The Relation Of Notch4Signaling And Coronary Artery Lesions Induced By Lactobacillus Casei Extract

Objective:To evaluate the effect of Notch4signaling on the coronary artery inflammation and lesions induced by LCWE in a mouse model of KD.Materials and Methods:Lactobacillus casei was purchased from CICC (China center of industrial culture collection) and harvested during the exponential growth phase for experiments.Part1:The establishment of an animal model of coronary arteritis induced by LCWEAfter being harvested by centrifugation, the bacteria were disrupted by overnight incubation in4%sodium dodecylsulfate. Sequential incubations with250mg/ml RNase, DNase I and trypsin were performed and the cell-wall fragment preparation was then sonicated. After sonication, the cell-wall fragments were spun, and the supernatant was retained. The L casei cell-wall content of the supernatant determined by colorimetric phenol-sulfuric acid extraction technique.4-6week old male BALB/c mice were injected with500ug of LCWE in PBS or PBS alone intraperitoneally. Aneasthetized mice were killed on days3,7,14and28after injection. Cardiac tissue and spleen were removed immediately following sacrifice and fixed in10%paraformaldehyde for24hours. Serial paraffin sections were subsequently stained with hematoxylin and eosin for light microscopy. Real-time PCR was performed to detect the expression of Thl/Th2cytokine profices.Part2:The relation of Notch4signaling and coronary artery lesionsThe eyeballs were extracted and blood was drawn at different points. Then cells were stained with antibodies to cd34-eFlour660, Flk-1FITC, Notch4-PE, cd45-PE-Cy5.5in dark. The number of circulating EPCs and expression of Notch4on the surcafe of EPCs were assessed by flow cytomtry. After design the primers of GAPDH, Notch4, RBP-JK, vcam-1and P-selectin, the expression was investigated Real-time PCR. Finally, vcam-1and P-Selectin expression on the endothelium were investigated using immunohistochemical staining of coronary roots and thoracic aorta sections.Result:Part1:The establishment of an animal model of coronary arteritis induced by LCWE1. The weight of mice treated with LCWE was significantly reduced on day3.2. A focal inflammatory infiltrate was evidently seen around the adventitia of coronary artery at each time points.3. The volume was enlargement and the weight was increased by gross anatomy method. Splenic corpuscle hyperplasia and fusion were evidently existed at each time points stained with hematoxylin and eosin for light microscopy.4. The expression of Thl/Th2cytokine profices was significantly increased, except for IL-6.Part2:Notch4and RBP-JK are associated with coronary artery lesions.1. The number of circulating EPCs was significantly higher at3day in the LCWE-treated mice compared to controls. The number of EPCs in the LCWE-treated mice became lower than controls on day14. The expression of Notch4on the surface of EPCs in the LCWE-induced mice was obvious higher than the expression of Notch4of controls on day7.2. The expression of P-selectin and RBP-JK of thoracic aorta from mice injected ip with LCWE on day14,28were significantly higher than that from mice injected ip with PBS. There was a significant correlation between thoracic aortic RBP-JK and P-selectin mRNA expression levels. There was also a significant correlation between thoracic aortic Notch4and vcam-1mRNA expression levels.3. In the sections from LCWE-treated mice, vcam-1was more strongly expressed in the coronary roots on day3,14,28and thoracic aorta on day3,7. Among these sections, the staining of the endothelial cells of coronary roots on day3was heaviest. Compared to the weak expression of P-selectin from PBS-control mice, it was obviously more intense positive staining for P-selectin observed in the coronary roots on day14,28and thoracic aorta on day14from mice treated with LCWE.Conclusion:1. The Notch4signaling pathway of EPCs contribute to the Lactobacillus casei Extract-induced coronary artery lesions.2. The Notch4signaling pathway in vascular endothelial cells is involved with CALs induced by LCWE.3. The changes of Notch4signaling pathway in vascular endothelial cells influence the function of coronary artery endothelial cells.