| Objectives Acute myocardial infarction(AMI) refers to coronary arterial blood supply sharply reduced or interruption, which leads to ischemic necrosis of the corresponding myocardial cell. In recent years, more and more attention has been focused on the study of the relationship between the gut and the heart. These studies showed that the impaired intestinal barrier function, namely the increase of intestinal permeability, is related to the development of cardiovascular disease. When the intestinal is ischemic and hypoxic, the expression of tight junction protein including occludin is changed and intestinal permeability is increased, which can cause intestinal bacteria metabolites, such as LPS and D-lactate can enter blood circulation, then activating the inflammatory responses, thereby influencing the process of inflammatory infiltration and tissue repair after myocardial infarction. Glutamine, as an important nutritional element of intestinal epithelial cells, can improve the intestinal barrier function. This study was sought to investigate the changes of intestinal permeability in mice after acute myocardial infarction and the characteristics of intestinal permeability changes in different intestinal segments, as well as the role of changes in the blood flow of the superior mesenteric artery and the expression of tight junction protein occludin on the intestinal barrier injury in the development of acute myocardial infarction, and the changes in circulating level of bacterial translocation markers, LPS and D-lactate, and the effects of glutamine on intestinal mucosal barrier injury in mice with AMI, which can provide experimental basis for the improvement of the intestinal barrier function as a new target for the treatment of acute myocardial infarction.Methods(1) Male C57BL/6 mice(6-8 week old) were randomly divided into sham operation group(Sham group) and acute myocardial infarction group(AMI group). AMI was induced by the ligation of left anterior descending coronary artery, while Sham group underwent similar procedure except for coronary ligation. Immediately, and 1d, 2d, 3d, 5d, 7d after surgery, mice were orally given FITC-dextran-4KD(FD4). Portal vein blood were collected four hours after the intervention, then the concentration of plasma FD4 was detected to assess the dynamic changes of intestinal permeability after AMI.(2) Male C57BL/6 mice(6-8 week old) were randomly divided into normal control group(Normal group), Sham group and AMI group. Intestinal permeability in different intestinal segments was evaluated by a FD4-based method on day 3 after AMI. After the mice were sacrificed, distal small intestine, cecum and colon were collected, hematoxylin and eosin staining was used to observe the pathological changes of intestinal mucosa.(3) Male C57BL/6 mice(6-8 week old) were randomly divided into Sham group and AMI group. Six hours, 1d, 3d, 7d after surgery, high frequency ultrasound imaging system was used to detect the cardiac function and superior mesenteric artery blood flow. The dynamic changes of colon permeability was evaluated by a FD4-based method. Specimens were collected after mice were killed, then the concentration of plasma LPS and D-lactate were detected, and 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to evaluate infarct area of the heart. The distribution of tight junction protein occludin in colon tissue was examined by immunofluorescence staining. Real-time polymerase chain reaction method was used to detect the expression of tight junction protein occludin m RNA in colon. Western Blot was used to detect the protein expression of occludin in colon.(4) Male C57BL/6 mice(6-8 week old) were randomly divided into Sham group, AMI group, AMI+saline group(AMI+NS group) and AMI+glutamine group(AMI+Glu group). Glutamine(0.02g/d) was administered by gavage immediately and on 1d, 2d, 3d after surgery in AMI+Glu group. Equal volume of saline were given in AMI+NS group. Superior mesenteric artery blood flow was detected on day 3 after AMI. Intestinal permeability was evaluated by a FD4-based method. The plasma was collected to detect the concentration of plasma LPS and D-lactate.Results(1) The results of intestinal permeability showed that the intestinal permeability in AMI group presented a trend of increase followed by a gradual decrease. The concentration of plasma FD4 started to increase on day 1 after AMI(4.52±0.54?g/m L vs 2.20±0.09?g/m L, P<0.01), reaching the peak level on 3d(5.76±0.89?g/m L vs 2.66±0.10?g/m L, P<0.01), then begin to decrease at 5d(5.20±0.64?g/m L vs 2.23±0.06?g/m L, P<0.01). The level of FD4 returned to baseline level on 7d.(2) The results of intestinal permeability in different intestinal segments showed that compared with the normal group, there was no significant difference in intestinal permeability of distal small intestine, the cecum and the colon in the Sham group. Compared with Sham group, intestinal permeability in AMI group as assessed by plasma FD4 level, was significantly increased in distal small intestine(9.76±0.32?g/m L vs 4.43±0.19?g/m L), cecum(1.82±0.08?g/m L vs 0.49±0.06?g/m L) and colon(5.41±0.34?g/m L vs 2.07±0.18?g/m L) respectively, P<0.001. The fold changes of intestinal permeability in the cecum underwent the most dramatic change, followed by the colon and distal small intestine. H.E. staining revealed that mucosa tissue in distal small intestine, the cecum and the colon presented with inflammatory cell infiltration and edema in AMI group.(3) The cardiac function revealed by high frequency ultrasound imaging system showed that the left ventricular end systolic diameter and left ventricular end diastolic diameter were significantly increased, while ejection fraction and fractional shortening decreased after AMI in mice. The superior mesenteric artery blood flow in AMI group underwent a decreasing trend from 1d after surgery(154.5±26.05mm/s vs 274.1±32.09mm/s, P<0.05), and reached the nadir level on 3d(108.0±5.90mm/s vs 251.5±46.31mm/s, P<0.01), and remained decreased on 7d(130.1±24.94mm/s vs 274.1±30.03mm/s, P<0.05). The colon permeability measured by FD4 level in AMI group started to increase on 1d after surgery(6.85±0.58?g/m L vs 3.22±0.39?g/m L, P<0.05), and remained elevated on 3d(8.53±1.84?g/m L vs 3.07±0.49?g/m L, P <0.05), and then followed by a decrease on 7d. Plasma LPS tests showed that the concentration of plasma LPS in AMI group started to increase on 1d after surgery(0.31±0.02EU/m L vs 0.23±0.02EU/m L, P<0.05), and increased significantly on 3d(0.46±0.03EU/m L vs 0.24±0.02EU/m L, P<0.01), then decreased to baseline on 7d. Plasma D-lactate tests showed that the concentration of plasma D-lactate in AMI group started to increase on 1d after surgery(5.26±0.39mg/L vs 3.91±0.47mg/L, P<0.05), and increased significantly on 3d(7.05±0.56mg/L vs 4.10±0.53 mg/L, P<0.01), then decreased to baseline on 7d. Immunofluorescence staining showed that the expression of tight junction protein occludin in the colonic mucosal surface and crypt was decreased in AMI group. The results of Real time-PCR and Western Blot showed that the expression of occludin protein was consist with the expression of occludin m RNA. The expression of occludin m RNA and protein levels in AMI group began to decrease on 1d, and then significantly downregulated on 3d, followed by regression to baseline level on 7d.(4) The results of intestinal permeability after glutamine administration showed that compared with the AMI+NS group, intestinal permeability in AMI+Glu group had a tendency to decrease, but the difference was not statistically significant(4.62±0.32?g/m Lvs 5.70±0.54?g/m L, P>0.05). The superior mesenteric artery blood flow in AMI+Glu group had a tendency to increase, and the concentration of plasma LPS and D-lactate had a tendency to decrease, but the difference was not statistically significant.Conclusions(1) Intestinal permeability undergoes a significant increase with pathological changes of intestinal mucosa after AMI in mice, and the most significant changes in gut permeability were observed on day 3 after AMI. The most dramatic change in segmental permeability is the cecum, followed by the colon and the distal small intestine.(2) The blood flow of superior mesenteric artery is decreased, accompanied by a parallel downregulation of intestinal tight junction protein occuldin after AMI in mice, which is associated with increased intestinal permeability and translocation of bacterial by-products LPS and D-lactate in peripheral circulation.(3) There is a tendency towards improvement in intestinal permeability after glutamine administration in mice after AMI. However, the dose-response relationship between glutamine treatment and potential therapeutic efficacy remains to be validated in future work. |
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