The Preliminary Study Of Interleukin -22 Effect On The Pathogenesis Of Atherosclerosis

With the aging of global population, cardiovascular and cerebrovascular disease has became one of major threats to the human health,with high morbidity and mortality. Atherosclerosis (AS) is a complicated multiple-factorial disease, which is the initiation or aggravation of these severe cardiovascular and cerebrovascular diseases. Its precise pathogenesis is still unknown. The research on the pathogenesis of AS is great useful in the developing strategies for its prevention and therapy.In recent years, scholars considered that immune inflammation played an important role in the AS development. It is the early sign of AS that endothelium lesion mediated by abnormal lipid metabolism. Chronic inflammatory reaction is the central part of its pathogenesis . One research has found that endothelial cell has direct contact with the dangerous factors of the blood, which is the first shield for the vascular pathological changes, with oxidized low density lipoprotein (ox-LDL) as one of the dangerous factors causing the lesion of endothelium. And then, damaged vascular endothelial cell had many changes on phenotype and function. Chemokines and cytokines secreted by pathological endothelium recruited inflammation cells, such as mononuclear macrophage.They located in the site of blood vessel lesion and amplified the inflammatory reaction. At the same time, it causes the apoptosis and necrosis of pathological endothelial cells, and breakdown integrality of endodermis shield, which will eventually intensify the formation of AS plaque.IL-22 is a cytokine found by Renauld in 2000, which is produced by immune cells such as activated T cells and NK cells and will take special effect on cells of nonhematopoietic origin, especially epithelial cells, but won't affect the function of immune cells. Recent researches have found that IL-22 may mediate either harmful or helpful inflammatory responses in many different diseases. IL-22 is known to be expressed in psoriasis, and its up-regulation often correlates with the expression of acute-phase protein and chemokine, keratinocytes proliferation and disease activity .In rheumatoid arthritis, IL-22 enhanced tissue inflammation by decrease the production of collagen and IgG. IL-22 mediates mucosal host defense against primary influenza virus infection and Gram-negative bacterial pneumonia . Sugimoto et al. found that IL-22 plays protective role on inflammatory bowel disease . However, more researches need to be taken for the biology effect of IL-22 on the cardiovascular system. In 2006, Chang et al. found that noncardiomyocytic noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells are also the targets of IL-22. In 2010, Gangemi found that IL-22 may have relation with chronic heart failure , which implies that IL-22 may participate in the inflammatory reaction of cardiovascular system.Previous researches have found that IL-22 played an important role in chronic infectious disease and autoimmune disease, suggested it may involed in the development of atherosclerosis (AS), a chronic inflammation disease caused by hyperlipemia. Based on the research above, we presume IL-22 may participate in the process of AS caused by dangerous factors in the blood circulation by taking effect in vascular endothelial cell or smooth muscle cell. Thus, what is the role of IL-22 in the AS? Is there any possibility that IL-22 works as the curative target and correlates with its effect on age-related diseases such as heart failure, chronic pulmonary disease, hepatic steatosis[15], inflammatory bowel disease, and bring good news for population aging.To prove these hypotheses above, we will carry out follwing experiment: (1) to detect the serum IL-22 level of coronary arteriosclerosis disease (CAD) patients and to analyze the associativity with disease. (2) to detect the express of IL-22 receptor in human vascular endothelial cell. (3) to screen the target molecules that IL-22 may affect in endothelial cell lesion process caused by ox-LDL, by using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and enzyme-linked immunosorbent assay(ELISA). (4) to investigate the effect of IL-22 on the molecular biology and function of endothelial cells stimulated by ox-LDL in vitro.Part 1. The expression of IL-22 in CAD patientsWe detect IL-22 mRNA expressed by peripheral blood mononuclear cells (PBMCs) from coronary arteriosclerosis disease (CAD) patients by RT-PCR. And the IL-22 concentration of the blood serum was detected by ELISA. And then, we analyze the association between IL-22 and AS based on it's expression of gene and protein, which will provide clues and evidence for further understanding of the effect of IL-22 in AS.The results have revealed that there are existing IL-22 mRNA in the peripheral blood mononuclear cells (PBMCs) of AS patients. After sorting the CAD patients based on the degree of coronary artery stenosis, we found that the concentration of serum IL-22 of the patients with early artery lesions (coronary artery stenosis <50%) is much higher than that of the no coronary artery stenosis group and advanced carotid artery disease group (coronary artery stenosis >50%).But there appears to be no correlation with the level of TCHO (total cholester), TG (triglycerid) and LDL-C (1ow density lipoprotein cholester).Part 2. The effect and mechanism of IL-22 participated in artherosclerosis induced by ox-LDLOur previous research revealed that there is relationship between IL-22 and AS. Thus does IL-22 have direct effect on the vascular endothelial cell? Is the high level of IL-22 related with the initial stage of AS and endothelium lesion caused by ox-LDL? Base on these questions, we detect the express of IL-22 receptor on the human vascular endothelial cell.Experiment 1. The expression of IL-22 receptor in the vascular endothelial cell of humanIL-22 receptor1 (IL-22R1) mRNA in ox-LDL stimulated human umbilical vein endothelial (CRL1730) cells was detected by real time fluorescent quantitation RT-PCR and flow cytometry. The results revealed that CRL1730 cells do express IL-22R and its expression is affected by the concentration of ox-LDL and its contact time. Consistent with the mRNA expression, IL-22R positive CRL1730 cell rate was great increase after 100μg/ml ox-LDL stimulated 24 hours (P<0.01).Experiment 2. The effect of IL-22 on the profile of molecule expression of human umbilical vein endothelial cell stimulated by ox-LDLThe previous experiment validated that IL-22 receptors are existed on vascular endothelial cell and change dynamically under the stimulation of 100μg/m ox-LDL. Then, what is the role of IL-22 in vascular endothelial cells, especially vascular endothelial cells stimulated by ox-LDL?Firstly, we select 7 genes (Bcl-2, COX-2, PGES, MCP-1, MMP1, ICAM and IL-6), which closely related with IL-22 function and endothelial cells lesion. Looking for the targets of IL-22, we detect the change of related molecule express in ox-LDL stimulated CRL1730 cells with or without IL-22 by RT-PCR and ELISA. Our research found that the level of Bcl-2 and ICAM of ox-LDL stimulated CRL1730 cells has increased significantly (P<0.05) in the presence of IL-22.Experiment 3. The effect of IL-22 on the apoptosis of human umbilical vein endothelial cell induced by ox-LDLFlow cytometry was applied to detect the effect of exogenous IL-22 on the apoptosis of CRL1730 cells induced by ox-LDL. After treated with or without IL-22 and ox-LDL, the relative expression of Bcl-2, Bcl-xl, Bcl-w, caspase 3 and STAT3 mRNA in CRL1730 cells were measured by real-time fluorescence quantitative RT-PCR, the phosphrylation status of STAT3 and the Bcl-2 protein level were detected by Western Blotting.We found that IL-22 significantly inhibited the apoptosis of CRL1730 cells induced by ox-LDL, and increased the relative expression of Bcl-2, Bcl-xl and Bcl-w, the well-known anti-apoptosis factors. By contrast, it decreased caspase 3 mRNA relative expression. In addition, we found phosphorylation of STAT3 in 1-2 hours and the sharp increase of Bcl-2 protein level after 4 hours when IL-22 was added to ox-LDL stimulated CRL1730 cells. Based on the research above, we speculate IL-22 inhibited the pro-apoptosis effect of ox-LDL on the CRL-1730 cell. This may be associated with the increased expression of Bcl-2, Bcl-xl and Bcl-w, decreased expression of caspase 3 and enhanced STAT3 phosphorylation.In conclusion, our research found that there is a relationship between IL-22 and the degree of coronary artery stenosis of AS patients.It is because IL-22 could repair the lesion blood vessel endothelial tissue and regain its integrity by inhibit apoptosis of endothelial cells and then slow down the atherosclerotic process indirectly.