Impact of cecal ligation and puncture-induced sepsis on the proliferation and differentiation of intestinal stem cells
Objective To analyze the impact of cecal ligation and puncture(CLP)-induced sepsis on the proliferation and differentiation of intestinal epithelial cells.Methods ① Animal experiment:sixteen male C57BL/6 mice were divided into sham operation group(Sham group)and CLP-induced sepsis model group(CLP group)by random number table method,with 8 mice in each group.After 5 days of operation,the jejunal tissues were taken for determination of leucine-rich-repeat-containing G-protein-coupled receptor 5(LGR5)and intestinal alkaline phosphatase(IAP)by polymerase chain reaction(PCR).The translation of LGR5 was detected by Western blotting.The expression of proliferating cell nuclear antigen(Ki67)was analyzed by immunohistochemistry.IAP level was detected by modified calcium cobalt staining and colorimetry.Immunofluorescence staining was used to detect the expression of Paneth cell marker molecule lysozyme 1(LYZ1)and goblet cell marker molecule mucin 2(MUC2).②Cell experiment:IEC6 cells in logarithmic growth stage were divided into blank control group and lipopolysaccharide(LPS)group(LPS 5 μg/mL).Twenty-four hours after treatment,PCR and Western blotting were used to analyze the transcription and translation of LGR5.The proliferation of IEC6 cells were detected by 5-ethynyl-2'-deoxyuridine(EdU)staining.The transcription and translation of IAP were detected by PCR and colorimetric method respectively.Results ① Animal experiment:the immunohistochemical results showed that the positive rate of Ki67 staining in the jejunal tissue of CLP group was lower than that of Sham group[(41.7±2.5)%vs.(48.7±1.4)%,P=0.01].PCR and Western blotting results showed that there were no statistical differences in the mRNA and protein expressions of LGR5 in the jejunal tissue between the CLP group and Sham group(Lgr5 mRNA:0.7±0.1 vs.1.0±0.2,P=0.11;LGR5/β-actin:0.83±0.17 vs.0.68±0.19,P=0.24).The mRNA(0.4±0.1 vs.1.0±0.1,P<0.01)and protein(U/g:47.3±6.0 vs.73.1±15.3,P<0.01)levels of IAP in the jejunal tissue were lower in CLP group.Immunofluorescence saining analysis showed that the expressions of LYZ1 and MUC2 in the CLP group were lower than those in the Sham group.②Cell experiment:PCR and Western blotting results showed that there was no significant difference in the expression of LGR5 between the LPS group and the blank control group(Lgr5 mRNA:0.9±0.1 vs.1.0±0.2,P=0.33;LGR5/β-actin:0.71±0.18 vs.0.69±0.04,P=0.81).The proliferation rate of IEC6 cells in the LPS group was lower than that in the blank control group,but there was no significant difference[positivity rate of EdU:(40.5±3.8)%vs.(46.5±3.6)%,P=0.11].The mRNA(0.5±0.1 vs.1.0±0.2,P<0.01)and protein(U/g:15.0±4.0 vs.41.2±10.4,P<0.01)of IAP in the LPS group were lower than those in the blank control group.Conclusion CLP-induced sepsis inhibits the proliferation and differentiation of intestinal epithelial cells,impairing the self-renewal ability of intestinal epithelium.
SepsisCecal ligation and punctureIntestinal epitheliumProliferationDifferentiation
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