谷氨酰胺对内毒素血症幼年大鼠心肌肌动蛋白及其mRNA表达的影响

Effects of glutamine on alpha-sarcomeric actin and its mRNA expression of myocardium in rats with endotoxemia

目的通过观察谷氨酰胺(glutamine, Gln)对内毒素血症幼年大鼠心肌肌动蛋白(alpha sarcomeric actin,α-SA)及其mRNA表达的影响,探讨Gln在大鼠内毒素血症中对心肌收缩功能的保护作用.方法选健康18日龄Wistar大鼠121只,按腹腔注射药物不同分为:0 h对照组(Ctrl:生理盐水,n=11),内毒素组(LPS, n=55)、谷氨酰胺组(Gln, n=55);后两组又分为2、4、6、24及72 h共5个时点,每个时点11只,在各指定时点分别将大鼠断头分离心脏,8只用逆转录-聚合酶链反应(RT-PCR)方法测定α-SA mRNA表达,另3只左心室肌用甲醛固定,制成石蜡切片,用免疫组化方法测定α-SA蛋白质表达.结果 (1)内毒素血症大鼠心肌α-SA及其 mRNA与对照组相比较均有显著下降(P<0.01),LPS组在6～24 h降至最低;而 Gln组则延至24 h为最低,72 h Gln组α-SA已与对照组差异不明显( P>0.05).(2)LPS与Gln组同一时点相比较,Gln组α-SA各时点均高于LPS组,而α-SA mRNA在4～ 72 h高于LPS组,早期相差不明显(P>0.05).结论 (1)α-SA及其mRNA表达在内毒素血症明显受到抑制,收缩蛋白合成减少,而非破坏增加,心肌收缩功能受损.(2)谷氨酰胺可以减轻LPS对α-SA及其mRNA表达的抑制作用,而且恢复较快.

Objective Endotoxemia is a serious syndrome resulting in multi-organ failure. Once it happens, the penetration of small intestine epithelium increases, body liquid losses, then effective circulating blood decreases and serious metabolic acidosis, serious hypotension, systolic failure, and even shock may occur. In this pathological process, endotoxin, tumor necrosis alpha and systolic dysfunction play important roles. Nowadays, many studies have been done to resolve the systolic dysfunction, but too much attention had been paid to the followings： the depressions of myocardium caused by tumor necrosis alpha, other inflammatory factors, endotoxin and metabolic acidosis; the disturbance of blood vessel-nerve regulations; nitric oxide （NO）/inducible nitric oxide synthase （iNOS） over-synthesis and the decreased density of β-receptors in the myocardium and/or their activities. Little attention has been paid to the relationship between alpha sarcmeric actin （α-SA） and systolic dysfunction during endotoxemia. Glntamine （Gin） can be metabolized into glutathione, an eliminator of free radical. It has been used in preventing myocardial damage from reperfusion. This study aimed to observe the dynamic changes of α-SA and mRNA expressions in rats with endotoxemia and examine the effects of Gin on them. Methods Classical rat model of endotoxemia was established by intraperitoneal injection of LPS （4 mg/kg, Escherichia coli O55：B5, Sigma）. 121 Wistar 18-day-rats were divided into three groups randomly, （1） 0 h control group （normal saline： 1 ml/kg, n=11）. （2） LPS group （LPS： 4 mg/kg, n=55）. （3） Gin group （LPS： 4 mg/kg and immediately 13.64% ; Gin： 1 ml/kg, Fresenus, n =55）, Furthermore, LPS and Gin groups were divided into 2,4,6, 24 and 72 h time points （n = 11 ）. Each time point of LPS and Gin as well as control rats were anaesthetized at each time point with 1% chloral hydrate injected intraperitoneally at the dosage of 1 ml/kg. Then rats were sacrificed at appoint time, and the hearts were isolated. Eight of them were put in 76℃ liquid nitrogen and then frozen in minute 80℃ icebox in order to measure the expression of α-SA mRNA by RTPCR. Three of them were fixed in 4% formaldehydum polymerisatum for 12 to 16 h, then the expression of α-SA was detected by immunohistochemistry. Results （ 1 ） Compared to 0 h, the expressions of α-SA and mRNA in LPS group were significantly depressed （P 〈0.01 ）. In LPS group, the lowest was at 6-24 h,while in Gin group, it was postponed to 24 h. At 72 h, there was no difference in expressions of α-SA between Gin and 0 h group （ P 〉 0. 05 ）. （2） Comparing at same time point, the expressions of α-SA were significant higher in Gin group than those in LPS group, while the expressions of α-SA mRNA in Gin group were high at 4 - 72 h. There was, however, no significant difference at early phase（ P 〉 0. 05）. Conclusion α-SA and its mRNA expression were depressed in LPS-induced endotoxemia, especially from 6 to 24 h. It could damage the systolic function, α-SA decrease in endotoxemia was due to the inhibited synthesis other than the promoted degradation. Glutamine could inhibit the effects of LPS on both α-SA and its mRNA expressions.