Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 7, 2015; 21(29): 8858-8867
Published online Aug 7, 2015. doi: 10.3748/wjg.v21.i29.8858
Overexpression of pim-3 and protective role in lipopolysaccharide-stimulated hepatic stellate cells
Lin-Hua Liu, Qi-Nan Lai, Jian-Yong Chen, Ji-Xiang Zhang, Bin Cheng
Lin-Hua Liu, Ji-Xiang Zhang, Bin Cheng, Department of Gastroenterology, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China
Qi-Nan Lai, Infectious Disease Hospital, Nanchang University, Nanchang 330006, Jiangxi Province, China
Jian-Yong Chen, Department of Gastroenterology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
Author contributions: Zhang JX designed the research; Liu LH, Lai QN and Cheng B performed the research; Chen JY analyzed the data; Liu LH wrote the paper.
Supported by National Natural Science Foundation of China, No. 81360074.
Conflict-of-interest statement: There was no conflict of interest in the study.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Ji-Xiang Zhang, MD, Department of Gastroenterology, The Second Affiliated Hospital of Nanchang University, No. 1 Minde Road, Nanchang 330006, Jiangxi Province, China.
Telephone: +86-791-86262262 Fax: +86-791-86262262
Received: February 20, 2015
Peer-review started: February 23, 2015
First decision: March 26, 2015
Revised: May 1, 2015
Accepted: May 27, 2015
Article in press: May 27, 2015
Published online: August 7, 2015

AIM: To investigate pim-3 expression in hepatic stellate cells (HSCs) stimulated by lipopolysaccharide (LPS), and its protective effect on HSCs.

METHODS: Rat HSC-T6 cells were stimulated by LPS. The effect of LPS on proliferation and apoptosis of HSC-T6 cells was investigated by methyl thiazoyltetrazolium (MTT) assay and flow cytometry after annexin V-fluorescein isothiocyanate/propidium iodide double staining. pim-3 mRNA and protein were detected by reverse transcriptase polymerase chain reaction and Western blotting at 48 h when HSC-T6 cells were stimulated with 1 μg/mL LPS for 0, 3, 6, 12, 24 and 48 h. The cells without stimulation served as controls. To study the effect of pim-3 kinase on HSC-T6 cells, si-pim3 (siRNA against pim-3) was transfected into HSC-T6 cells. HSC-T6 cells were subjected to different treatments, including LPS, si-pim3, or si-pim3 plus LPS, and control cells were untreated. Protein expression of pim-3 was detected at 48 h after treatment, and cell proliferation at 24 and 48 h by MTT assay. Apoptosis was detected by flow cytometry, and confirmed with caspase-3 activity assay.

RESULTS: LPS promoted HSC-T6 cell proliferation and protected against apoptosis. Significantly delayed upregulation of pim-3 expression induced by LPS occurred at 24 and 48 h for mRNA expression (pim-3/β-actin RNA, 24 or 48 h vs 0 h, 0.81 ± 0.20 or 0.78 ± 0.21 vs 0.42 ± 0.13, P < 0.05), and occurred at 12 h and peaked at 24 and 48 h for protein expression (pim-3/GAPDH protein, 12, or 24 or 48 h vs 0 h, 0.68 ± 0.12, 1.47 ± 0.25 or 1.51 ± 0.23 vs 0.34 ± 0.04, P < 0.01). pim-3 protein was ablated by si-pim3 and upregulated by LPS in HSC-T6 cells at 48 h after treatment (pim-3/GAPDH: si-pim3, si-pim3 plus LPS or LPS vs control, 0.11 ± 0.05, 0.12 ± 0.05 or 1.08 ± 0.02 vs 0.39 ± 0.03, P < 0.01). Ablation of pim-3 by si-pim3 in HSC-T6 cells partly abolished proliferation (OD at 24 h, si-pim3 group or si-pim3 plus LPS vs control, 0.2987 ± 0.050 or 0.4063 ± 0.051 vs 0.5267 ± 0.030, P < 0.01; at 48 h 0.4634 ± 0.056 or 0.5433 ± 0.031 vs 0.8435 ± 0.028, P < 0.01; si-pim3 group vs si-pim3 plus LPS, P < 0.01 at 24 h and P < 0.05 at 48 h), and overexpression of pim-3 in the LPS group increased cell proliferation (OD: LPS vs control, at 24 h, 0.7435 ± 0.028 vs 0.5267 ± 0.030, P < 0.01; at 48 h, 1.2136 ± 0.048 vs 0.8435 ± 0.028, P < 0.01). Ablation of pim3 with si-pim3 in HSC-T6 cells aggravated apoptosis (si-pim3 or si-pim3 plus LPS vs control, 42.3% ±1.1% or 40.6% ± 1.3% vs 16.8% ± 3.3%, P < 0.01; si-pim3 vs si-pim3 plus LPS, P > 0.05), and overexpression of pim-3 in the LPS group attenuated apoptosis (LPS vs control, 7.32% ± 2.1% vs 16.8% ± 3.3%, P < 0.05). These results were confirmed by caspase-3 activity assay.

CONCLUSION: Overexpression of pim-3 plays a protective role in LPS-stimulated HSC-T6 cells.

Keywords: Pim-3, Lipopolysaccharide, Hepatic stellate cell, Si-pim3

Core tip: Hepatic stellate cell (HSC)-T6 cells stimulated by lipopolysaccharide (LPS) showed overexpression of pim-3 kinase. Overexpression of pim-3 in LPS-stimulated HSC-T6 cells protected against apoptosis and promoted proliferation. Knockdown of pim3 gene abolished proliferation of HSC-T6 cells and led to apoptosis. Overexpression of pim-3 induced by LPS play a protective role in rat hepatic stellate cells.