花椒对利血平所致虚寒证大鼠骨骼肌Cox4i1表达及甲基化的影响  

Effects of Zanthoxylum bungeanum on the expression of Cox4i1 and methylation in skeletal muscle of deficient cold syndrome rats induced by Reserpine

作　　者：章明[1] 翁美芝[1] 吴超[1] 周莹[1] 黄鑫[1] 尹春胜 彭淑红[1] 朱卫丰[1] ZHANG Ming;WENG Mei-zhi;WU Chao;ZHOU Ying;HUANG Xin;YIN Chun-sheng;PENG Shu-hong;ZHU Wei-feng(Jiangxi University of TCM, Nanchang 330004, Chin)

机构地区：[1]江西中医药大学,南昌330004

出　　处：《中华中医药杂志》2018年第4期1532-1537,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.81260648);江西省教育厅基金资助项目(No.GJJ150844);江西中医药大学项目(No.2014BS013)~~

摘　　要：目的:探讨热性中药花椒对利血平所致虚寒证大鼠骨骼肌Cox4i1表达及甲基化的影响。方法:利血平作用于SD雄性大鼠造成虚寒证模型后,用花椒水煎剂按0.54、5.40g/kg剂量灌胃大鼠12d,取骨骼肌;Elisa法测定COX4I1蛋白浓度;Real-time PCR法检测大鼠骨骼肌中Cox4i1、Dnmt1、Dnmt3a、Dnmt3b、Tet1、Tet2和Tet3基因m RNA表达水平;BSP法检测Cox4i1基因DNA甲基化状态;Western blot法检测骨骼肌中TET3和DNMT3A蛋白表达情况。结果:模型组COX4I1蛋白量和m RNA水平均比空白组显著增加(P<0.01);花椒低、高剂量组能显著降低模型组大鼠Cox4i1 m RNA水平(P<0.01,P<0.05)。模型组Cox4i1甲基化频率有降低趋势,且接近显著水平;花椒低剂量组能显著增加Cox4i1甲基化频率(P<0.05)。模型组Dnmt1、Tet1、Tet2 m RNA水平显著增加(P<0.05);花椒低剂量组能显著降低Dnmt3a、Dnmt3b、Dnmt1、Tet1、Tet2和Tet3 m RNA水平(P<0.05,P<0.01);花椒高剂量组能显著降低Dnmt3b、Tet3、Tet2 m RNA水平(P<0.05,P<0.01)。花椒高、低剂量组对于DNMT3A和TET3蛋白表达量均未有显著影响。结论:低剂量花椒可能通过同时降低DNA甲基化酶和去甲基化酶的表达水平,而发挥甲基化的作用,使Cox4i1 DNA甲基化频率增加,从而使m RNA水平降低。Objective： To investigate the effect of Zanthoxylum bungeanurn on the expression of Cox4il and methylation in skeletal muscle of deficient cold syndrome rats induced by Reserpine. Methods＇. Male SD rats were treated by Reserpine to establish the deficiency-cold syndrome model. Then skeletal muscle was collected after the oral administration with Zanthoxylum bungeanum with the dose of 0.54g/kg or 5,4g/kg for 12d. Elisa method was adopted to measure the content of COX411 protein and real-time PCR was used to detect mRNA levels of Cox4il, Dnmtl, Dnmt3a, Dnmt3b, Tetl, Tet2 and Tet3. Cox4il methylation was detected by bisulfite-sequencing PCR （BSP）, Protein levels of TET3 and DNMT3A were measured using Western blot, Results： Both mRNA and protein levels of Cox4il in model group were increased compared with control group （P〈0,01）, but mRNA level of Cox4il was decreased by low-dose or high dose of Zanthoxylum bungeanum （P〈0.01, P〈0,05） compared with model group. Methylation frequency of Cox4il in model group was lower than control group, but that was higher in low-dose group than model group （P〈0.05）. MRNA levels of Dnmtl, Tetl and Tet2 were raised in model group （P〈0.05）, However, low-dose Zanthoxylum bungeanum could reduce mRNA levels of Dnmt3a and Dnmt3b （P〈0.05） as well as mRNA levels of Dnmtl, Tetl, Tet2 and Tet3 （P〈0.01）, and high-dose Zanthoxylum bungeanum could also decrease mRNA levels of Dnmt3b, Tet3 and Tet2 （P〈0.05, P〈0.01）.Protein levels of DNMT3A and TET3 revealed less change both in low-dose and high-dose groups. Conclusion： The increased methylation frequency and the lowered mRNA level of Cox4il show that low-dose Zanthoxylum bungeanum might play 0, certain role on DNA methylation by lowering the mRNA levels of DNA methyLation enzymes and DNA demethylation enzymes.

关 键 词：花椒 虚寒证 Cox4i1 DNA甲基化 利血平 DNA去甲基化 

分 类 号：R285.5[医药卫生—中药学]