谷氨酰胺对脓毒症幼年大鼠心肌基质金属蛋白酶及其组织抑制因子的影响

Effects of glutamine on matrix metalioproteinase-3 and tissue inhibitor of metalloproteinase-3 expressions in myocardium of rats with sepsis

目的探讨谷氨酰胺对脓毒症大鼠心肌胶原的保护机制。方法选健康18日龄Wistar大鼠121只,每个时间点随机取11只按腹腔注射药物不同分为：(1)生理盐水对照组(0h)；(2)内毒素(LPS)；(3)谷氨酰胺治疗(Gln)组。后两组又分为2、4、6、24及72 h时点。在各时点分离心脏,8只用逆转录-聚合酶链反应(RT-PCR)方法测定基质金属蛋白酶组织抑制因子3(TIMP-3) mRNA表达,另3只用于石蜡切片,用免疫组化方法测定金属心肌基质蛋白酶3(MMP-3)及其TIMP-3,以及用原位杂交方法测定MMP-3 mRNA表达。结果(1)LPS组各时点MMP-3及其mRNA表达较0h组增强,在6 h达高峰,到72h虽然有所下降仍高于对照组,P＜0．01；Gln组各时点MMP-3 mRNA及其蛋白质表达也较0 h组增强,但较LPS组各时点为弱,最高点在24 h,P＜0．01；(2)LPS组各时点TIMP-3以及mRNA表达较0 h组明显减弱,在6 h最低,P＜0．01；Gln组各时点TIMP-3以及mRNA表达也较0 h组减弱,但较LPS组为强,最低点在24 h,P＜0．01。(3)脓毒症组比Gln组心脏,超微结构改变明显。结论(1)在脓毒症时MMP-3及其mRNA表达增强,而TIMP-3及其mRNA表达受抑制。(2)谷氨酰胺可以减轻这种影响,而且使这种损伤作用时间向后推迟。

Objective The underlying mechanisms for cardiac dysfunction in sepsis include the inhibitory effect of endotoxin and inflammatory factors on myocardium and the decrease in cardiac myocardial cells in number. However, whether there is ventricular remodeling resulted from the abnormalities of extracellular collagen metabolism and whether glutamine （Gln ） can protect myocardium from LPS-induced damage as in reperfusion are unknown. The aim of the present study was to examine the effects of Gln on the expressions of matrix metalloproteinase-3 （ MMP-3 ）, tissue inhibitor of metalloproteinase-3 （ TIMP-3 ） and their mRNA in myocardium of rats with sepsis. Methods Classical rat model of sepsis was established by intraperitoneal injection of lipopolysaccharide （LPS） （4 mg/kg, from Escherichia coli O55 ：B5, Sigma ）. from 121 Wistar rats aged 18 days were divided into three groups randomly, 0 h control group （ normal saline： 1 ml/kg, n = 11 ）, LPS group （ LPS ： 4 mg/kg, n = 55 ） and Gln group （ LPS： 4 mg/kg and immediately 13. 64% glutamine 1 ml/kg, Fresenus, n =55）. Furthermore, LPS and Gin groups were examined at 2 h, 4 h, 6 h, 24 h and 72 h time points （ n = 11 ）. On each time point, rats of LPS and Gln groups as well as control group were anesthetized with 1% chloral hydrate injected intraperitoneally at a dosage of 1 ml/ kg. Then, rats were sacrificed, and the hearts were isolated. Eight of them were frozen at minus 80℃ to measure the expression of TIMP-3 mRNA by using RT-PCR. The expressions of MMP-3 and TIMP-3 were observed with immunohistrochemistry and the expression of MMP-3 mRNA was observed by using in situ hybridizatiorr Results （ 1 ） Compared to 0 h, the mRNA expressions of MMP-3 and TIMP-3 in LPS group significantly increased （P 〈0. 01 ） with the peak at 6--24 h. While, in Gln group, they were significantly higher than those in controls but significantly lower than those in LPS group with the peak at 24 h （ P 〈 0.01 ）. Even at 72 h, they were still higher than those at 0 h （P 〈0. 05 and P 〈0.01 ）. （2） Compared to 0 h, the expressions of MMP-3 and TIMP-3 in LPS group were significantly lower at any other time point with the lowest at 6 h （P 〈 0. 01 ）. In Gln group, these expressions were also significantly lower than those in controls, but significantly higher than those in LPS group with the lowest being postponed to 24 h （P 〈 0. 01 ）. （3） The ultra structure changed obviously. Z line was unclear and the ridge of mitochondrion disappeared. While, in Gin group, the myocardial injury was slight compared to that in LPS group. Conclusions MMP-3 mRNA expression was increased and TIMP-3 mRNA expression was depressed in LPS-induced sepsis. Myocardial extracelluar matrix was damaged in sepsis. Glutamine might decrease the effects of LPS on MMP-3 and TIMP-3 expressions and postpone the time of myocardial matrix injury.