Overexpression of pim-3 and protective role in lipopolysaccharide-stimulated hepatic stellate cells.
OBJECTIVE
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.


CONCLUSIONS
Overexpression of pim-3 plays a protective role in LPS-stimulated HSC-T6 cells.
Lin-Hua Liu, Ji-Xiang Zhang, Bin Cheng, Department of Gastroenterology, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China.
Lin-Hua
LH
Liu
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
QN
Lai
Lin-Hua Liu, Ji-Xiang Zhang, Bin Cheng, Department of Gastroenterology, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China.
Jian-Yong
JY
Chen
Lin-Hua Liu, Ji-Xiang Zhang, Bin Cheng, Department of Gastroenterology, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China.
Ji-Xiang
JX
Zhang
Lin-Hua Liu, Ji-Xiang Zhang, Bin Cheng, Department of Gastroenterology, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China.
Bin
B
Cheng
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000201:enzymology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
D008070:Lipopolysaccharides; D012333:RNA, Messenger; C113398:Pim3 protein, rat; D017346:Protein Serine-Threonine Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3
Hepatic stellate cell; Lipopolysaccharide; Pim-3; Si-pim3