扶正化瘀方干预肝硬化大鼠血浆蛋白质组的初步研究

Effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis:preliminary experimental study with rats

目的利用蛋白质组技术比较 CCl_4诱导肝硬化大鼠血浆蛋白质变化并研究扶正化瘀方对肝硬化大鼠血浆蛋白质的影响。方法将雄性 SD 大鼠随机分为3组,分别给予 CCl_1(CCl_4/橄榄油:1体积比=1:1)腹腔注射、CCl_4腹腔注射+扶正化瘀方灌胃、橄榄油腹腔注射,8周后通过Masson 三色染色法用图像分析仪自动分析肝脏胶原面积。应用双向凝胶电泳技术经银染显色和PDQuest7.3软件分析凝胶图像,对在三组中表达均有差异的蛋白质点用基质辅助激光解吸电离飞行时间串连质谱进行鉴定。结果扶正化瘀方干预组肝纤维化程度明显轻于模型组,胶原纤维面积在肝内沉积少(8.9%±3.7%vs 12.4%±4.7%,P<0.05)。实验去除血浆中的高丰度蛋白质可获得重复性较好的血浆蛋白质图谱。实验共鉴定出10个明显差异表达的蛋白质。在这些蛋白质中,血浆谷胱甘肽过氧化物酶及其前体、前白蛋白、结合珠蛋白、载脂蛋白 A-IV 前体、补体4、α抑制剂重链4和微管相关调节激酶1在肝硬化大鼠血浆中表达减少,扶正化瘀方干预组表达增加,肝脏再生相关蛋白和波形纤维蛋白在肝硬化大鼠血浆中表达增加,扶正化瘀方干预组表达减少。结论 CCl_4诱导肝硬化大鼠血浆中与氧化应激、细胞增值和转化相关蛋白质表达明显改变。扶正化瘀方可通过促进蛋白质合成、抗氧化、调控细胞增值和转化等多方面发挥抗纤维化的作用。

Objective To study the effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis. Methods Twenty-six male S-D rats were randomly divided into three groups, cirrhotic model group （ n = 10 ）, treated with CCl4 （ CCl4/olive oil ： v/v = 1 ： 1 ）, Fuzheng Huayu Decoction intervention group （n = 10）, treated with CCl4 ＋ Fuzheng Huayu Decoction, and normal control group （n =6）, treated with olive oil only. After 8 weeks, blood sample was collected from the vena cava inferior to undergo bidimensional electrophoresis （2DE） and analysis by PDQuest 7.3 software. Differential protein spots were cut, enzyme hydrolysis was conducted, and peptide fragments extracted from the mixture underwent mass spectrometry with MALDI-TOF-TOF-MS. The liver fibrogenesis was assessed by digital image analysis instrument of Masson＇s trichrome stained sections. Results The fibrosis area of the Fuzheng Huayu Decoction was 9%±4% , significantly smaller than that of the cirrhotic model group （ 12% ± 5%, P 〈 0.05）. Ten markedly changed protein spots were identified by MALDI-TOF-TOF-MS. Eight of the 10 proteins, including plasma glutathione peroxidase, plasma glutathione peroxidase precursor, prealbumin, haptoglobin, apolipoprotein A-IV precursor, complement CA, inter-alpha-inhibitor H4 heavy chain, and serine/threonine-protein kinase MARK1 （microtubule- affinity regulating kinase 1 ） were expressed very lowly in the cirrhotic model group while were expressed highly in the Fuzheng Huayu Decoction group. The expression of liver regeneration-related protein LRRG03 and vimentin increased in the cirrhotic model group, and reduced in the Fuzheng Huayu Decoction group. Conclusion Some proteins related to oxidative stress, cell proliferation and transformation have changed in the plasma of cirrhosis induced by CCl4. Fuzheng Huayu Decoction promotes protein synthesis and plays an anti-fibrotic role by antioxidation and accommodation of cell proliferation and transformation.