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

来源 :World Journal of Gastroenterology | 被引量 : 0次 | 上传用户:aaatzh
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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 m RNA 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/m L 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(si RNA 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 LPSoccurred at 24 and 48 h for m RNA 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. 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 were investigated by methyl thiazoyltetrazolium (MTT) assay and flow cytometry after annexin V-fluorescein isothiocyanate / propidium iodide double staining. Pim-3 m RNA and protein were detected by reverse transcriptase polymerase chain reaction and Western blotting at 48 h when The cells without stimulation served as controls. To study the effect of pim-3 kinase on HSC-T6 cells, HSC-T6 cells were stimulated with 1 μg / mL LPS for 0, 3, 6, 12, 24 and 48 h. 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 si-pim3 (si RNA against pim-3) was transfected into HSC-T6 cells expression of pim-3 was detected at 48 h after treatment, and cell proliferatio Significantly delayed upregulation of pim-3 expression induced by npH 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. LPSoccurred at 24 and 48 h for m RNA expression, and occurred at 12 h (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 < 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, ± 0.05 or 1.08 ± 0.02 vs 0.39 ± 0.03, P <0.01). Ablation of pim-3 by si-pim3 in partly abolished proliferation of HSC-T6 cells (OD at 24 h, si-pim3 group or si-pim3 plus LPS vs control, 0.2987 ± 0.050 or 0.4063 ± 0.051 vs 0.5 267 ± 0.030, PP <0.01; at 48 h, 0.4634 ± 0.056 or 0.5433 ± 0.031 vs 0.8435 ± 0.028, P <0.01; and overexpression of si-pim3 group vs si-pim3 plus LPS, P <0.01 at 24 h and P <0.05 at 48 h) 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 < 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 < Results were confirmed by caspase-3 activity assay. CONCLUSION: Overexpression of pim-3 plays a protective role in LPS-stimulated HSC-T6 cells.
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