PLK1 Inhibits Lps-induced Intestinal Barrier Dysfunction Through Modulating NF-κB Pathway

OBJECTIVE To investigate the role of PLK1 and NF-κB signaling pathway in intestinal barrier dysfunction during sepsis by in vivo and in vitro experiments.METHODS In vivo experiment: Forty male C57 BL rats were randomly divided into two groups(20 rats/group): sham control(C group), sepsis(LPS group). Endotoxemic rat model was induced by intraperitoneal injection of lipopolysaccharide(LPS, 40 mg/kg). After 12 hours of modeling, determin the TNF-α and diamine oxidase(DAO) levels in serum of rats, then all rats were euthanized by a cervical dislocation, and intestinal samples were prepared in vitro. The intestinal samples were fixed by 4% paraformaldehyde, made into paraffin section.Observing the pathological changes of intestinal through HE staining. TUNEL method is used to detect apoptosis of intestinal cell. The expression levels of PLK1 were detected by Western blotting and immunohistochemical methods. In vitro experiments: Different concentrations(0,10,20,30mg / ml) of LPS was added to the medium of HT29 cells for 24 hours,then the cells were collected for Annexin V / Propidium Iodide staining,following with flow cytometry analysis.Cell protein was extracted to detect the expression of PARP1, Caspase3, PLK1, IkB-a using Western blotting. Different concentrations(0,2,10,20 ng / ml) of PDTC was added to the medium of HT29 cells for 24 hours, then the cells were collected for Annexin V / Propidium Iodide staining,following with flow cytometry analysis.Cell protein was extracted to detect the expression of Caspase3, IkB-a using Western blotting. PLK1 inhibitor BI2536(50n M) was added to the medium of HT29 cellsfor 24 hours,then extract the cell protein to detect the expression of PARP1, PLK1,, IkB-a using Western blotting, Immunofluorescence assay was used to detect NF-κB protein localization.Overexpression PLK1 in HT29,then adding LPS(30mg / ml) to the medium of HT29 cells for 24 hours, the cells were collected for Annexin V / Propidium Iodide staining,following with flow cytometry analysis.Cell protein was extracted to detect the expression of PARP1, Caspase3 using Western blotting.RESULTS In vivo experiment:(1) Compared with C group, LPS group rats were apathetic, slow movement.There are a large number of purulent fluid in the abdominal cavity and intestinal edema in LPS group rats.The serum TNF-α levels were significantly increased in LPS group rats(P <0.001).(2) Compared with C group, LPS group rats were intestinal mucosal atrophy and ntestinal villi los. The level of serum DAO level is significantly increased(P <0.001).(3) Compared with C group, the intestinal epithelial cells of LPS group rats presents obviously apoptosis,the expression of PARP1 and Caspase3 also reduced.(4) Compared with C group, the expression of PLK1 and I?B-? in LPS rats intestinal tissue were downregulation.In vitro experiments:(1) LPS treat HT29 cells can induce cell apoptosis, and the percentage of apoptotic cells increased with increasing concentrations of LPS.(2) Different concentrations of LPS treated HT29 cells 24 hours, compared with the control group, the nuclear NF-?B increased, while the expression level of I?B-? downregulation.(3) With PDTC(10 ng / ml, 20 ng / ml) pretreated HT29 cells for four hours, compared with none pretreatment group, the proportion of HT29 cell apoptosis induced by LPS was significantly reduced(P <0.01 or P <0.001).(4) Different concentrations of LPS treated HT29 cells 24 hours, compared with the control group, the expression levels of PLK1 protein was down-regulated.(5) BI2536(50n M) treated HT29 cells for 24 hours, compared with the control group, the nuclear NF-?B increased,while the expression of IKB-?, PARP1 downregulation.(6) Overexpression PLK1 in HT29, compared with the control group, LPS(30?g / ml) treatment induced apoptosis proportion was significantly lower(P <0.01).CONCLUSION During sepsis, the decrease expression of PLK1 and activation of NF-κB signaling pathway play an important role in intestinal barrier dysfunction.By negative regulation of NF-κB pathway, PLK1 cause the apoptosis of intestinal epithelial cells, increasing intestinal permeability, resulting in intestinal barrier dysfunction.