PLK1 protects against sepsis-induced intestinal barrier dysfunction

Abstract

Sepsis and sepsis-associated intestinal barrier dysfunction are common in intensive care units, with high mortality. The aim of this study is to investigate whether Polo-like kinase 1 (PLK1) ameliorates sepsis-induced intestinal barrier dysfunction in the intestinal epithelium. The mouse intestinal barrier was disrupted after Lipopolysaccharide (LPS) injection due to intestinal epithelial cell apoptosis and proliferation inhibition, accompanied by decreased PLK1. In HT-29 intestinal epithelial cells, LPS stimulation induced cell apoptosis and inhibited cell proliferation. Overexpression of PLK1 partly rescued the apoptosis and proliferation inhibition in HT29 cells caused by LPS. Finally, LPS stimulation promoted the reduction of PLK1, resulting in apoptosis and proliferation inhibition in intestinal epithelial cells, disrupting the intestinal epithelial barrier. These findings indicate that PLK1 might be a potential therapeutic target for the treatment of sepsis-induced intestinal barrier dysfunction.

Figure 1

Sepsis-induced intestinal barrier dysfunction. (A) Intestinal tissue from the control and sepsis groups at 12 h after injection of LPS (haematoxylin and eosin staining); sections are representative of ten animals in each group. (B) DAO (a marker of intestinal permeability) concentrations in serum 12 h after administration of LPS. DAO concentrations are expressed as the mean ± SD. ***P < 0.001, control versus sepsis (n = 10).

Figure 2

Sepsis suppresses intestinal epithelial-cell proliferation and induces apoptosis. (A) TUNEL assay was used to determine apoptosis in intestinal epithelium. The green-stained (fluorescein isothiocyanate (FITC)) cells are apoptotic cells. (B) Intestinal tissue protein of each group was extracted, and western blot was used to determine the levels of Ki67 and caspase3. Actin was used as control. The graph represents the relative band densities. Values are mean ± SEM (n = 3). ***P < 0.001 versus control group.

Figure 3

Sepsis induces PLK1 down-regulation in intestinal epithelium. (A) Immunohistochemical staining for PLK1 in intestinal tissue sections of control and septic animals. (B) Western blot analysis of PLK1 in intestinal tissue of control and septic mice. ERK1/2 was used as control. The graph represents the relative band densities. Values are mean ± SEM (n = 3). ***P 0.001 versus control group.

Figure 4

LPS suppresses proliferation in HT29 Cells. (A) The cell-proliferative inhibition effect of LPS in HT29 cells (CCK-8 assay). ***P < 0.001 compared with control group. (B) The levels of PLK1 and Ki67 in HT29 cells after treatment with 30 μg/mL LPS for 24 h. The graph represents the relative band densities. Values are mean ± SEM (n = 3). ***P < 0.001 versus control group.

Figure 5

LPS induces apoptosis in HT29 cells. (A) HT29 cells were exposed to various concentrations of LPS for 24 h. Apoptosis was analysed by Annexin V-FITC/PI double-labelling assay. (B) The degree of apoptotic cell death was quantified. Data represent the mean ± SD (**P < 0.01, ***P < 0.001 compared with control group.). (C) The levels of PLK1 and caspase-3 in HT29 cells after treatment with LPS at various concentrations for 24 h. The graph represents the relative band densities. Values are mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 versus control group.

Figure 6

Over-expression of PLK1 partly rescues the apoptosis and proliferation inhibition caused by LPS in HT29 cells HT29 cells were transfected with pcDNA-PLK1-myc or the control cDNA for 24 h, then exposed to LPS (30 μg/ml) for 24 h. (A) A representative result of apoptosis analysed by Annexin V-FITC/PI double-labelling assay in HT29 cells after the above treatments. (B) Percentages of apoptotic cells in HT29 cells after the above treatments. Values are mean ± SEM (n = 3). **P < 0.01, ***P < 0.001. (C) The absorbance at 450 nm of HT29 cells after the above treatments. Values are mean ± SEM (n = 3). **P < 0.01, ***P < 0.001. (D) The levels of Ki67 and caspase3 after the above treatments. The graph represents the relative band densities. Values are mean ± SEM (n = 3). ***P < 0.001 versus control group. **P < 0.01 versus LPS group.