基于CCL4肝损伤大鼠模型的护肝片降ALT作用机制研究

发布时间：2018-03-02 05:27

  本文关键词： 护肝片 CCL_4 ALT Ca~(2+) 线粒体　出处：《黑龙江中医药大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：研究护肝片降低CCL4肝损伤模型大鼠血清丙氨酸氨基转移酶活性的作用机制 方法：将Wistar大鼠适应性饲养7天后,随机分为空白对照组、模型对照组、护肝片治疗组,每组.8只。各组大鼠正常饮食,光照时间7:00am-7:00pm。空白对照组和模型对照组灌胃给予适宜体积的生理盐水；护肝片治疗组按1700mg/kg灌胃给予护肝片混悬液,1次/天,连续9天。末次给药后1h,模型组及药物治疗组予大鼠腹腔注射20%CCl4橄榄油溶液(0.2ml/100g)；空白对照组腹腔注射等体积的生理盐水。造模24h后采集观察指标。比色法测定血清及肝组织ALT活性；Western blotting测定肝组织ALT-1、ALT-2相对含量；RT-PCR测定肝细胞ALT mRNA表达情况；紫外-可见分光光度法测定线粒体肿胀敏感性及线粒体Na+-K+ATPase、Ca2+—Mg2+ATPase活性荧光分光光度法测定肝细胞线粒体膜电位、肝细胞内及肝细胞内游离钙离子浓度,酶联免疫法测定线粒体渗透性转换孔(MPTP)开放程度。 结果： 1.护肝片对CCL4肝损伤模型大鼠ALT活性及含量的影响：与空白组比较,模型组大鼠血清及肝组织ALT活性显著升高(P0.01)；与模型组比较,护肝片治疗组大鼠血清及肝组织ALT活性水平显著降低(P0.01)。与空白组比较,模型组肝脏ALT-1含量升高0.47倍,ALT-2升高0.83倍.药物组ALT-1含量升高0.42倍,ALT-2升高0.54倍。结合肝组织ALT活性测定结果(与空白组比较,模型组肝ALT活性升高1.72倍,药物组升高1.43倍)可以看出,尽管ALT-1、ALT-2含量变化幅度要小于活性的,但两者变化趋势一致。 2.护肝片对肝组织ALT-1、ALT-2mRNA表达的影响研究结果：模型组肝组织ALT-1mRNA表达通量为空白组的0.42；药物组为0.52。药物组ALT-2mRNA表达通量为空白组的0.48；药物组为0.47。 3.护肝片对CCL4肝损伤模型大鼠肝细胞线粒体功能及细胞内游离钙离子浓度的影响研究结果：与空白组比较,模型组大鼠肝细胞内游离钙离子浓度、肝组织MPTP开放程度显著增加,膜电位、ATPase活性及对钙离子诱导肿胀敏感性的均显著降低；与模型组比较,药物组大鼠肝细胞内游离钙离子浓度及肝组织MPTP开放程度显著降低；膜电位、ATPase活性及对钙离子诱导肿胀敏感性的均显著升高。 4.护肝片对CCL4肝损伤模型大鼠血清ALT代谢的影响研究结果：空白组大鼠血清在0～84h内呈轻微上升趋势,但仍处于正常范围内；模型组及模型组大鼠血清在0～84h内呈下降趋势,但二者下降趋势相近。药物组ALT活性下降一半时间(t1/2)位于72～84h之间,而模型组ALT活性尚未降低至50%。说明药物组ALT活性下降速率高于模型组 结论： 1、护肝片具有降低CCL4肝损伤模型血清及肝组织ALT活性的作用；ALT-1、ALT-2含量升高是肝组织ALT活性升高的物质基础；护肝片可减少损伤肝组织ALT-1、ALT-2含量的升高。 2、CCL4肝损伤模型大鼠肝组织ALT-1、ALT-2mRNA表达降低,护肝片使损伤肝组织的ALT-1mRNA表达有所恢复。 3、初步确定护肝片降低血清ALT活性的机制为保护线粒体功能,进而减少损伤肝细胞细胞内钙超载现象,保护肝细胞。 4、护肝片能促进CCL。肝损伤模型大鼠血清中ALT的代谢。
[Abstract]:Objective: To study the mechanism of liver protection tablets to reduce the activity of serum alanine aminotransferase in rat model of CCL4 liver injury
Methods: Wistar rats were fed for 7 days, were randomly divided into control group, model control group, Hugan tablets in the treatment group, each group of.8 rats. The rats with normal diet, light time 7:00am-7:00pm. blank control group and model control group were lavaged with appropriate volume of saline; huganpian treatment group by 1700mg/kg perfusion Administration of Hugan tablets suspension, 1 times / day for 9 consecutive days. After the last administration of 1H, the model group and the treatment group was injected intraperitoneally to rats 20%CCl4 olive oil solution (0.2ml/100g); the control group were injected with saline. After 24h model acquisition observed. The determination of ALT the activity of serum and liver tissue Western blotting colorimetric method; Determination of liver tissue ALT-1, ALT-2 relative content; Determination of RT-PCR liver cells ALT mRNA expression; UV Vis spectrophotometric method for the determination of the sensitivity of mitochondrial swelling and mitochondrial Na+-K+ATPase, Ca2+ Mg2+ATPase activity spectrofluorometry was used to measure the mitochondrial membrane potential of liver cells, the concentration of free calcium in hepatocytes and hepatocytes, and the permeability of mitochondrial permeability transition pore (MPTP) was measured by enzyme-linked immunosorbent assay (ELISA).
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