核因子-κB与内毒素血症致幼鼠肝损伤的关系及谷氨酰胺的干预作用研究

发布时间：2018-04-21 08:39

本文选题：内毒素血症 + 肝损伤　；参考：《广西医科大学》2009年硕士论文

【摘要】： 目的:通过建立幼年大鼠内毒素血症肝损伤模型,检测肝脏核因子-κB(NF-κB)的活化水平及肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的含量,探讨NF-κB活化与肝损伤的关系,并给予谷氨酰胺(Gln)干预,探索Gln对肝脏是否具有保护作用。方法:健康18日龄wistar大鼠72只,随机分为正常对照组(NS组,n=8)、内毒素组(LPS组,n=32)和谷氨酰胺干预组(Gln组,n=32)。给予LPS组腹腔注射内毒素(5mg/kg),NS组腹腔注射等容量生理盐水,Gln组腹腔注射内毒素(5mg/kg),同时给予谷氨酰胺(2g/kg)灌胃。其中LPS组和Gln组按注射后2h、6h、12h、24h 4个时点分为四个亚组,每个亚组8只。分别于各时点活杀动物取血,比色法测定丙氨酸转氨酶(ALT)活性;取肝脏标本制备石蜡切片和组织匀浆,光镜和电镜下观察肝组织的病理改变情况;免疫组化染色法检测肝脏NF-κB的活化水平;酶联免疫吸附测定(ELISA)法测定肝组织匀浆TNF-α和IL-6的含量,并对结果进行统计学分析。结果:(1)LPS组血清ALT水平于2h开始升高,12h达峰值,其中在6h、12h、24h ALT水平比NS组显著升高(p＜0.05),Gln组ALT水平也于12h达峰值,比NS组显著升高(p＜0.05),在12h、24h均比LPS组显著降低(p＜0.05)。(2)LPS组肝组织NF-κBp65蛋白的表达以2h为最多,随后逐渐下降,24h又有所升高,各时点的核阳性细胞百分比均比NS组显著升高(p＜0.05),Gin组变化趋势与LPS组相仿,各时间点均显著高于NS组(p＜0.05),其中在2h、6h均比LPS组阳性表达显著降低(p＜0.05)。(3)LPS组与Gln组肝组织的TNF-α、IL-6含量均于6h达到峰值,与NS组相比有显著性差异(p＜0.05)。其中LPS组6h、12h的TNF-α含量及各时点的IL-6含量均比NS组显著升高(p＜0.05);Gin组6h的TNF-α含量及6h、24h的IL-6含量均比NS组显著升高(p＜0.05),12h的TNF-α含量及2h、12h、24h的IL-6含量均比LPS组显著降低(p＜0.05)。(4)光镜下观察LPS组肝组织呈细胞空泡样变,炎性细胞浸润、片状出血坏死、肝小叶结构破坏等病理改变,以6h组的损伤最为明显;电镜下观察LPS组肝细胞呈空泡变性,线粒体肿胀,线粒体嵴数量减少,内质网扩张,染色质边集。Gln组上述病理改变明显减轻。结论:(1)经LPS作用后,大鼠血清ALT升高,肝组织病理改变明显,说明成功建立了内毒素导致肝损伤的动物模型。(2)NF-κB的活化参与了内毒素血症肝损伤的病理过程,其机制可能是通过介导细胞因子TNF-α和IL-6等发挥作用使肝脏发生炎性损伤。(3)谷氨酰胺可以减轻LPS所致的肝损伤,其机制可能是通过抑制NF-κB的活化而下调TNF-α和IL-6的表达。
[Abstract]:Objective: to study the relationship between the activation of NF- 魏 B and the levels of tumor necrosis factor- 伪 (TNF- 伪) and interleukin-6 (IL-6) in the liver injury model of young rats with endotoxemia, and to explore the relationship between the activation of NF- 魏 B and liver injury. Glutamine glutamine (Glutamine) was given to investigate whether Gln has protective effect on liver. Methods: 72 healthy 18-day-old wistar rats were randomly divided into normal control group (n = 8), endotoxin group (n = 32) and glutamine intervention group (n = 32). The LPS group was given intraperitoneal injection of endotoxin 5 mg / kg and the NS group was given intraperitoneal injection of endotoxin 5 mg / kg and glutamine 2 mg / kg respectively. The LPS group and the Gln group were divided into four subgroups according to the time points of 2 h, 6 h, 12 h and 24 h after injection, with 8 rats in each subgroup. The activity of alanine aminotransferase (alt) was determined by colorimetric method, paraffin sections and tissue homogenates were prepared from liver samples, and the pathological changes of liver tissues were observed under light and electron microscopy. The activation level of NF- 魏 B in liver was detected by immunohistochemical staining, and the contents of TNF- 伪 and IL-6 in liver homogenate were determined by Elisa, and the results were analyzed statistically. Results the serum ALT level began to increase at 2 h and reached the peak at 12 h, and the level of ALT reached the peak at 12 h after 6 h of lipopolysaccharide concentration in the control group compared with that in NS group (P < 0.05). Compared with NS group, the expression of NF- 魏 Bp65 in liver tissue was significantly decreased at 12 h and 24 h compared with that in LPS group. The expression of NF- 魏 Bp65 in liver tissue was the highest at 2 h, then decreased gradually at 24 h, and then increased at 24 h. The percentage of nuclear positive cells at each time point was significantly higher than that in NS group. The change trend of Gin group was similar to that of LPS group. The positive expression of TNF- 伪 and IL-6 in liver tissue of 0.05).(3)LPS group and Gln group reached the peak at 6 h after 2 h, and there was significant difference compared with NS group (P < 0.05). The content of TNF- 伪 in LPS group was significantly higher than that in NS group at 6h and 12h, and the content of TNF- 伪 at 6h and IL-6 at 6h in LPS group was significantly higher than that in NS group. The content of TNF- 伪 in LPS group was significantly higher than that in NS group at 6h or 12h, and the content of IL-6 at 2h 12h was significantly lower than that in LPS group (P < 0.05U. 4) under light microscope. In LPS group, the liver tissue showed vacuolar degeneration. The pathological changes, such as inflammatory cell infiltration, flake hemorrhage and necrosis, damage of hepatic lobule structure, were most obvious in the 6h group, the liver cells in LPS group showed vacuolar degeneration, mitochondria swelling, mitochondrial cristae decreased, endoplasmic reticulum dilated, and so on. The above pathological changes in chromatin edge set. GLN group were obviously alleviated. Conclusion (1) after treated with LPS, the level of serum ALT in rats was increased, and the pathological changes of liver tissue were obvious, which indicated that the activation of NF- 魏 B in lipopolysaccharide induced liver injury was involved in the pathological process of endotoxemia liver injury. The mechanism may be that glutamine can attenuate liver injury induced by LPS by mediating cytokines such as TNF- 伪 and IL-6. The mechanism may be to down-regulate the expression of TNF- 伪 and IL-6 by inhibiting the activation of NF- 魏 B.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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