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Construction of a recombinant pIRES2-EGFP-ARTS plasmid and its effect on LX-2 cells
Feifan Xu1, Yuanlong Han1, Dandan Zhu2
1Clinical Laboratory, The Sixth People's Hospital of Nantong, Nantong, Jiangsu 226011, P.R. China.
Abstract:
The inhibition of the activation of hepatic stellate cells (HSCs) and the induction of their apoptosis have been investigated as potential strategies to counteract the development and progression of liver fibrosis. Previous research has suggested that apoptosis‑related protein in the transforming growth factor‑β signaling pathway (ARTS) may serve a significant role in numerous cell types; however, little is known regarding its roles in HSCs. Total RNA was extracted from LX‑2 cells, and the human full‑length ARTS gene was obtained by reverse transcription‑polymerase chain reaction and inserted into the pIRES2‑EGFP cloning vector. Subsequently, the recombinant pIRES2‑EGFP‑ARTS plasmid was transfected into LX‑2 cells by FuGENE 6 transfection reagent, and the expression of ARTS was detected by western blotting and fluorescent microscopy. In addition, the effects of pIRES2‑EGFP‑ARTS on the activation, apoptosis, viability and migration of LX‑2 cells were assessed by western blot analysis, TUNEL staining, an MTT assay, and scratch and Transwell assays, respectively. The present results demonstrated that the pIRES2‑EGFP‑ARTS vector expressing human ARTS was successfully constructed, and the overexpression of ARTS contributed to enhance the apoptosis and inhibit the activation of human LX‑2 HSCs. The present findings suggested that ARTS overexpression may have potential as a novel therapeutic strategy to reverse hepatic fibrosis.
Insights
Overexpressing the apoptosis-related protein (ARTS) enhances hepatic stellate cell (HSC) apoptosis and inhibits their activation, offering a potential strategy for reversing liver fibrosis.
Area of Science:
- Hepatology
- Cell Biology
- Molecular Medicine
Background:
- Liver fibrosis is a significant health concern, with HSC activation and apoptosis being key therapeutic targets.
- The role of the apoptosis-related protein in the transforming growth factor-β signaling pathway (ARTS) in HSCs remains largely unexplored.
Purpose of the Study:
- To investigate the role of ARTS in human LX-2 hepatic stellate cells (HSCs).
- To assess the potential of ARTS overexpression as a therapeutic strategy for liver fibrosis.
Main Methods:
- Constructed a recombinant pIRES2-EGFP-ARTS plasmid and transfected it into LX-2 cells.
- Detected ARTS expression using Western blotting and fluorescent microscopy.
- Evaluated HSC activation, apoptosis, viability, and migration using Western blot, TUNEL staining, MTT assay, and Transwell assays.
Main Results:
- Successfully constructed and expressed the pIRES2-EGFP-ARTS vector in LX-2 cells.
- ARTS overexpression significantly enhanced apoptosis in LX-2 HSCs.
- ARTS overexpression inhibited the activation and migration of LX-2 HSCs.
Conclusions:
- ARTS overexpression effectively promotes HSC apoptosis and inhibits activation.
- ARTS holds potential as a novel therapeutic target for reversing liver fibrosis.

