| Background and Objective:Atrial fibrillation(AF) is the most sustaining arrhythmia in clinic, with the aging population, the incidence rate is rising year by year, has become an important public health threat to human health. But at present, atrial fibrillation and maintenance the exact molecular mechanism of occurrence and maintenance for AF is not clear.As previously known, atrial electrical remodeling and structural remodeling is the main molecular mechanism for the occurrence and development of atrial fibrillation, but the significance of atrial structural remodeling in atrial fibrillation focus more and more attention in recent years, especially the related mechanisms of oxidative stress and atrial fibrosis, becoming a hot topic.The body can produce antioxidant enzymes such as superoxide dismutase(SOD), malondialdehyde(MDA) to remove redundant ROS in normal in order to keep the dynamic balance of free radicals.but under oxidative stress,excessive oxidation will caused cell damage and overproduction of ROS, induced sarcoplasmic reticulum,increased cytosolic calcium concentration, dissolved atrial muscle, lead to structural remodeling which showed myocardial fibrosis in cell ultrastructure. myocardial fibrosis has been proved to be one of the important reasons causing recurrence of atrial fibrillation after treatment.The myocardial fibroblasts are the major effector cells of myocardial fibrosis, but its origin is unclear,and the composition changes of fibroblast which from different origin are not clear under a variety of pathological conditions. Therefore, confirm its origin,blocking and transforming atrial fibrosis process is a hot scientific problem in the field of basic research of atrial fibrillation in recent years.Recent studies indicate that the endocardial and microvascular endothelial cells to mesenchymal transition(End MT) may be an important source of myocardial fibroblast,morever,End MT is involved in the proliferation of atrial fibroblast under oxidative stress,and atrial fibroblasts have stronger ability in promoting fibrosis in pathological status.Endothelial cells transform into fibroblast cells(End MT) promote the atrial fibrosis within stimulating factors may participate in occurence and maintenance of atrial fibrillation.This study intends to observe the expression of the oxidative stress markers in the right atrium and serum of two groups,and to investigate the role and significance of oxidative stress in the atrial structural remodeling.Observe the expression of α-SMA, S100A4, CD31 change to verify End MT involved in atrial fibrosis as well as their significance in atrial fibrosis in clinical intervention.The results expected for provide new therapeutic targets to atrial fibrillation. Methods:Part one:changes and significance of oxidative stress biomarkers in atrial structural remodeling in patientsFrom February 2014 to May 2014, 44 cases with rheumatic heart valve disease was made a diagnosis of mitral valve disease by ultrasound at cardiacvascular surgery of Xinqiao Hospital.Those patients was divided into two groups according to the medical history and preoperative ECG recordings.chronic atrial fibrillation group(AF,n=26),based on the duration of AF≥six months, mean age was( 53.30±8.81) years. Sinus rhythm group(SR,n=18), defined as no atrial fibrillation anytime,mean age was( 50.37±13.03) years.Exclusion criteria: ① Patients with coronary heart disease, diabetes, liver and kidney dysfunction, thyroid function hyperfunction or weakened, malignant tumor, cardiomyopathy; ② no operative during six months and no taking of any type of ACEI and ARB class of drugs; ③ the moderate tricuspid regurgitation and above(defined as tricuspid regurgitation area is larger than 3cm2); the duration of AF is less than 6 months; age was less than 18 years or older than 80 years;Collected tissues sample:At the beginning of cardiopulmonary bypass operation, cut a part of the right atrial appendage tissue, cut off the excess blood and adipose tissue,then implantate into-80 ℃ refrigerator for testing. Selected 5 cases from each group randomly in 4% formaldehyde Solution to fix 24-36 h,embedded in paraffin,serial sections(6-8pieces, section thickness 5um) for masson, immunohistochemical and immunofluorescence staining.Observe atrial fibrosis,the expression of NOX2 protein in right atrial tissue, the level of oxidative stress in the tissue and serum were assayed by the ELISA method.Collected blood sample: collect from patients venous blood in 3ml in the day before operation, at room temperature for 2 hours,then 1000 x g centrifugation for 15 minutes in 4 ℃, take the supernatant,labeled and packed in EP tubes, placed at-80 ℃ to save, but should avoid repeated freezing and thawing.Masson staining : detected atrial fibrosis.Immunohistochemistry and immunofluorescence staining: detected the expression of NOX2 protein in two patients in atrial tissue.ELISA method: measured the level of oxidative stress in rightl appendage tissue and serum.Part two: the significance of End MT involved in atrial fibrosis for atrial structureral remodeling of valvular heart disease with AF patientsFrom July 2014 to October 2014,a total of 42 patients with rheumatic heart disease underwent mitral valve replacement, according to the history and preoperative ECG recordings were divided into 2 groups: chronic atrial fibrillation group(AF,n=22), sinus rhythm group(SR,n=20) Evaluation preoperative heart function through echocardiography.The inclusion and exclusion criteria was the same as the part one.Collect tissue sample:Take right atrial tissue about 200 mg before the cardiopulmonary bypass, cut off excess fatty tissue, the same sample is divided into three parts;one part in 4% formaldehyde Solution to fix 24-36 h,embedded in paraffin,serial sections(6-8pieces, section thickness 5um) for masson and trinitrophenol-sirius red,immunohistochemistry staining,one part in-80 ℃refrigerator rapidly,for extracting total RNA and protein; one part embed in OCT, then place-80℃ refrigerator, or immediately frozen sections for immunofluorescence staining.masson and trinitrophenol-sirius red staining:obverse the cardiac fibrosis and calculate collagen volume fraction(CVF).RT-q PCR:detect the expression of α-SMA、CD31、S100A4 m RNA in two patients in right atrial tissue.Western blot:detect the difference expression of α-SMA and S100A4 proteins in two groups patients in right atrial tissue.Immunohistochemistry and double immunofluorescence technique:detect the difference expression of α-SMA,CD31,Vimentin,S100A4 proteins in two groups patients in right atrial tissue. Result:1. part one oxidative stress and atrial remodeling(1) Comparison of two groups with general situation.26 cases in AF group,7 males and19 females;18 cases in SR group,6males and 12 females.the left atrial diameter(LADd) in AF group was significantly higher than that in SR group [(55.57±10.76) vs(43.94±6.21), P<0.05]; left ventricular ejection fraction(LVEF) in AF group was significantly lower than that in group SR [(63.12±7.66) vs(67.83±4.62), P<0.05], the incidence of left atrial thrombus and cerebral infarction in AF group was significantly higher than that of SR group [11% vs 6%; 2% vs 0%, P<0.05].(2) The results of immunohistochemistry: positive cell area of AF group was higher than that of SR group [(32005.72±8885.11) vs(6438.32±2849.63), P<0.05].Compared to SR Group,the integral optical density of AF group was higher [(4705559±1225040) vs(1213255±421952, P<0.05]).(3) The results of ELISA:ROS、SOD、MDA、AOPP protein expression in right atrial appendage tissue of AF group was significantly higher than that of SR group [(18.53±3.47) vs(13.65±3.69), P<0.01]; [(13.32±1.25) vs(10.65±1.15),P<0.05 ], [(15.41±1.69) vs(11.46±1.83),P<0.01 ],[(29.69±2.09) vs(21.94±1.94),P<0.01 ]. Compared to SR group,ROS proteins of AF in serum was significantly higher [(34.6±2.61) vs(17.8±1.03), P<0.05],AOPP proteins in the two groups was no significant difference [(13.64±2.56) vs(13.27±3.38),P>0.05]2. part two End MT involved in atrial structural remodeling(1) For masson and trinitrophenol-sirius red staining:compared to SR group, right atrial tissue collagen fibers hyperplasia in AF group was significantly higher,especially for type I collagen, collagen volume fraction(CVF) increased significantly(P < 0.05);(2) Laser scanning confocal microscope found: in AF group,S100A4, CD31 two kinds of protein fluorescence express in myocardial vascular endothelial, while in SR group, only CD31 protein expression, S100A4 protein expression was no expression or weaken.(3) Immunohistochemical staining :showed the expression of S100A4, α-SMA, Vi protein in myocardial tissue of right atrial in each two groups,but SR group was significantly weaker than that in AF group.(4) Real-time quantitative PCR(RT-q PCR) analysis revealed:compared to SR group, expression of S100A4, α-SMA m RNA in right atrial tissues were significantly higher in AF group(P < 0.05),but expression of CD31 m RNA in right atrial tissues were lower in AF group.(5) Western blot result analysis:compared to SR group,expression of S100A4, α-SMA proteins in right atrial tissues were significantly higher in AF group(P < 0.05); Conclusions:1. Compared with the two group patients, atrial fibrillation patients left atrial diameter increased, ejection fraction reduced. incidence of left atrial thrombus and cerebral embolism enhanced.2. Oxidative stress biomarkers increased, especially in atrial tissue,which proved is closely relationship for oxidative stress and atrial fibrosis,and suggested the possible as follow was exist.NADPH oxidase /NOX2 lead to oxidative stress /inflammation which caused the occurrence of atrial structural remodeling, enhancing susceptibility to atrial fibrillation.2. End MT is involved in atrial structural remodeling.endothelial cells in atrial tissue can be converted into fibroblast to participate in atrial fibrosis. fibroblast specific biomarkers such as α-SMA,FSP-1(also called S100A4)and vimentin,which proteins and m RNA expression increased significantly in the right atrial tissue, and endothelial cell markers CD31 which m RNA and proteins expression were significantly decreased in right atrial tissue. |
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