摘要
由于肝脏功能的复杂性,肝损伤机理也呈现出复杂性；不同原因造成的肝损伤及具体机理不同。所以对于每种原因引起的肝损伤,都需要进行大量详尽的研究才可能阐明其机制。
我们前期研究用DMN造成肝损伤模型,其病理损害特点实际上与重型肝炎一致。表现为肝组织大片出血坏死灶密集地遍布于肝组织,大部分出血坏死灶内肝细胞结构完全消失。
本文中,我们研究了DMN诱导的肝损伤在病理损害较为严重的造模后48h时的肝组织基因表达谱变化,发现了该模型肝损伤中变化显著的基因；然后观察了中药复方肝毒清对这些变化显著的基因mRNA量的影响。1DMN诱导的小鼠肝损伤肝组织基因表达谱的变化
昆明小鼠20只,体重18±2g,雄性。购买于广州中医药大学实验动物中心。随机取出10只小鼠作为空白对照组,腹腔注射生理盐水,10ml/kg；其余10只小鼠均腹腔注射DMN造模,造模剂量为15mgDMN/kg。至造模48h时分别取肝组织,每只小鼠取50mg,投入Trizol中,于-70℃冷冻保存。与上海康成生物科技有限公司联系好后送交该公司业务代表进行基因表达谱分析。
实验结果显示,在小鼠44170个基因中,共发现有2331个基因表达的差异在2倍以上,其中1131个基因上调,1200个基因下调。而差异在6倍以上的基因有99个,其中53个上调,46个下调。这些6倍以上表达的基因涉及21个信号通路。其中有可能与肝组织损害相关的信号通路有12个,包括PPAR信号通路、脂肪酸代谢通路、p53信号通路、药物代谢通路、花生四烯酸代谢通路、甘油脂代谢通路、外源化学物质p450代谢通路、细胞外基质受体相互作用通路、TGF-β信号通路、细胞周期信号通路、MAPK信号通路。
2DMN致小鼠肝损伤肝组织基因表达谱中显著改变的基因的验证
昆明小鼠24只,体重18±2g,雄性。随机取出12只小鼠作为空白对照组,腹腔注射生理盐水,10ml/kg；其余12只小鼠均腹腔注射DMN造模,造模剂量为15mgDMN/kg。造模后48h处死所有小鼠,留取肝组织标本,进行定量PCR检测,主要验证前面分析到的可能与肝组织严重损害有关的信号通路中显著改变超过6倍以上的基因表达15个。在检测的15种mRNA中,所有RNA均定量PCR检测结果与基因表达谱检测结果基本一致。
3中药复方对DMN诱导的小鼠肝损伤相关显著升高的mRNA表达的影响
昆明小鼠60只,分为空白对照组、模型对照组、中药高剂量组(20g生药/kg.次)、中药中剂量组(10g/kg.次)、中药低剂量组(5g/kg.次)、联苯双酯组(150mg/kg.次),模型组灌胃生理盐水100ml/kg.次。每组10只。除空白对照组外均腹腔注射DMN,15ul/kg,同时开始分组给药,每12h给药一次。48h后采集血清冻存于-70℃冰箱中,肝组织标本留取50mg左右投入含1mlTrizol的离心管中。本实验结果显示,复方肝毒清对DMN诱导的小鼠肝损伤升高的血清谷丙转氨酶具有明显的降低作用,这又一次显示了复方肝毒清具有确切的护肝作用；本实验结果还显示,在所检测的15个基因的mRNA中,复方肝毒清具有明显影响的有9个,它们分别是：CPT1C、CYP7A1、CDKN1A、THBS1、GSTA1、FMO3、MGLL、TGFB3、HSPB1。
这些实验结果说明,肝毒清对DMN诱导的小鼠肝损伤肝组织的作用涉及多个肝损害相关的信号途径,但肝毒清仍然对一部分肝损害相关的基因mRNA未见明显影响。
全文结论：
1DMN诱导的小鼠肝损伤模型肝组织基因表达谱显示有2331个基因表达的差异在2倍以上,其中1131个基因上调,1200个基因下调。而差异在6倍以上的基因有99个,其中53个上调,46个下调。这些6倍以上差异表达的基因涉及21个信号通路。其中可能与肝损害关系密切的为涉及12个信号通路的15条基因。
2复方肝毒清对所检测的15个基因的mRNA中的9个有显著影响,它们分别是：CPT1C、CYP7A1、CDKN1A、THBS1、GSTA1、FMO3、MGLL、TGFB3、HSPB1。显示了复方肝毒清改善DMN诱导的小鼠肝损害机制涉及多条信号通路的多条基因。
Because of the complexity of liver organ, the mechanism of liver injury also complex. Different kind of liver injury showed different mechanisms. So for every kind of liver injury, the mechanism need plenty of research work.
We had use DMN injected to the mice to modeling the severe hepatitis. The liver pathological changes are similar to human severe hepatitis. The main liver pathological changes include focal zone of massive bleeding and necrosis closely distributed in liver tissue. Most of liver tissue structure within the focal zone of massive bleeding and necrosis are disappeared.
In this research, we analyzed the changes of gene expression profile of DMN induced mouse severe hepatitis model 48h after DMN modeling. The expression significantly changed genes were identified in this model. Then the effects of Chinese herb complex Gan Du Qaing to the significantly changed genes were evaluated. 1The changes of gene expression profile of DMN induced mouse severe hepatitis model
20 Kunmin mice, body weight 18±2g, male, bought form centers of experimental animal of Guangzhou University of Chinese Medicine.10 mice were randomly taken as blank control group which were peritoneally injected with saline in dose of 10ml/kg. Another 10 mice were peritoneally injected with DMN in the dose of 15mg/kg for modeling.50 mg liver tissues of every mouse was harvested at 48h after DMN injection and put into lml of Trizol reagent and then settled in-70℃refrigerator. The liver tissues then send to ShangHai KangChen Bio-tech Inc to conduct analysis of gene expression profile.
The results showed that 2331 kind of genes were identified to be changed for 2 more times in 44170 kinds of tested genes. There are 1331 genes up-regulated and 1200 genes down-regulated. There are 99 genes changed for 6 more times include 53 genes up-regulation and 46 genes down-regulation. These changed genes involved in 21 signaling pathways. There are 12 signaling pathways may related to liver injury which include PPAR, Fatty acid metabolism, p53 signaling, Drug metabolism, Arachidonic acid metabolism, Glycerolipid metabolism, Metabolism of xenobiotics by cytochrome P450, ECM-receptor interaction, TGF-beta signaling, Cell cycle and MAPK signaling pathways. 2The verification of significant changed expression of genes in the gene expression profile of DMN induced mouse severe hepatitis model
24 Kunmin mice, body weight 18±2g, male.12 mice were randomly taken as blank control group which were peritoneally injected with saline in dose of 10ml/kg. Another 12 mice were peritoneally injected with DMN in the dose of 15mg/kg for modeling. All of the mice were sacrificed and liver samples were collected for quantitative PCR testing at the time point of 48h after modeling. The verification experiments concentrated on the 6 more times changed 15 genes which closely related to liver injury signaling pathway. The results showed that all of the verified genes are meet with the results of the gene expression profile.
3The effect of Chinese herb complex to the genes closely related to liver injury signaling pathway in DMN induced mouse severe hepatitis model.
60 kunming mice were randomly divided into blank control group, model control group, Chinese medicine High (20g/kg.time), middle(10g/kg.time), low dose group(5g/kg.time) and Bifendate control group (150mg/kg.time). There are 10 mice in every group. All of the mice except blank group were injected with DMN in the dose of 15ul/kg for a single dose and start drug administration simultaneously. The drugs were administrated one time every 12 hours. After 48h of drug administration, The mice were sacrificed and serum and liver tissue were collected. Serum was put into-70℃refrigerator. The liver tissue samples were put into 1ml of Trizol reagent for mRNA testing. The results showed that the Chinese herb complex Gan Du Qing can significantly decrease the level of glutamate-pyruvate transaminase in serum which verified the liver protection activity of Gan Du Qing again. The results also showed that the Chinese herb complex Gan Du Qing can significantly affect the mRNA levels of 9 genes in 15 tested genes. The 9 genes are CPT1C、CYP7A1、CDKN1A、THBS1、GSTA1、FMO3、MGLL、TGFB3、HSPB1.
These results revealed that the mechanisms of effects of the Chinese herb complex Gan Du Qing to the DMN induced mice severe hepatitis involved in multi-pathways of liver injury. But the Gan Du Qing doesn't affect the mRNA levels of some related genes of liver injury.
Conclusion:
1. The results of gene expression profile testing showed that there are 2331 genes were identified to be changed for 2 more times in 44170 kinds of tested genes. There are 1331 genes up-regulated and 1200 genes down-regulated. There are 99 genes changed for 6 more times include 53 genes up-regulation and 46 genes down-regulation. These changed genes involved in 21 signaling pathways.15 genes are closely related to liver injury signaling pathway.
2. The Chinese herb complex Gan Du Qing can significantly affect the mRNA levels of 9 genes in 15 tested genes. The 9 genes are CPT1C、CYP7A1、CDKN1A、THBS1、GSTA1、FMO3、MGLL、TGFB3、HSPB1. These results revealed that the mechanisms of effects of the Chinese herb complex Gan Du Qing to the DMN induced mice severe hepatitis involved in multi-pathways of liver injury.
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