利用基因芯片技术研究针刺治疗小鼠高胆固醇血症的分子机理
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摘要
采用国际上认可的高胆固醇饲料导致的高胆固醇血症小鼠模型,在生化水平上肯定针刺疗效的同时,利用基因芯片技术高通量、高度并行性、高灵敏度的优势,从整体基因组的角度对针刺降脂的分子机理进行研究。
将C57BL/6J近交系小鼠随机分为正常组、高胆固醇血症组、针刺预防组(简称针预组)。除正常组给予基础饲料以外,其余小鼠给予高胆固醇饲料,针预组小鼠同时进行电针丰隆穴治疗。2周后,高胆固醇血症组小鼠的血浆总胆固醇水平较正常组升高2倍以上,达到6.87±0.36mmol/L,表明高胆固醇血症造模成功。然后将该组的小鼠随机分为模型组、针刺治疗组(简称针治组)、针刺非穴位组(简称针非组)、药物治疗组(简称药物组)。针治组开始电针丰隆穴治疗,针非组电针非穴位,药物组用辛伐他汀治疗,针预组继续电针治疗。各组在进行以上各种处理时继续给予高胆固醇饲料,正常组给予基础饲料,不做处理。16天后,处死小鼠,血浆用于血液生化检测,光镜观察肝脏病理改变,检测肝脂含量,提取肝脏RNA用于基因芯片和实时定量RT-PCR分析。
对6组小鼠的12项血液生化指标分析结果表明,血浆总甘油三酯的比较无统计学意义,各组小鼠除血浆总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇3项指标以外的其余8项指标均无显著差异。模型组小鼠的上述3项指标和2项肝脂指标(肝脏总胆固醇和总甘油三酯)与正常组小鼠相比有显著升
The molecular mechanism of the effect of electro-acupuncture (EA) induced hypocholesterolemia on the hypercholesterolemia mice induced by hypercholesterolemic diet was studied by using cDNA microarray.Male C57BL/6J mice were first randomly divided into three groups: normal group (NG), hypercholesterolemia group and EA preventive group (EPG). NG mice were fed normal chow, hypercholesterolemia group were fed a hypercholesterolemic diet (HD), EPG mice were fed the same HD and received EA treatment on Fenglong acupoint (ST40). After 2 weeks , the plasma cholesterol levels in hypercholesterolemia mice markedly raised to 6.87 ± 0.36 mmol/L, which was more than two-fold high in the NG mice. It indicated that the hypercholesterolemia model was successfully constructed. The mice of hypercholesterolemia group were then randomly divided into model group (MG), EA treatment group (ETG), EA non-acupoint group (ENG) and drug control group (DG). Mice of ETG received EA treatment, ENG received EA on non-acupoint, DG received simvastatin treatment, EPG received continuous EA treatment. During the treatments, the mice were continuously fed HD. NG mice were still fed normal chow, and received no treatment. After 16 days, all mice were killed, plasma was obtained for biochemical
analysis, liver were excised for the analysis of histology, lipid content, cDNA microarray and real-time quantitative RT-PCR.The result from 12 items of plasma biochemical analysis indicated that plasma total triglyceride (TG) level had no statistically significance, and there were 8 items with no significant difference among 6 groups. However, the levels of total cholesterol (TC), high density cholesterol , and low density cholesterol of plasma, TC and TG of liver in MG mice were significantly elevated compared with those of NG, and those of EPG, ETG and DG were decreased when compared with MG mice. The hepatic steatosis of MG mice was severe, and that of EPG, ETG and DG was ameliorated. It indicated that EA at the Fenglong acupoint had the same hypocholesterolemic effect as that of simvastatin. All above-mentioned levels of ENG mice were not significantly different from those of MG. The results indicated that acupoint is important and necessary in the hypocholesterolemic treatment.Although EA or simvastatin had significant hypocholesterolemia effect, the above-mentioned levels of EPG, ETG and DG were not restored to the normal level. The microarray analysis of the expression of 16,463 murine genes was studied in the 6 groups' mice. The most number of expressed gene detected was 5,766(35%), the least number was 3,104 (19%) . The numbers of significantly expressed genes were different in various combinations of hybridizations as follows: NG/MG, 98 genes; EPG/MG, 101 genes; ENG/MG, 63 genes; ETG/MG, 29 genes; DG/MG, 27 genes. The function of the significantly expressed genes were associated with metabolism(including cholesterol metabolism), response to stress, immunity, signal transduction, transcription regulation, transport, cell apoptosis, cell cycle, development, cell organization, cell differentiation, cell adhesion.The result of clusters analysis of microarray data indicated that the various combinations of hybridization were clustered into different branch in the denbrogram: ENG/MG, a single branch; EPG/MG and DG/MG, one branch; EPG/MG and NG/MG, one branch.
The result of the function analysis of differentially expressed genes in the EPG and ETG indicated that EA not only affected the gene expression of cholesterol metabolism, but also that of signal transduction, cell apoptosis and response to stress.4 differentially expressed genes in the cDNA microarray and 10 other genes associated with the liver cholesterol metabolism were analyzed by using real-time quantitative RT-PCR in their expression level. The result not only confirmed the reliability of microarray data, but also indicated that the molecular mechanism of hypocholesterolemia effect of EA was different from that of simvastatin. EA effect was associated with higher expression levels for LXRcc and genes encoding bile acid synthetic enzymes. DG treatment had the same lower plasma cholesterol levels as EPG, but it was associated with the lower FXR mRNA level and higher expression levels for genes encoding membrane-associated bile acid transporters.This work is the first time to study the molecular mechanism of the effect of EA-induced hypocholesterolemia. The results in the present study provide more clues on not only how hypocholesterolemia is induced by EA, but also the exploration of target gene of treating hypercholesterolemia.
将C57BL/6J近交系小鼠随机分为正常组、高胆固醇血症组、针刺预防组(简称针预组)。除正常组给予基础饲料以外,其余小鼠给予高胆固醇饲料,针预组小鼠同时进行电针丰隆穴治疗。2周后,高胆固醇血症组小鼠的血浆总胆固醇水平较正常组升高2倍以上,达到6.87±0.36mmol/L,表明高胆固醇血症造模成功。然后将该组的小鼠随机分为模型组、针刺治疗组(简称针治组)、针刺非穴位组(简称针非组)、药物治疗组(简称药物组)。针治组开始电针丰隆穴治疗,针非组电针非穴位,药物组用辛伐他汀治疗,针预组继续电针治疗。各组在进行以上各种处理时继续给予高胆固醇饲料,正常组给予基础饲料,不做处理。16天后,处死小鼠,血浆用于血液生化检测,光镜观察肝脏病理改变,检测肝脂含量,提取肝脏RNA用于基因芯片和实时定量RT-PCR分析。
对6组小鼠的12项血液生化指标分析结果表明,血浆总甘油三酯的比较无统计学意义,各组小鼠除血浆总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇3项指标以外的其余8项指标均无显著差异。模型组小鼠的上述3项指标和2项肝脂指标(肝脏总胆固醇和总甘油三酯)与正常组小鼠相比有显著升
The molecular mechanism of the effect of electro-acupuncture (EA) induced hypocholesterolemia on the hypercholesterolemia mice induced by hypercholesterolemic diet was studied by using cDNA microarray.Male C57BL/6J mice were first randomly divided into three groups: normal group (NG), hypercholesterolemia group and EA preventive group (EPG). NG mice were fed normal chow, hypercholesterolemia group were fed a hypercholesterolemic diet (HD), EPG mice were fed the same HD and received EA treatment on Fenglong acupoint (ST40). After 2 weeks , the plasma cholesterol levels in hypercholesterolemia mice markedly raised to 6.87 ± 0.36 mmol/L, which was more than two-fold high in the NG mice. It indicated that the hypercholesterolemia model was successfully constructed. The mice of hypercholesterolemia group were then randomly divided into model group (MG), EA treatment group (ETG), EA non-acupoint group (ENG) and drug control group (DG). Mice of ETG received EA treatment, ENG received EA on non-acupoint, DG received simvastatin treatment, EPG received continuous EA treatment. During the treatments, the mice were continuously fed HD. NG mice were still fed normal chow, and received no treatment. After 16 days, all mice were killed, plasma was obtained for biochemical
analysis, liver were excised for the analysis of histology, lipid content, cDNA microarray and real-time quantitative RT-PCR.The result from 12 items of plasma biochemical analysis indicated that plasma total triglyceride (TG) level had no statistically significance, and there were 8 items with no significant difference among 6 groups. However, the levels of total cholesterol (TC), high density cholesterol , and low density cholesterol of plasma, TC and TG of liver in MG mice were significantly elevated compared with those of NG, and those of EPG, ETG and DG were decreased when compared with MG mice. The hepatic steatosis of MG mice was severe, and that of EPG, ETG and DG was ameliorated. It indicated that EA at the Fenglong acupoint had the same hypocholesterolemic effect as that of simvastatin. All above-mentioned levels of ENG mice were not significantly different from those of MG. The results indicated that acupoint is important and necessary in the hypocholesterolemic treatment.Although EA or simvastatin had significant hypocholesterolemia effect, the above-mentioned levels of EPG, ETG and DG were not restored to the normal level. The microarray analysis of the expression of 16,463 murine genes was studied in the 6 groups' mice. The most number of expressed gene detected was 5,766(35%), the least number was 3,104 (19%) . The numbers of significantly expressed genes were different in various combinations of hybridizations as follows: NG/MG, 98 genes; EPG/MG, 101 genes; ENG/MG, 63 genes; ETG/MG, 29 genes; DG/MG, 27 genes. The function of the significantly expressed genes were associated with metabolism(including cholesterol metabolism), response to stress, immunity, signal transduction, transcription regulation, transport, cell apoptosis, cell cycle, development, cell organization, cell differentiation, cell adhesion.The result of clusters analysis of microarray data indicated that the various combinations of hybridization were clustered into different branch in the denbrogram: ENG/MG, a single branch; EPG/MG and DG/MG, one branch; EPG/MG and NG/MG, one branch.
The result of the function analysis of differentially expressed genes in the EPG and ETG indicated that EA not only affected the gene expression of cholesterol metabolism, but also that of signal transduction, cell apoptosis and response to stress.4 differentially expressed genes in the cDNA microarray and 10 other genes associated with the liver cholesterol metabolism were analyzed by using real-time quantitative RT-PCR in their expression level. The result not only confirmed the reliability of microarray data, but also indicated that the molecular mechanism of hypocholesterolemia effect of EA was different from that of simvastatin. EA effect was associated with higher expression levels for LXRcc and genes encoding bile acid synthetic enzymes. DG treatment had the same lower plasma cholesterol levels as EPG, but it was associated with the lower FXR mRNA level and higher expression levels for genes encoding membrane-associated bile acid transporters.This work is the first time to study the molecular mechanism of the effect of EA-induced hypocholesterolemia. The results in the present study provide more clues on not only how hypocholesterolemia is induced by EA, but also the exploration of target gene of treating hypercholesterolemia.
引文
1.常小荣,周国平,严洁,林亚萍,易受乡.1988.隔药饼灸对高脂血症患者血清HDL-C、LDL-C及其它脂质含量的影响.针刺研究.13:272-274.
2.陈晓萍.1991.耳穴贴压治疗高脂血症.上海中医药杂志.25:27-29.
3.韩舰华.1988.不同时辰电针“足三里”对实验性高脂血症家兔的疗效观察.针刺研究.4:278-280.
4.李怡.2002.针灸治疗高脂血症近况.四川中医.20:23-24.
5.彭悦.1986.氮氖激光照射内关穴治疗高脂血症50例临床观察.中国针灸.6:1-2.
6.任敬威.1996.针刺足三里、内关穴治疗高脂血症45例报告.甘肃中医.9:15-16.
7.孙国杰.1997.针灸学.上海科学技术出版社.P56.
8.谭震华,冯起国,王耀斌.2000.针灸治疗高脂血症和动脉粥样硬化的研究进展.辽宁中医杂志.27:64-67.
9.唐寒松.1998.针灸从脾论治高脂血症的探讨.中国针灸.18:703-704.
10.王华,1997.丹参穴位注射治疗高脂血症40例临床分析.中国针灸.17:269-271.
11.王玉堂.1990.针刺丰隆穴降血脂47例临床观察.中国针灸.10:21-23.
12.徐叔云,卞如廉,陈修.2002.药理实验方法.人民卫生出版社.
13.杨丹红,许文波,江庆淇,2000.穴位注射对高脂血症动物模型的降脂作用研究.针灸临床杂志.16:34-36.
14.赵和熙.1984.针刺内关穴对高血脂的影响.中国中西医结合杂志.4:666-668.
15. Black, D. M. 2002. A general assessment of the safety ofHMG-CoA reductase inhibitors (statins). Curr. Atheroscl. Rep. 4: 34-41.
16. Chittur S. V. 2004. DNA microarrays: tools for the 21st Century. Comb. Chem. High Throughput Screen. 7: 531-537.
17. Clarke, P. A, R. T. Peele and P. Workman. 2004. Gone expression microarray technologies in the development of new therapeutic agents. Eur. J. Cancer. 40: 2560-2591.
18. Export Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Export Panel on Detection, Evaluation, and treatment of High Blood Cholesterol in Adults (Adult Treatment Panel Ⅲ). 2001. JAMA. 285: 2486-2497.
19. Ezzo, J., B. Berman, V. A. Hadhazy, A. R. Jadad, L. Lao and B. B. Singh. 2000. Is acupuncture effective for the treatment of chronic pain? A systematic review. Pain. 86: 217-225.
20. Han, J. S. 2004. Acupuncture and endorphins. Neuroscience lett. 361: 258-261.
21. http://www.satcm.gov.cn/
22. http://xz365.com.cn/
23. Knopp, R. H. 1999. Drug therapy: drug treatment of lipid disorders. N. Engl. J. Med. 341: 498-511.
24. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel Ⅲ). Third report of the National Cholesterol Education Program (NCEP) Export Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults(Adult Treatment Panel Ⅲ) final report. 2002. Circulation. 106: 3143-3421.
25. Safeer, R. S. and C. L. Lacivita. 2000. Choosing drug therapy for patients with hyperlipidemia. Am. Fam. Physician. 61: 3371-3382.
26. Scott, C. L. 2003. Diagnosis, prevention, and intervention for the metabolic syndrome. Am. Cardiol. 92: 352-421.
27. Vanholder, R., M. S. Sever and E. Erek. 2000. Rhabdomyolysis. J. Am. Soc. Nephrol. 11: 1553-1561.
2.陈晓萍.1991.耳穴贴压治疗高脂血症.上海中医药杂志.25:27-29.
3.韩舰华.1988.不同时辰电针“足三里”对实验性高脂血症家兔的疗效观察.针刺研究.4:278-280.
4.李怡.2002.针灸治疗高脂血症近况.四川中医.20:23-24.
5.彭悦.1986.氮氖激光照射内关穴治疗高脂血症50例临床观察.中国针灸.6:1-2.
6.任敬威.1996.针刺足三里、内关穴治疗高脂血症45例报告.甘肃中医.9:15-16.
7.孙国杰.1997.针灸学.上海科学技术出版社.P56.
8.谭震华,冯起国,王耀斌.2000.针灸治疗高脂血症和动脉粥样硬化的研究进展.辽宁中医杂志.27:64-67.
9.唐寒松.1998.针灸从脾论治高脂血症的探讨.中国针灸.18:703-704.
10.王华,1997.丹参穴位注射治疗高脂血症40例临床分析.中国针灸.17:269-271.
11.王玉堂.1990.针刺丰隆穴降血脂47例临床观察.中国针灸.10:21-23.
12.徐叔云,卞如廉,陈修.2002.药理实验方法.人民卫生出版社.
13.杨丹红,许文波,江庆淇,2000.穴位注射对高脂血症动物模型的降脂作用研究.针灸临床杂志.16:34-36.
14.赵和熙.1984.针刺内关穴对高血脂的影响.中国中西医结合杂志.4:666-668.
15. Black, D. M. 2002. A general assessment of the safety ofHMG-CoA reductase inhibitors (statins). Curr. Atheroscl. Rep. 4: 34-41.
16. Chittur S. V. 2004. DNA microarrays: tools for the 21st Century. Comb. Chem. High Throughput Screen. 7: 531-537.
17. Clarke, P. A, R. T. Peele and P. Workman. 2004. Gone expression microarray technologies in the development of new therapeutic agents. Eur. J. Cancer. 40: 2560-2591.
18. Export Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Export Panel on Detection, Evaluation, and treatment of High Blood Cholesterol in Adults (Adult Treatment Panel Ⅲ). 2001. JAMA. 285: 2486-2497.
19. Ezzo, J., B. Berman, V. A. Hadhazy, A. R. Jadad, L. Lao and B. B. Singh. 2000. Is acupuncture effective for the treatment of chronic pain? A systematic review. Pain. 86: 217-225.
20. Han, J. S. 2004. Acupuncture and endorphins. Neuroscience lett. 361: 258-261.
21. http://www.satcm.gov.cn/
22. http://xz365.com.cn/
23. Knopp, R. H. 1999. Drug therapy: drug treatment of lipid disorders. N. Engl. J. Med. 341: 498-511.
24. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel Ⅲ). Third report of the National Cholesterol Education Program (NCEP) Export Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults(Adult Treatment Panel Ⅲ) final report. 2002. Circulation. 106: 3143-3421.
25. Safeer, R. S. and C. L. Lacivita. 2000. Choosing drug therapy for patients with hyperlipidemia. Am. Fam. Physician. 61: 3371-3382.
26. Scott, C. L. 2003. Diagnosis, prevention, and intervention for the metabolic syndrome. Am. Cardiol. 92: 352-421.
27. Vanholder, R., M. S. Sever and E. Erek. 2000. Rhabdomyolysis. J. Am. Soc. Nephrol. 11: 1553-1561.