基于iTRAQ技术研究丹参酮Ⅱ A对高脂血症大鼠肝脏蛋白质的影响

Effects of Tanshinone ⅡA on Liver Proteome Expression of Hyperlipidemia Rats by iTRAQ Technology

目的应用相对和绝对定量同位素标记技术(iTRAQ)研究丹参酮ⅡA对高脂血症大鼠肝脏蛋白质组表达的影响,探讨丹参酮ⅡA对肝脏保护的作用。方法 24只SD大鼠随机分为空白对照组、高脂血症组和丹参酮组,每组8只。空白对照组每日给予普通大鼠饲料喂饲。高脂血症组和丹参酮组给予高脂饲料喂饲,4周后丹参酮组予丹参酮ⅡA磺酸钠10 mg/(kg·d)腹腔注射,空白对照组、高脂血症组给予等体积的磷酸盐缓冲生理盐水腹腔注射。用药8周后检测大鼠血脂、肝功能情况以及肝脏组织形态学变化。分离提取肝脏总蛋白,以iTRAQ试剂标记后进行质谱检测并以软件分析差异表达的蛋白质,用Western blot方法验证部分差异蛋白的表达趋势。结果与空白对照组比较,高脂血症组血清TC、TG、LDL-C、ALT、AST水平升高(P<0.01),HDL-C水平降低(P<0.01);与高脂血症组比较,丹参酮组血清中ALT、AST水平降低(P<0.05)。iTRAQ技术分析发现:丹参酮组肝脏中CES1、Apoa1、Acots2、3-KAT及细胞色素P450家族Cyp3a2、Cyp3a18、Cyp4a3、Cyp2b2、Cyp2c23这9个差异蛋白表达量上调,Tkt及eNT表达下调。Western blot验证差异蛋白CES1、Apoa1、Acots2变化趋势与蛋白质组结果相一致。结论丹参酮ⅡA可能通过促进胆固醇逆向转运、脂肪酸氧化,改善肝脏代谢功能,减少肝脏脂质沉积,达到其肝脏保护作用。

Objective To study the effects of Tanshinone ⅡA on the liver proteome expression of hyperlipidemia rats by isobaric tags for relative and absolute quantitation （iTRAQ） technology, and to observe its protection for liver. Methods Totally 24 SD rats were randomly divided into the blank control group （Cgroup）, the high fat hyperlipidemia group （HFH group）, and Tanshinone IIA treatment group （TAN group）, 8 in each group. Rats in the C group were fed with common forage. High fat forage was fed to rats in the HFH group the TAN group. Sodium Tanshinone ⅡA sulfonate （10 mg · kg-1 ·d-1） was intra- peritoneally injected to rats in the TAN group 4 weeks later. Equal volume of phosphate buffered saline was intraperitoneally injected to rats in the C group and the HFH group. After 8-week medication, blood lipid levels, liver function, and histomorphological changes of liver were detected. The total protein of liver was extracted and labeled by iTRAQ. Then differentially expressed proteins were detected by mass spec- trometric detection and analyzed by software. The tendency of partial differentially expressed proteins were detected by Western blot. Results Compared with the C group, the levels of TC, TG, LDL-C, ALT, and AST increased （P 〈0.01 ）, and the HDL-C level decreased （P 〈0.01 ） in the HFH group. Compared with the HFH group, the levels of ALT and AST decreased （P 〈0.05） in the TAN group, iTRAQ technology analysis showed that 9 differentially expressed proteins such as CES1, Apoal, Acots2-KAT, Acots3- KAT, and cytochrome P450 family proteins （Cyp3a2, Cyp3a18, Cyp4a3, Cyp2b2, Cyp2c23） were up- regulated in the TAN group. Expressions of Tkt and eNT were down-regulated in the TAN group. Results proved by Western blot （changing tendency of differentially expressed proteins such as CES1, Apoal, Acots2） were consistent with proteome expression results. Conclusions Tanshinone ⅡA might improve liver metabolism and reduce liver lipid deposition possibly by promoting counter transport of cholesterol and fatty acid oxidation, thus protecting the liver.