The Role And Mechanism Of PLK1 Mediated MTOR Signaling Pathway Regulating Autophagy In Sepsis Intestinal Barrier Dysfunction

Objective Through in vivo and in vitro experiments,the mutual regulatory relationship between PLK1,mTOR and autophagy during sepsis was investigated,and its role and mechanism in intestinal injury were studied.Methods In vivo experiments: 30 well-developed six-week-old female C57 mice were randomly divided into 3 groups(n = 10): sham operation group(SHAM),sepsis group(CLP),and PKI sepsis group(PLK1 transgenic mice were modeled with CLP),and CLP(Cecal Ligation and Puncture,cecal ligation and puncture)was used to prepare the sepsis model.Blood samples were sacrificed 24 hours after modeling,and the serum TNF-? level was measured by Elisa.Then,the small intestine 5cm near the blind end was taken for tissue embedding,sectioning,and HE staining to observe the pathological changes of the small intestine.Small intestinal epithelial cells p-mTOR,autophagy molecules Lc3,and p62 were measured using immunohistochemistry.PLK1 and tight junction proteins ZO-1 and occuludin of small intestinal epithelial cells were determined by western blot.In vitro experiments: Colon epithelial cells NCM460 were used as model cells.After treatment with LPS(30 ?g / ml)for 36 hours,cells were collected to detect proteins such as PLK1,p-mTOR,autophagy molecules Lc3,p62,and cell tight junction proteins ZO-1 and occuludin.Expression changes;CO-IP(co-immunoprecipitation)was performed under the same experimental conditions todetect the direct interaction between PLK1 and p-mTOR.PLK1-specific inhibitor BI2536(50nM)was used for 24 hours,and siRNA was knocked down for 48 hours to determine its regulation of mTOR signaling pathway and autophagy.The PLK1 overexpression plasmid vector pCDNA3.1-PLK1-myc was created and transfected into NCM460 cells for recovery experiments for verification.Results In vivo experiments: 1.After CLP modeling of mice,the intestinal cavity of the mice is obviously edema,brittle and easy to break.HE staining shows that the villi of the small intestine tissue obviously shrink and fall,and the morphological integrity is damaged.Elisa test found TNF-? levels.Significantly increased.2.After CLP modeling in mice,the tight junction proteins ZO-1 and occludin were significantly down-regulated.3.Twenty-four hours after CLP modeling in mice,the WB test revealed that the Lc3 ? / ? ratio was reduced and the expression of p62 protein was up-regulated,indicating autophagy inhibition.4.After CLP modeling in mice,the expression of PLK1 protein was detected to be down-regulated;mTOR signaling pathway was activated.5.After CLP modeling,compared with wild-type mice,PLK1 +/ + mice(PLK1 overexpressing transgenic mice)can restore the down-regulation of PLK1 expression caused by CLP;can restore the down-regulation of ZO-1 and occludin proteins caused by CLP.In vitro experiments: 1.LPS treatment resulted in significant down-regulation of the tight junction proteins ZO-1 and occludin.2.LPS treatment of cells resulted in upregulation of p62 expression and a decrease in Lc3 I / II ratio,indicating that autophagy tended to inhibit.3.LPS treatment resulted in down-regulation of PLK1 expression and up-regulation of p-mTOR and p-s6 in NCM460 cells.4.Overexpression of PLK1 can significantly restore the up-regulation of p-mTOR and the inhibition of autophagy caused by LPS treatment.5.CO-IP detection revealed that there was a direct interaction between PLK1 and m TOR in NCM460 cells.Conclusion In sepsis,the expression of PLK1 is down-regulated,and the activation of the mTOR signaling pathway leads to the inhibition of autophagy,which in turn causesthe balance between apoptosis and autophagy to be broken,and finally leads to intestinal barrier dysfunction;Toxic intestinal mucosal barrier dysfunction plays an important role.