脾阴虚证脾失健运大鼠回肠组织中相关蛋白表达差异及其调控机制的实验研究

Differential Expression of Related Proteins in the Ileum of Rats with Spleen Yin Deficiency and Spleen Failure Movement and the Mechanism of Regulation

目的:探讨脾阴虚证脾失健运状态下,大鼠回肠组织中相关差异蛋白的表达及其调控机制。方法:根据分层随机原则,将36只SPF级健康Sprague-Dawley(SD)大鼠,随机分成健康对照组、脾阴虚证模型组、中药反证组共3组,每组各12只。采用经典复合因素法塑造脾阴虚证脾失健运大鼠模型。行逆转录聚合酶链反应(RT-PCR)法及蛋白免疫印迹(Western blot)法,分别对课题组前期蛋白组学鉴定出的过氧化氢酶(Catalase,CAT)、顺乌头酸水合酶(Aconitate hydratase,ACO2)、钙联结蛋白(Calnexin,Canx)、Papss 2(Papss 2 69KD protein)、热休克蛋白90(heat shock protein 90KD,HSP 90)5个特异性表达蛋白进行检测。结果:(1)半定量分析比较各组大鼠回肠组织差异表达蛋白mRNA表达水平发现,与健康对照组比较,脾阴虚模型组回肠组织中CAT、Canx、ACO2及Papss 2蛋白的mRNA表达均显著上升(P<0.05),而HSP 90蛋白的mRNA表达显著下调(P<0.05),中药反证组各指标均未出现明显差异(P>0.05);(2)半定量分析比较各组大鼠回肠组织差异表达蛋白表达水平发现,与健康对照组比较,脾阴虚模型组回肠组织中CAT、Canx、ACO2及Papss 2蛋白含量均显著上升(P<0.05),其中以CAT上调的最为显著(P<0.01),而HSP 90蛋白含量显著下调(P<0.05),中药反证组各指标均未出现明显差异(P>0.05)。结论:脾阴虚脾失健运状态下,大鼠回肠组织中可能存在以线粒体损伤为基础的细胞膜过氧化损伤、Ca2+稳态失衡、类乙醇毒害、过度硫酸化、神经元损伤及能量和遗传物质变化等一系列病理改变。

Objective：To investigate the expression of differentially expressed proteins in the ileum of rats with spleen yin deficiency syndrome and spleen failure movement, and to explore the regulatory mechanism. Methods： According to the random principle, 36 SPF health Sprague Dawley （SD） rats were randomly divided into normal control group, spleen yin deficiency model group, TCM proof group 3 group, with 12 rats in each group. The model of spleen yin deficiency in rats was established by the classical composite factor method. For reverse transcriptase polymerase chain reaction （ RT - PCR） and protein immunoblotting （Westernblot） method, respectively on catalase ourprevious proteomics identified （Catalase, CAT）, aconitate hydratase （Aconitatehydratase, ACO2）, calcium binding protein （Calnexin, Canx）, Papss2 （Papss269KDprotein）, heat shock protein 90 （heatshockprotein90KD, HSP90） 5 specific expression protein were detected. Results： ①The expression level of mRNA protein expression analysis found that the semi quantitative differences in ileum tissue of rats, compared with normal control groups, CAT, Canx, ACO2 and Papss2 protein in spleen yin deficiency model group, the expression of mRNA in ileum increased significantly （ P 〈 0. 05 ） , and HSP90 protein expression were significantly reduced （ mRNA P 〈 0. 05 ） , the indexes of TCM proof groups had no obvious differ- ence between disproof （ P 〉 0. 05 ） ; ②Expression analysis of protein expression level found semi quantitative differences in ileum tissue of rats, compared with the normal control groups, spleen yin deficiency model group, Canx, CAT in ileal tis- sue ACO2 and Papss2 protein were significantly increased （P 〈 0. 05）, and the increase of CAT was the most significant （P 〈 0. 01 ） . However, the content of HStO0 protein significantly decreased （P 〈 0. 05 ）, the indexes of TCM proof groups had no obvious difference between disproof （ P 〉 0. 05 ）. Conclusion ： The spleen yin deficiency condition in rat ile- um may exist in mitochondrial damage cell membrane based oxidative damage, Ca2 ＋ homeostasis, ethanol toxicity, ex- cessive sulfation, neuronal damage and energy and genetic material change and a series of pathological changes.