Continuous Blood Purification Therapy Ameliorates Endothelial Permeability In Severe Acute Pancreatitis Patients By Regulating ROCK-mediated Intercellular Tight Junction Proteins

Background: Severe acute pancreatitis（SAP） is one of the acuteabdominal diseases, which can develop to systemic inflammatory responsesyndrome (SIRS) rapidly, even multiple organ dysfunction syndrome(MODS). The endothelial cell was the inner barrier of blood vessels, andendothelial hyperpermeability maybe play a key role in the pathogenesis ofMODS. Tight junction proteins are the important compont of theintercellular junctional complex, which also play a important role in theendothelial barrier stability. Continuous blood purification (CBP) has beenwidely used in treating critically patients including SIRS or MODS, whichcould ameliorates the prognosis obviously. We found CBP could ameliorateendothelial permeability in our early research, but further work was requiredto determine that whether the effect of CBP was relatant to intercellular tightjunction proteins. The aim of the present study was to probe the effect ofintercellular tight junction proteins on CBP therapy ameliorating endothelial barrier function in SAP patients.Method: Human umbilical vein endothelial cells (HUVECs) wereexposed to serum samples taken from patients with SAP at specific timeperiods during CBP, or pretreated with Rho kinase(ROCK) inhibitorY-27632（5μM、10μM、20μM）followed by treatment with serum. Then,the permeability of FITC-BSA in endothelial cell was detection byFluorescence reader. The distribution and reorganization of tight junctionproteins Occludin and Claudin-1were observed by immunofluorescencestaining, the expression levels of Occludin and Claudin-1were also detectedby Werstern blot, and the F-actin stress fiber was observed by confocal laserscanning microscopy.Results: Immunofluorescent staining revealed that Occludin andClaudin-1were mainly distributed around the endothelial cell andconstructed smooth and continuous linkbelts in areas of cell-to-cell contact.The endothelial cell permeability was significantly increased and theexpression of tight junction proteins Occludin and Claudin-1wassignificantly decreased, and the breakdown and reorganization of Occludinand Claudin-1was found, the marked redistribution of the actin cytoskeletonwith the increased formation of dense F-actin stress fibers both in numberand density was found in all patients, with the serum from patients with SAPcompared with healthy control. On the other hand, using the serum after CBPto stimulate the endothelial cells, the permeability was significantly decreased and the breakdown and reorganization of Occludin and Claudin-1was improved, the expression was significantly increased. There wassignificant difference between these groups. Also, the formation of F-actinstress fibers was decreased. Furthermore, in the group that was pretreated bydifferent concentrations of Y-27632, the permeability was significantlydecreased and the breakdown and reorganization of Occludin and Claudin-1was improved compared with Pre-CBP group, the expression wassignificantly increased, the formation of F-actin stress fibers was decreased,and the effects were dose dependent compared with Pre-CBP group.Conclusion: The serum of SAP patients could provoke the significantincrease of endothelial permeability and after CBP treatment the serumcould decrease the permeability. The inhibition of ROCK-mediated tightjunction proteins Occludin and Claudin-1and F-actin remodeling might bethe mechanism.