A Study Of The Effect Of S100A12-RAGE Pathway On Immune Coronary Artery Lesions

Kawasaki disease (KD) is an acute vasculitis of infants and children predominantly affecting small-and medium-sized arteries, and KD has become the main cause for acquired heart diseases in young children.Many scholars have chosen lots of experimental approaches to clarify the pathogenesis for Kawasaki disease, but the results were not satisfactory. More and more study found that S100A12-RAGE axis is responsible for vascular inflammation. However, the role of S100A12 and RAGE in KD coronary artery lesions remains to be further clarified. Ulinastatin, as an endogenous protease inhibitor, can inhibit neutrophil elastase, while it may be a protective role in many diseases.To produce an experimental coronary artery lesions mimic Kawasaki disease and clarify the relevance of the internal mechanism between immunity coronary artery damage and Kawasaki disease coronary artery lesions; to observe the effect of S100A12-RAGE pathway on the immune coronary artery injury and the relationships between the neutrophil elastase inhibitor (ulinastatin), S100A12-RAGE pathway and coronary artery damage horse serum-induced. Experimental animals were purebred Landrace,1.5-3 months old, healthy and clean, male, and weight of 9-12Kg.Part I:"Kawasaki disease-like" coronary arteritis induced by horse serum11 purebred piglets were randomly divided into four groups (5,2,2,2), and 10ml/kg of horse serum was infused i.v. three times at 5-d interval. Suppose the first time for the injection of horse serum as day 0, observe the clinical performances during the experiment. On days 15,20,25and 30, the animals were sacrificed respectively and the fresh coronary artery specimens carried with myocardial tissues were removed, following, neutral formalin-fixed, paraffin embedded and sliced, and HE stained, then light microscopy.PartⅡ:The relevance between ulinastain, S100A12-RAGE pathway and coronary artery lesions induced by horse serum1. Animal ModelDivided into three groups, namely, on experimental day 0 (self-control), days 10 (the immunization-finished group, equivalent of the first course of 5 days, the acute phase), days 20 (equivalent to the first 15 days of the course, the subacute stage). Group of days 20 was further divided into two groups, and it was the ulinastatin group and the positive control group.1) Experimental group (10):①Ulinastatin group (5):From days 11, ulinastatin 10,000 U/kg, diluted with normal saline(NS) to 20ml, intravenous injection, once a day x 3 days.②Positive control group (5):From days 11, normal saline 20ml, intravenous injection, once daily×3 every day.2) Normal control group (2):the same environment and feeding conditions as experimental group, not to any intervention.Intravenous blood (10ml) was collected on days 0,10,20, severally. All animals were sacrificed at days 20.2. Blood routine tests for white blood cell counts, hemoglobin levels, platelet counts, serum albumin levels, C-reactive protein levels and erythrocyte sedimentation rates.3. Detections for CECs/MNC, S100A12-CECs/CECs, FI-S100A12-CECs, RAGE-CECs/CECs, and FI-RAGE-CECs with flow cytometry.4. Paraffin sections, HE stains, and immunohistochemistry of S100A12 and RAGE.PartⅠ:"Kawasaki disease-like" coronary arteritis induced by horse serum1. Some similar clinical manifestations to Kawasaki disease:fever (chills while temperature rising), systemic polymorphic rashes, red lips, red eyes, etc.2. General views of the naked eye:no gross morphological abnormalities; neither bead-like expansion or tumor-like phenomenon on coronary arteries or its major branches.3. HE stains:on days 15, the main manifestation was myocarditis, and no finding for vascular damage; on days 20, coronary arteries had some serious lesions, such as endothelial cells infiltrations, intimal disruption, intimal thickening, interior elastic fiber layers destruction, and so on; on days 25 and 30, the injury of coronary arteries were similar to days 20.PartⅡ:The relevance between ulinastain, S100A12-RAGE pathway and coronary artery lesions induced by horse serum1. Blood routine tests:on days 0 (the control group),10 (the immunization-finished group),20 (the ulinastatin group),20 (the positive control group), the indexes were as follows respectively. White blood cell counts (×109/L):16.74±6.27,24.35±7.79,17.06±2.67,13.12±2.79; hemoglobin levels (g/L):103.60±10.71,106.60±10.53,103.60±8.44,104.00±6.36; platelet counts (×109/L):1068.6±447.06,293.50±116.31, 422.75±88.51,452.50±67.69; serum albumin levels (g/L):39.33±5.04,28.86±3.77, 36.52±2.58,34.62±4.03; C-reactive protein levels (g/L):all were lower than 1; erythrocyte sedimentation rates (mm/h):2.11±1.27,2.11±0.60,4.20±2.17,2.40±0.89.2. On days 0 (the control group),10 (the immunization-finished group),20 (the ulinastatin group),20 (the positive control group), the indicators were as follows respectively. CECs/MNC (%):0.49±0.44,0.58±0.38,0.33±0.13,0.39±0.16; S100A12-CECs/CECs (%):87.61±8.72,91.33±9.67,89.31±8.96,94.02±2.83; FI-S100A12-CECs:200.28±79.75,232.46±93.27,225.55±73.64,206.09±96.59; RAGE-CECs/CECs (%):96.16±10.42,82.60±15.57,68.75±17.77,62.73±31.58; FI-RAGE-CECs:264.38±210.31,122.98±76.16,185.25±103.46,151.21±78.54.3. HE and immunohistochemical stains1) HE stains:results of positive control group were the same as the cases on the days 20 in Part I; ulinastatin-infused short-term could not prevent coronary artery endothelial damage, and after the interventions by ulinastatin, the inflammation at the skin rashes did not subside quickly.2) Immunohistochemical stains of S100A12:semi-quantitative counting method to calculate the S100A12-positive cells per field, the average numbers of the ulinastatin group:8.61±2.76; the positive control group:18.00±22.03; the normal control group: 9.46±5.64. There was no significant statistical difference in comparison between three groups, however, at the sites of inflammatory cells infiltrations in the positive control group, S100A12 had a high expression level, inversely, on vascular endothelial cells there even no S100A12 positive expression.3) Immunohistochemical stains of RAGE:RAGE was mainly expressed in the cytoplasm, such as on the myocardial tissue the nuclears dyed lightly while cytoplasms were strong stain; on coronary endothelial cells, coronary artery smooth muscle cells and myocardial cells, the immunoreactive scores (IRS) of RAGE were as follows, respectively, the ulinastatin group:11.11±1.76,0.78±0.67,5.56±2.13; the positive control group:12.00 ±0.00,2.70±3.34,7.60±1.27; the normal control group:6.50±3.54,1.50±0.71,3.50±3.54.1. S100A12-RAGE pathway is involved in immunity coronary artery lesions induced by horse serum, and the attack of S100A12 may be the direct cause of vascular endothelial cells shed and endothelial injury.2. When there are immune vasculitises, the levels of S100A12 and RAGE on circulating endothelial cells and tissues change with the development of disease, while the change is in an unstable dynamic balance.3. To some extent, ulinastatin slows the infiltrations of inflammatory cells, but it seems that ulinastatin used short-term can not prevention the progression of immunity coronary artery lesions; for S100A12-RAGE pathway, the regulation of ulinastatin is slightly weak; it is necessary to adjust usage and dosage of ulinastatin and add samples.4. Horse serum-induced coronary artery injury model mimic Kawasaki disease is not a suitable model. Kawasaki disease is known to be a systemic vasculitis resulted from kinds of immune factors, while foreign protein immune-induced animal model of allergic-type III can not fully explain the machinism of Kawasaki disease.