Chlorogenic acid alleviates sepsis-induced endothelial barrier dysfunction and the underlying mechanism


 The authors have withdrawn this preprint due to author disagreement.

Chlorogenic acid alleviates sepsis-induced endothelial barrier dysfunction and the underlying mechanism Results FITC-BSA leakage from mesenteric micro-vessels was significantly increased after septic shock, which was significantly improved by CGA. Liver and kidney blood flow were increased by 41.2% and 38.4%, respectively, after CGA administration compared with the septic shock group. Hemodynamic status was significantly decreased after septic shock, and significantly improved by CGA. The 72-h survival rate of septic shock rats in the CGA group (50%) was significantly higher than the septic shock group (6.25%). CGA improved the tight junction opening after septic shock and also significantly upregulated the expression of ZO-1 and VE-cadherin. CGA also protected endothelial hyperpermeability against lipopolysaccharide-stimulated VEC injury by increasing the expression of ZO-1 and VEcadherin in vitro.

Conclusion
CGA was beneficial for endothelial barrier function in rats with septic shock, which is the major contribution to CGA with respecting to improving hemodynamic status and organ perfusion. The underlying mechanism involved CGA up-regulation of ZO-1 and VE-cadherin.

Background
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infections that can affect multiple organs in the body. Sepsis is a major cause of worldwide mortality, 1 which is most often attributed to multi-organ dysfunction syndrome (MODS). Severe vascular endothelial barrier failure is associated with sepsis. Hyperpermeability of vascular endothelial cells (VECs) is identified as the key factor in the progression to MODS in septic patients. 2 Thus, recovery of endothelial barrier integrity is essential for maintaining vascular homeostasis and tissue fluid balance for treatment of severe sepsis and related multiple organ dysfunction.
Sepsis-induced dysfunction of VECs could include barrier dysfunction, inflammation and coagulation disorders. These abnormalities may lead to severe hypotension, tissue edema, leukocyte adhesion, and hemostasis imbalance and vasomotor tone alteration. 3,4 Various options are available for treating VEC injury, such as inhibition of inflammatory signaling pathways, tissue factor natural inhibitors, and cellular therapy, but the clinical efficacy is not fully established. 5,6 In our study, we reported that CGA could improve sepsis-induced vascular endothelial permeability.

Preparation of septic shock model in rats
Healthy adult Sprague-Dawley (SD) rats, 12-14weeks old for both sexes and weighing 180-220 g, were anesthetized with sodium pentobarbital (45 mg/kg ip; Sigma, St. Louis, MO, USA). One hundred and twenty rats were randomly divided into three groups (40 per group), and designated as the sham, sepsis, and CGA groups. The sepsis rat model was induced by cecal ligation and puncture (CLP), as described previously. 16 In brief, an abdominal midline incision was made and the cecum was exposed and ligated. The ligated cecal stump was twice-punctured at a 1-cm distance with a triangular needle (the needle size was approximately 1.5 mm in diameter). Feces were allowed to flow into the abdominal cavity. After closure of the abdomen, the rats were returned to the cages and allowed food and water ad-libitum. The rats in the sham group underwent sham operations following the same procedure as the sepsis group but without CLP and treatment. The rats in the CGA group received a cauda vein injection of CGA (20 mg/kg; Sigma) immediately after CLP establishment. CGA was dissolved in normal saline (NS) to prepare a solution with a concentration of 10 mg/mL. After 24 h, and in separate experiments, the dynamics of bovine serum albumin labeled with fluoresceineisothiocyanate (FITC-BSA) leakage from the mesenteric micro-vessels were determined by intravital microscopy. The liver and kidney blood flow, hemodynamic parameters, and blood gases were observed. The mesenteric micro-vessels and superior mesenteric vein (SMV) were collected for histologic evaluation and protein level determination.
Cell culture and treatment VECs were obtained from the mesenteric vein of SD rats by enzymatic digestion as described by our research team. 17 VECs were cultured in endothelial cell medium (ECM, Cat. No.1001; ScienCell, USA).
VECs were seeded on inserts of a 6-cell culture plate (0.4-µm pore size, #3450; Corning, USA). VECs under lipopolysaccharide (LPS) stimulation (100 ng/ml, Escherichia coli serotype O111:B4; Sigma) were co-cultured with CGA (20 µmol/L; Sigma) [8] . Liver and kidney blood flow Liver and kidney blood flow were measured by a Laser-Doppler Perfusion Imager (PeriCam PSI ZR, Sweden). Briefly, the liver and kidneys were exposed and a computer-controlled optical scanner was positioned above the surface of the liver and kidneys at a distance of 14 cm. A color-coded image to denote the specific relative perfusion level was displayed on a video monitor, and all images were evaluated with PIMsoft software (PeriCam PSI ZR, Sweden).

Hemodynamic parameters and blood gases
The rats were anesthetized with 3% sodium pentobarbital, and the left femoral artery and left ventricle were catheterized with a polyethylene(PE) catheter for measurement of the mean arterial pressure (MAP) and hemodynamic parameters, including the left ventricular systolic pressure (LVSP) and maximum rate of increase and decrease of the left intraventricular pressure [± dp/dt max] (SP844, Power Lab; AD Instruments, Castle Hill, NSW, Australia). After instrumentation, the rats were allowed to equilibrate for 10 min, 0.3-0.5 mL of blood was collected for measurement of blood gas using a blood gas analyzer (ABL90 Flex; Radiometer, Denmark). To avoid additional blood loss in the rats, an equal volume of blood was reinfused after each blood sample was obtained.

Immunohistochemistry Rat Mesentery Tissue
The rat mesenteric micro-vessels in each experimental condition were harvested for examination by immunohistochemistry. Briefly, mesenteric tissues were aseptically harvested from the gut using micro-scissors and forceps after superficially dripping 4% phosphate-buffered paraformaldehyde for

Role of CGA in protecting the endothelial barrier function in vivo and in vitro
To determine the effects of CGA on vascular leakage following sepsis, the dynamics of FITC-BSA leakage from the mesenteric micro-vessels was measured. Albumin leakage from the mesenteric micro-vessels increased in the septic shock group; the rate of increase was 243.3%. CGA treatment significantly ameliorated the albumin leakage; the rate of decrease was 37.9% compared with the LPS group ( Fig. 1A and B).
To determine the effect of CGA on endothelial barrier function in vitro, we next investigated the role of CGA in regulating the TEER and the endothelial monolayer permeability to FITC-BSA. The data showed that the TEER of VECs was significantly decreased and the permeability of FITC-BSA was increased 24 h after LPS stimulation. CGA significantly increased the TEER and reduced the permeability of FITC-BSA by 34.3% and 44.8%, respectively, compared to the LPS group ( Fig. 1C and D).

Mechanism underlying CGA protection of the endothelial barrier function in vivo and in vitro
To determine the potential involvement of tight and adherens junctions with ZO-1 and VE-cadherin in the endothelial barrier after septic shock, we examined the patterns of protein expression by 8 immunohistochemistry and western blotting. Examination by confocal microscopy revealed a decrease in the expression of ZO-1 and VE-cadherin in mesenteric micro-vessels after septic shock, which was relieved by treatment with CGA ( Fig. 2A). This result was supported by the western blot findings (Fig. 2B). Further study also showed that CGA effectively prevented the LPS-induced decrease in ZO-1 and VE-cadherin expression in vitro. These results indicated that CGA improved the vascular endothelial barrier function by increasing the expression of ZO-1 and VE-cadherin.

Effects of CGA on tight junctions in endothelial cells of the mesentery microvessels
Because fluid leakage for macromolecules such as albumin is carried out via widening of the intercellular cleft, we assessed the effect of CGA on tight junctions in endothelial cells of mesenteric micro-vessels using electron microscopy. The results showed that endothelial cells formed smooth, continuous, and high-density tight junctions in the sham-group. After sepsis was established, the integrity of tight junctions was disrupted and usually coincided with erythrocyte diapedesis. The sepsis-induced alterations in the endothelium ultrastructure were significantly reduced by treatment with CGA injection (Fig. 3).

Effects of CGA on the liver and kidney perfusion in rats with septic shock
To determine whether or not CGA ameliorated the micro-circulation by improving vascular leakage, liver and kidney perfusion were measured. The results showed that both liver and kidney perfusion were significantly decreased after septic shock. Specifically, liver and kidney perfusion decreased by 73.8% and 78.3%, respectively, compared with the sham-operated group. Liver and kidney blood flow increased by 41.2% and 38.4%, respectively, compared with the septic shock group (Fig. 4A and B).
These results suggest that CGA significantly protects vital organ perfusion by decreasing vascular leakage.

Effects of CGA on hemodynamic parameters in rats with septic shock
To evaluate whether CGA is beneficial for hemodynamics, changes in MAP, LVSP, and ± dP/dtmax were monitored. The hemodynamic parameters significantly decreased after septic shock; the MAP decreased to 63 mmHg (Fig. 5A) and LVSP decreased to 84 mmHg in the septic shock group (Fig. 5B).
The blood lactic acid (Lac) level is an important prognostic index, which reflects tissue oxygen delivery, metabolic state, and organ dysfunction. The results showed that the Lac level after septic shock increased significantly. CGA antagonized the septic shock-induced increase in the Lac level and the level of Lac decreased from 3.57 mmol/L in the septic shock group to 2.54 mmol/L in the CGA group (Fig. 5E) . 6A and B).

Discussion
Vascular endothelial barrier dysfunction is the major cause of sepsis-induced organ dysfunction.
Previous studies have shown that CGA protects against vital organ injury, such as acute liver and acute kidney injury, by inhibiting inflammasome activation. 18,19 The present study showed that CGA significantly improved vascular leakage in septic shock rats, then enhanced tissue perfusion in vital organs including the heart, and thus improved hemodynamic status and survival time. Improvement in the endothelial barrier by CGA was closely related to increased expression of ZO-1 and VEcadherin.
Sepsis is defined as a "dysregulated host immune response to infection leading to organ failure".
Recently, endothelial system dysfunction has been suspected to be detrimental to the recovery of septic patient and contribute to the high morbidity and mortality. 2,20 Previous studies have demonstrated that the inter-alpha-inhibitor ameliorates endothelial inflammation in septic human patients, 21 indicating that improvement in the endothelial barrier function may be an important measure for protecting organ function during sepsis. In this study we used CLP to induce sepsis, and found that CGA significantly improved vascular leakage and vital organ function, thus, prolonging survival by protecting endothelial barrier function in septic shock, which may provide a new measure for the clinical treatment of septic patients.
It is well-known that the tight junction (TJ) protein, ZO-1, and the adherens junction (AJ) protein, clinical situations but has lower stability and poor control. 15,29,30 In the present study we used the CLP model, which better mimics the pathologic and physiologic processes in patients with sepsis.
Moreover, the severity of sepsis was controlled in our laboratory. In addition, we found that the greater omentum encased the punctured cecum and affected the success rate of the model.