The Research On The Mechanism Of Rat Myocardial Damage Caused By Lipopolysaccharide And The Protection Of Glutamine To It

Infective shock is a serious syndrome including multi - organ failure and one of common disease. When it happened, serious hypotension caused by increasing of penetration and the lose of body liquid, then effective circulation blood decrease, furthermore, serious metabolize acidosis make systolic decline, at last developing to the shock. The endotoxin released by Gram negative bacilli is major responsabal for it. The key of the circulation failure is that myocardial damage caused by tumor necrosis alpha and endotoxin. Much attention have been paid on protect myocardium and amend circulation in order to improve the recovery by pediatrician. Nowadays, the studies of myocardial damage are focus on the relationship between serum inflammation factor, the regulation of blood vessel - nerve, NO, iNOS and the blood vessel and/or systolic function, little attention has been paid to the relationship of inflammation factor between serum and tissue. As to extra cellular signal regulation, alpha sarcmeric actin and matrix metalloproteinase studies mainly in hypertension, myocardial infarct and burning animals. Weather these changes happened during endotoxemia is not clear. But it has been showed that matrix metalloproteinase damage can result left ventricular rebuilding after myocardial infarct. Interleukin 8 and 10 are novelty and representative indexes for inflammation and anti — inflammation, they were chosed as damage and anti - damage factors. This study is on the assumption that tumor necrosis factor alpha and interleukin 1,6,8 and other infection factors can be induced by lipopolysarccprotein injure organs, at meanwhile re-leased free radix make myocardial damage. On the other hand, the synthesis depression of protective factor, such as interleukin 10 and insulin growth factor -1, make the recovery more difficult. Endotoxin depressed the synthesis of alpha sarcomeric actin ( a - SA) and /or increased the losing of it, at same time too much cardiac hypertrophy and hyperplasia resulted the systolic function deline. The endothelial cells of cardial vessel was destructed by endotoxin, cardial vessel narrowed and blood supply decreased, which caused myocardial energy accommodate decline. The increasing of matrix metalloproteinase (MMP) expression destruct myocardial collagen result the weakening of the support,to cardiac myocytes and vessel, enhance the rebuilding of left ventricle, the heart lost its inherence geometry figure and the systolic function declined, then heart failure happened. During the struggle of damage and anti damage, the heart, a motor organ, go through these key mechanisms and resulted to unreversible damage.During the treatment of shock, the efficient drug to protect myocardium, except antibiotics, is dexamethasone, especially at depressing inflammation and decreasing the synthesis of NO and iNOS. But docters often hesitate when they choose dexamethasone for its side effects. Recently, a novel drug, glutamine was found which can produce glutathione, an eliminator of free radix, such as H2O2, LOOP". Furthermore, it can clean out free radix without depending on any enzyme and reduce myocardial apoptosis and necrosis. On the other hand, glutamine have a self protection by accelerating the synthesis of heat shock pro-tein70 ( HSP70) , which can diminish the damage of organs, including heart. It was found that before the heart - lung bypass in rat, infusing glutamine for one week significantly decrease serum interleukin 6,8 and significantly decrease myocardial apoptosis and necrosis. Furthermore it can diminish the myocardial damage caused by doxorubicin. However, untill now there is no report about if glutamine has these effects on myocardium during endotoxemia, if it has, then how to act, they are not clear yet.Classic endotoxemia model were established by injecting LPS intraperitone-ally. This study will testify that during endotoxemia, glutamine can depress inflammation/ promote recovery, diminish myocardial apoptosis, increase the synthesis of a - SA, weaken the damage of vessel inner membrance, restrain theinjury of endotoxin to myocardial collagen fibre, decrease the ventricular rebuilding, offer some guiding to clinical about myocardial protection.Materials and Methods1. Animal model132 healthy Wistar rats (18 day -old, weight 29. 3 ±5. 4g,ignoring sex) were separated into 11 groups randomly:Oh control group (normal saline is injected intraperitoneally 1 ml/kg) ; UPS group (UPS is injected intraperitoneally 4mg/kg) ; Gin group (UPS 4mg/kg and 13.46%glutamine 1 ml/kg are injected intraperitoneally ). The rats both IPS and Gin were sacrificed at the time points of 2X4N6X24 and 72h (n = 12) after injection.2. Sample collection and tissue preparationEach group of LPS and Gin as well as control rats were anaesthetized at each time point with 1 % chloral hydroate injected intraperitoneally at the dosage of 1 ml/kg. The rat was sacrificed, opened thorax and isolated heart, then stored respectively according to the following methods:2. 1 All eight samples of mixed blood were collected in vessel,then spun in centrifuge, absorb the serum and put it in minus 30 °C ice - box in order to ra-dioimmunology analysis.2. 2 Eight samples of heart were isolated in Eppendorf vessel without Rnase, then put in liquid nitrogen and stored at minus 80^ freezer for measur-ment of RT - PCR.2. 3 Three samples of heart were fixed in 4% formaldehydum polymerisation for Immunohistochemistry and In situ hybridization.2.4 One heart (lmm3 myocardium) of left ventricular is fixed in put in 2. 5% glutaral for transmission electron microscope.3. Experiment Methods3.1 The changes of the heart pathologyGross exam; After the opening of thorax and peritoneal cavity, the lung, the heart, the liver, small intestine, stomach and kidney were check for the sign of bleeding. Microscope exam; the fixed tissue was dehydrated, embedded inwax, sectioned, stained with HE staining method. Transmission electron microscope exam: the fixed tissue is washed, fixed, dehydrated by ethanol concentration gradient, immersed and embedded in Epon 812, ultra thin sectioned, strained with acetic uranium and lead citrate. The section is observed under the ? transmission electron microscope.3. 2The measurement of serum IL - 8 and IL - 10 concentrations: Radioim-munology.3.3 The detection at protein level of myocardial TNF - a: Immunohisto-chemistry.3.4 The measurement of ERK -2 and p38 expressions in the cardiac myocyte.3.5 The detection at mRNA level of ERK -2: In situ hybridization. The detection at protein level of p38: Immunohistochemistry.3. 6 The measurement of a - SA and PEC AM — 1 expressions in the cardiac myocyteThe detection at mRNA level of a - SA: RT - PCR. The detection at protein level of a - SA: Immunohistochemistry. The detection at protein level of PEC AM - 1; Immunohistochemistry.3. 7 The measurement of MMP -3 and TIMP -3 expressions in the cardiac myocyteThe detection at mRNA level of MMP - 3: In situ hybridization. The detection at protein level of MMP - 3: Immunohistochemistry. The detection at mRNA level of TIMP - 3 : RT - PCR. The detection at protein level of TIMP - 3: Immunohistochemistry.Results1. The pathological findings of the heart in endotoxemia rat At the organ level: Paleness can be seen on the surface of the heart. At the microscope level: The arrangement of myocardium is sparsity and disorder at 6 to 24 hour of endotoxemia. While in glutamine group, this change is not significant. At the electron microscope level: The arrange of cardiac myocyte is in or-der, the Z line in the cardiac myocytes were clear in 0 hour control group; while in LPS group, its arrangement is sparsity and disorder, some fibres have lost, the ridge of mitochondrion disappear and most mitochondria within cardiac myocytes were vacuole denaturalization and appeared necrosis, the Z line in myocar-dial fibers were disclear, the nucleus chromatin was condense at the side of nuclear at 6 to 24 hour of endotoxemia. While in glutamine group, the myocardial injury was slightly than that in LPS group.2. The dynamic changes of serum IL - 8 and IL - 10 concentrations.The concentration of serum IL - 8 is obviously higher than other points at 2 hour of endotoxemia ( P < 0. 01) , then gradually decline. While in glutamine group, the concentrations at every point time are lower than those in LPS group, and the peak was delayed to 6 hour (P < 0.01). The concentrations of serum IL — 10 are decline in LPS group, the lowest point is at 4 hour of endotoxemia (P <0.01) , then gradually ascendant, till72 hour, it has recovered; while in glutamine group, the concentrations at every point time are higher than that in LPS group ( P < 0. 01) , and recovery is at 24 hour, which is earlier than in LPS group.3. The dynamic changes of TNF - a at protein level in myocardium with endotoxemiaIt can be seen that the positive expression of TNF - a is in the plasma of cardiac myocyte by Immunohistochemistry. The expressions increased at 2 hour and 24 hour and peaked at 24 hour of endotoxemia, which is significant higher than that in control (P <0.01) ; while in glutamine group, the expressions of TNF - a increased only at 6 hour of endotoxemia, which is significant higher than that in control but significant lower than that in LPS group ( P <0.01).4. The expression of ERK -2 at mRNA level in myocardium with endotoxemia:It is shown that the positive expression of ERK - 2 mRNA is mainly in the plasma of cardiac myocyte by In situ hybridization, the expressions increased at every point time, the peak is at 6 hour of endotoxemia, which is significant higher than that in control (P <0.01) ; while in glutamine group, this expression increased mildly, it was significant lower than that in LPS group (P <0.01).5. The expression of p38 at protein level in myocardium with endotoxemia It is shown that the positive expression of p38 is mainly in the plasma ofcardiac myocyte by Immunohistochemistry, the expressions increased at every point time, the peak is at 24 hour of endotoxemia, which is significant higher than that in control (P <0. 01) ; while in glutamine group, the expressions of p38 increased mildly, which is significant higher than that in control but significant lower than that in LPS group (P < 0.01).6. The dynamic variation of a - SA at mRNA level in myocardium with endotoxemia :It is shown that the most condensed positive expression of a - SA mRNA by RT - PCR in control, while it was significantly weaken at every point time in endotoxemia , the lowest point is at 6 to 24 hour, even at 72 hour it was still significantly weaken than that in control (P <0.01). In glutamine group, this expression was significantly weaken at every point time than that in control but significant stranger than that in LPS group, and the lowest point delayed to 24 hour (P <0.05 orP<0.01). The expressions of myocardial a - SA at protein level in myocardium with endotoxemia: It is shown that positive expression of a — SA by Immunohistochemistry in the plasma of myocardium in control, while it was significantly weaken in LPS group at every point time of endotoxemia than that in control, the lowest point is at 24 hour, the trend of variation is parallel to the changes of a - SA mRNA (P < 0.01). In glutamine group, the changes of a -SA at protein level is also parallel to the expression of a - SA mRNA (P <0. 01) and is milder than that in LPS group ( P <0.01) , till 72 hour, it has recovered nearly to control (P > 0.05).7. The expression of PEC AM - 1 at protein level in the endothelial cells of myocardial vessel with endotoxemia:It can be seen that the plasma of myocardial vessel endothelial cells remained unstained at any point time by Immunohistochemistry.8. The dynamic variations of MMP -3 at mRNA and protein levels in the cardiac myocyte with endotoxemia:It is shown that positive expression of MMP - 3 mRNA by In situ hybridiza-significant stronger at every point time in LPS group than that in control (P <0. 01), the peak is at 6 to 24 hour, there is no significant difference (P >0.05) , till 72 hour it was still significant stronger than that in control (P < 0. 01). In glutamine group, the expression of MMP - 3 mRNA was significantly weaken at every point time than that in LPS group, the peak delayed to 24 hour (P <0. 01). Positive expression of MMP - 3 can be seen around cardiac myocyte and vessel with endotoxemia by Immunohistochemistry, which significant thicker at every point time than that in control ( P <0.01) , the peak is at 24 hour, till 72 hour it was still significant thicker than that in control group ( P < 0. 01). In glutamine group, the trend of MMP - 3 expression is similar to the change of MMP - 3 mRNA expression, it is significant milder at every point time than that in LPS group, and the peak is at 24 hour (P < 0.01).9. The dynamic variation of TIMP - 3 at mRNA and protein levels in the myocardium with endotoxemia.It can be seen that the thickest expression of TIMP - 3 mRNA by RT - PCR in control, it is decline in endotoxemia, the lowest point is at 6 hour, there is significant difference (P < 0. 01) , till 72 hour it has recovered partly but still significantly weaken than that in control (P<0.01). In glutamine group, the expression of TIMP - 3 mRNA was significantly weaken than that in control, but stronger than that in LPS group, and the lowest point is at 24 hour (P <0.01). The expression of myocardial TIMP - 3 at protein level in endotoxemia: it can be seen that positive expression of TIMP - 3 by Immunohistochemistry at the surrounding of cardiac myocyte and vessel in control, but it was weakened in endotoxemia than that in control ( P <0.01) , the lowest point is at 6 to 24 hour, the variation of TIMP - 3 express is similar to its change of TIMP - 3 mRNA, till 72 hour it was still significant lower than that in control ( P <0.01). In Gin group, the variation of TIMP - 3 expression is also similar to its change of TIMP -3mRNA, it is significant stronger at every point time than that in LPS group (P...