knocking down CTGF. The incorporation of [3H]proline into RPE cells was also increased by CTGF. In addition, 8‑Bromoadenosine cAMP inhibited CTGF‑stimulated collagen synthesis and transient transfection of RPE cells with a CTGF antisense oligonucleotide inhibited TGF‑β1‑induced collagen synthesis. The phosphorylation of PI3K and AKT in RPE cells was promoted by CTGF and TGF‑β1 and the latter promoted the expression of CTGF. The results of the present study indicated that CTGF may promote EMT and ECM synthesis in PVR via the PI3K/AKT signaling pathway and suggested that targeting CTGF signaling may have a therapeutic or preventative effect on PVR.

Project description:Normal function of the retinal pigment epithelium (RPE) is essential for maintaining the structural integrity of retinal photoreceptors and the visual process. Sustained oxidative damage of the RPE due to aging and other risk factors contributes to the development of age-related macular degeneration (AMD). The transcription factor NF-E2-related factor 2 (Nrf2) is a central regulator of cellular antioxidant and detoxification responses. Enhancing Nrf2 function protects RPE cells from oxidation-related apoptosis and cell death. Previously, we demonstrated that Nrf2 activation can be induced by endoplasmic reticulum (ER) stress; however, the mechanisms are not fully understood. In the present study, we examined the role of X box-binding protein 1 (XBP1), an ER stress-inducible transcription factor, in regulation of Nrf2 in the RPE. We found that RPE-specific XBP1 conditional knockout (cKO) mice exhibit a significant reduction in Nrf2 mRNA and protein levels, along with decreased expression of major Nrf2 target genes, in the RPE/choroid complex. Using primary RPE cells isolated from XBP1 cKO mice and human ARPE-19 cell line, we confirmed that loss of XBP1 gene or pharmacological inhibition of XBP1 splicing drastically reduces Nrf2 levels in the RPE. Conversely, overexpression of spliced XBP1 results in a modest but significant increase in cytosolic and nuclear Nrf2 protein levels without affecting the transcription of Nrf2 gene. Moreover, induction of ER stress by tunicamycin and thapsigargin markedly increases Nrf2 expression, which is abolished in cells pretreated with XBP1 splicing inhibitors 4μ8C and quinotrierixin. Mechanistic studies indicate that quinotrierixin reduces Nrf2 expression likely through inhibition of protein translation. Finally, we demonstrate that overexpression of Nrf2 protected RPE cells against oxidative injury but appeared to be insufficient to rescue from XBP1 deficiency-induced cell death. Taken together, our results indicate that XBP1 modulates Nrf2 activity in RPE cells and that XBP1 deficiency contributes to oxidative injury of the RPE.

Dataset Information

Protective effects of triptolide against oxidative stress in retinal pigment epithelium cells via the PI3K/AKT/Nrf2 pathway: a network pharmacological method and experimental validation.

Purpose

Methods

Results

Conclusion

Publications

Protective effects of triptolide against oxidative stress in retinal pigment epithelium cells via the PI3K/AKT/Nrf2 pathway: a network pharmacological method and experimental validation.

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