海藻玉壶汤中海藻不同品种与甘草加减应用对甲状腺肿大模型大鼠甲状腺Bax、Bcl-2 mRNA表达的影响
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摘要
目的探讨含高剂量反药组合的海藻玉壶汤中海藻不同品种与甘草加减应用对甲状腺肿大模型大鼠甲状腺Bax、Bcl-2 mRNA表达的影响。方法选取Wistar大鼠144只,雌雄各半,随机分为空白组、模型组、阳性药优甲乐组(优甲乐组)、含羊栖菜的海藻玉壶汤全方组(羊栖菜全方组)、含海蒿子的海藻玉壶汤全方组(海蒿子全方组)、含羊栖菜的海藻玉壶汤去甘草组(羊栖菜去甘草组)、含海蒿子的海藻玉壶汤去甘草组(海蒿子去甘草组)、海灌玉壶汤去海藻组(全方去海藻组)、海藻玉壶汤去海藻甘草组(全方去海藻甘草组),共计9组。造模期间,除空白组外,其余各组大鼠灌服丙基硫氧嘧啶(PTU)10 mg/kg,每日1次,共给造模药14 d。造模后给予治疗药物,周期为28 d,其中,空白组与模型组给予等量蒸馏水灌胃,阳性药组灌服优甲乐,给药剂量为20μg/kg,每日1次,其余各给药组给予相应药液灌胃,大鼠每日给药1次,给药剂量为海蒿子/羊栖菜去甘草组12.6 g/kg,全方去海藻组13.5 g/kg,海蒿子/羊栖菜全方组18.0 g/kg,全方去海藻甘草组8.1 g/kg。在给药期间,为了稳定模型,除空白组外,每隔1 d,其余各组灌服PTU,剂量同前。给药结束后计算各组大鼠甲状腺系数,观察甲状腺组织病理形态,用RT-PCR法检测甲状腺组织中Bax、Bcl-2 mRNA的表达。结果与空白组比较,各组大鼠甲状腺系数明显升高(P<0.01);与模型组比较除优甲乐组外其余各给药组甲状腺系数明显降低(P<0.01)。甲状腺组织病理形态观察显示模型组与优甲乐组较空白组变化明显,甲状腺组织小叶排列紊乱,滤泡大小不一,滤泡上皮细胞弥漫性增生和肥大,其余各给药组有纠正作用,其中全方(海蒿子/羊栖菜)组组织病理形态最接近空白组。与空白组比较,模型组Bax mRNA表达降低(P<0.05);与模型组比较海藻玉壶汤全方及加减应用各组Bax mRNA表达升高(P<0.05,P<0.01)。各组Bcl-2 mRNA表达无差异。与空白组比较,模型组Bax/Bcl-2降低(P<0.05),与模型组比较,海藻玉壶汤全方及加减应用各组数值均升高(P<0.05,P<0.01)。结论含高剂量反药组合的海藻玉壶汤中不同品种海藻与甘草加减应用,对甲状腺肿大模型大鼠甲状腺系数及甲状腺组织形态有纠正作用;其作用机制可能与其升高Bax/Bcl-2从而促进甲状腺细胞凋亡相关。
Objective To investigate the effects of different species of Haizao(haihaozi, Sargassum Pallidum or yangqicai, Sargassum Fusiforme) in Haizao Yuhu(HZYH) Decoction with or without gancao(Radix Glycyrrliizae) on the expressions of Bax, Bcl-2 mRNA in goiter rats. Methods Rats(n =144) were randomly divided into 9 groups: blank group, model group, positive drug euthyrox(20 μg/kg euthyrox) group, HZYH Decoction with yangqicai without gancao group(12.6 g/kg), HZYH Decoction with haihaozi without gancao group(12.6 g/kg), HZYH Decoction without haizao group(13.5 g/kg), HZYH Decoction group with haihaozi(18.0 g/kg), HZYH Decoction group with yangqicai(18.0 g/kg), and HZYH Decoction without Haizao and Gancao group(8.1 g/kg). During the modeling period, except for the blank group, the other groups of rats were given propyl thiouracil 10 mg/kg once a day for 14 days. The investigational treatment was administered after modeling for 28 days. Among them, the blank group and the model group were given the same amount of distilled water for gavage. The rat model of goiter was induced with propyltluouracil(PTU). Except for the blank group, the others were correspondingly given interventional medicine respectively. Thyroid index of rats and pathological morphology in thyroid tissue were evaluated. The gene expressions of Bax and Bcl-2 were respectively detected. Results The levels of thyroid index in model group and eutbyrox group were significantly increased compared with blank group(P<0.01). Compared with model group, thyroid index decreased in all other groups(P<0.01). Pathological observation of thyroid tissue showed that model group and euthyrox changed significantly compared with blank group. The lobulations of thyroid gland was disordered; the follicle size was different; the thyroid follicular epithelium cells presented with diffuse hyperplasia and hypertrophy. These changes were rectified to different degrees in other groups. The histopathological morphology of HZYH Decoction with haihaozi or yangqicai group was the closest to blank group. The expression of Bax mRNA in model group was decreased compared with blank group(P<0.05). Its expressions were increased in HZYH Decoction with or without haizao and gancao groups compared with model group(P<0.05, P<0.01). There was no difference in the expression of Bcl-2 mRNA between groups. Bax/Bcl-2 in model groups was decreased compared with blank group(P<0.05). Bax/Bcl-2 were increased in HZYH Decoction with or without haizao and gancao groups compared with model group(P<0.05, P<0.01). Conclusion Different varieties of haizao in Haizao Yuhu Decoction containing high-dose antagonistic medicinal combination with modification have corrective effect on thyroid index and pathological morphology in thyroid tissue in goiter models. Its mechanism may be related with upregulation of Bax/Bcl-2, which promotes apoptosis of thyroid cells.
Objective To investigate the effects of different species of Haizao(haihaozi, Sargassum Pallidum or yangqicai, Sargassum Fusiforme) in Haizao Yuhu(HZYH) Decoction with or without gancao(Radix Glycyrrliizae) on the expressions of Bax, Bcl-2 mRNA in goiter rats. Methods Rats(n =144) were randomly divided into 9 groups: blank group, model group, positive drug euthyrox(20 μg/kg euthyrox) group, HZYH Decoction with yangqicai without gancao group(12.6 g/kg), HZYH Decoction with haihaozi without gancao group(12.6 g/kg), HZYH Decoction without haizao group(13.5 g/kg), HZYH Decoction group with haihaozi(18.0 g/kg), HZYH Decoction group with yangqicai(18.0 g/kg), and HZYH Decoction without Haizao and Gancao group(8.1 g/kg). During the modeling period, except for the blank group, the other groups of rats were given propyl thiouracil 10 mg/kg once a day for 14 days. The investigational treatment was administered after modeling for 28 days. Among them, the blank group and the model group were given the same amount of distilled water for gavage. The rat model of goiter was induced with propyltluouracil(PTU). Except for the blank group, the others were correspondingly given interventional medicine respectively. Thyroid index of rats and pathological morphology in thyroid tissue were evaluated. The gene expressions of Bax and Bcl-2 were respectively detected. Results The levels of thyroid index in model group and eutbyrox group were significantly increased compared with blank group(P<0.01). Compared with model group, thyroid index decreased in all other groups(P<0.01). Pathological observation of thyroid tissue showed that model group and euthyrox changed significantly compared with blank group. The lobulations of thyroid gland was disordered; the follicle size was different; the thyroid follicular epithelium cells presented with diffuse hyperplasia and hypertrophy. These changes were rectified to different degrees in other groups. The histopathological morphology of HZYH Decoction with haihaozi or yangqicai group was the closest to blank group. The expression of Bax mRNA in model group was decreased compared with blank group(P<0.05). Its expressions were increased in HZYH Decoction with or without haizao and gancao groups compared with model group(P<0.05, P<0.01). There was no difference in the expression of Bcl-2 mRNA between groups. Bax/Bcl-2 in model groups was decreased compared with blank group(P<0.05). Bax/Bcl-2 were increased in HZYH Decoction with or without haizao and gancao groups compared with model group(P<0.05, P<0.01). Conclusion Different varieties of haizao in Haizao Yuhu Decoction containing high-dose antagonistic medicinal combination with modification have corrective effect on thyroid index and pathological morphology in thyroid tissue in goiter models. Its mechanism may be related with upregulation of Bax/Bcl-2, which promotes apoptosis of thyroid cells.
引文
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[11] 修琳琳.含反药组合的海藻玉壶汤中海藻不同品种与甘草加减对甲状腺肿大大鼠生物效应影响及机制探讨[D].北京:北京中医药大学,2017:59.Xiu LL.Biological effects and its mechanism of differ ent species of Sargassum and Glycyrrhiza uralensis in Haizao Yuhu Decoction in goiter rats with anti-drug combination[D].Beijing:Beijing University of Traditional Chinese Medicine,2017:59.
[12] 李娜,钟赣生,张晨,等.海藻玉壶汤中海藻不同品种与甘草加减应用对甲状腺肿大大鼠氧化应激及肝脏Nrf2/Ho-1通路的影响[J].北京中医药大学学报,2019,42(3):190-197.Li N,Zhong GS,Zhang C,et al.Influence of different special of Haizao and modified administration of Gan Cao in Haizao Yuhu Tang on oxidative stress and liver Nrf2/Ho-1 pathualg in rats with goiter[J].Journal of Bei jing University of Traditional Chinese Medicine,2019,42(3):190-197.
[13] 丁选胜,李欧.海藻、甘草及其不同比例配伍后的水提取物对实验性大鼠甲状腺肿的影响[J].中药药理与临床,2001,17(6):32-33.Ding XS,Li O.Effects of seaweed,licorice and different proportions of water extracts on experimental rat goiter[J].Pharmacology and Clinics of Chinese Materia Medica,2001,17(6):32-33.
[14] 朱春根,谢东浩,徐卫东,等.海藻甘草不同比例配伍对甲状腺肿大鼠甲状腺功能和形态的影响[J].齐齐哈尔医学院学报,2011,32(22):3610-3612.Zhu CG,Xie DH,Xu WD,et al.Effect of algae and liq uorice in various proportions on the thyroid function and morphology in goiters SD Rats[J].Journal of Qiqihar Medical College,2011,32(22):3610-3612.
[15] 谢东浩,马利华,徐卫东,等.海藻、甘草不同比例配伍对甲状腺相关调控因子Fas和Bcl-2的表达研究[J].南京中医药大学学报,2010,26(6):450-452.Xie DH,Ma LH,Xu WD,et al.Effect of combination of Herba Sargassii and Radix Glycyrrhizae Uralensis in different proportion on the expression of thyroid-related regulatory factors Fas and Bcl-2[J].Journal of Nanjing TCM University,2010,26(6):450-452.
[16] 高天舒,齐腾澈.海藻玉壶汤及其拆方对大鼠碘缺乏致甲状腺肿的干预作用[J].中医杂志,2012,53(19):1671 -1676.Gao TS,Qi TC.Intervention effect of Haizaoyuhu De coction and Its decomposed recipes for goiter caused by iodine deficiency[J].Journal of Traditional Chinese Medicine,2012,53(19):1671-1676.
[17] 陈杰,李甘地.病理学[M].北京:人民卫生出版社,2005:35-37.Chen J,Li GD.Pathology[M].Beijing:People’s Medical Publishing House,2005:35-37.
[18]Oltvai ZN,Milliman CL,Korsmeyer SJ.Bcl-2 het erodimerizes in vivo with a conserved homolog,Bax that accelerates programmed cell death[J].Cell,1993,74(4):609-619.
[2] 陈绍红,钟赣生,刘佳,等.国家级名老中医临证应用海藻甘草反药组合的处方特征[J].中国临床医生杂志,2015,43(6):89-93.Chen SH,Zhong GS,Liu J,et al.National famous old Chinese medicine clinical application prescription of sar gassum and uralensisa anti-drug combination[J].Chi nese Clinical Doctor feature,2015,43(6):89-93.
[3] 陈实功.外科正宗[M].北京:中国中医药出版社,2011:112.Chen SG.Orthodox Manual of External Diseases[M].Beijing:China Press of Traditional Chinese Medicine,2011:112.
[4] 黄晓朋,俞旭君,陈晓洋,等.海藻玉壶汤临床应用举隅[J].实用中医药杂志,2014,30(4):343-344.Huang XP,Yu XJ,Chen XY,et al.Example of clinical application of Haizao Yuhu Decoction[J].Journal of practical Traditional Chinese Medicine,2014,30(4):343 -344.
[5] 李怡文,钟赣生,王茜,等.含反药配伍的海藻玉壶汤临床应用分析[J].南京中医药大学学报,2011,27(4):317 -318.Li YW,Zhong GS,Wang Q,et al.Clinical application anal ysis of Haizao Yuhu Decoction with antagonistic medic inal compatibility[J].Journal of Nanjing TCM University,2011,27(4):317-318.
[6] 陈藏器.本草拾遗[M].尚志钧,点校.合肥:安徽科学技术出版社,2002:97.Chen ZQ.A Supplement to Materia Medica[M].proof rea ded by Shang ZJ.Hefei:Anhui Science and Technology Publishing House,2002:97.
[7] 曹琰,段金廒,郭建明,等.不同产地海蒿子、羊栖菜中无机元素的含量分析与评价[J].科技导报,2014,32(15):15-24.Cao Y,Duan JA,Guo JM,et al.Analysis and evaluation of inorganic elements in Sargassum pallidum and Sar gassum fusiforme from different regions[J].Science and Technology Review,2014,32(15):15-24.
[8] 崔征,李玉山,肇文荣,等.中药海藻及数种同属植物的药理作用[J].中国海洋药物,1997,63(3):5-8.Cui Z,Li YS,Zhao WR,et al.A pharmacological study of curde drug sargassum and same genus plants[J].Chi nese Journal of Marine Drugs,1997,63(3):5-8.
[9] 吴越,曲敏,佟长青,等.羊栖菜多糖对小鼠抗疲劳作用的研究[J].食品工业科技,2013,34(8):350-352.Wu Y,Qu M,Tong CQ,et al.Study on the effect of pol ysaccharide from Hizikia fusiformis of anti-fatigue of mice[J].Science and Technology of Food Industry,2013,34(8):350-352.
[10] 张华峰,高征,罗亚飞,等.海蒿子活性多糖降血脂作用的研究[J].中成药,2009,31(12):1925-1927.Zhang HF,Gao Z,Luo YF,et al.Study on the effect of artemisia argyi active polysaccharide on blood lipid[J].Chinese Traditional Patent Medicine,2009,31(12):1925 -1927.
[11] 修琳琳.含反药组合的海藻玉壶汤中海藻不同品种与甘草加减对甲状腺肿大大鼠生物效应影响及机制探讨[D].北京:北京中医药大学,2017:59.Xiu LL.Biological effects and its mechanism of differ ent species of Sargassum and Glycyrrhiza uralensis in Haizao Yuhu Decoction in goiter rats with anti-drug combination[D].Beijing:Beijing University of Traditional Chinese Medicine,2017:59.
[12] 李娜,钟赣生,张晨,等.海藻玉壶汤中海藻不同品种与甘草加减应用对甲状腺肿大大鼠氧化应激及肝脏Nrf2/Ho-1通路的影响[J].北京中医药大学学报,2019,42(3):190-197.Li N,Zhong GS,Zhang C,et al.Influence of different special of Haizao and modified administration of Gan Cao in Haizao Yuhu Tang on oxidative stress and liver Nrf2/Ho-1 pathualg in rats with goiter[J].Journal of Bei jing University of Traditional Chinese Medicine,2019,42(3):190-197.
[13] 丁选胜,李欧.海藻、甘草及其不同比例配伍后的水提取物对实验性大鼠甲状腺肿的影响[J].中药药理与临床,2001,17(6):32-33.Ding XS,Li O.Effects of seaweed,licorice and different proportions of water extracts on experimental rat goiter[J].Pharmacology and Clinics of Chinese Materia Medica,2001,17(6):32-33.
[14] 朱春根,谢东浩,徐卫东,等.海藻甘草不同比例配伍对甲状腺肿大鼠甲状腺功能和形态的影响[J].齐齐哈尔医学院学报,2011,32(22):3610-3612.Zhu CG,Xie DH,Xu WD,et al.Effect of algae and liq uorice in various proportions on the thyroid function and morphology in goiters SD Rats[J].Journal of Qiqihar Medical College,2011,32(22):3610-3612.
[15] 谢东浩,马利华,徐卫东,等.海藻、甘草不同比例配伍对甲状腺相关调控因子Fas和Bcl-2的表达研究[J].南京中医药大学学报,2010,26(6):450-452.Xie DH,Ma LH,Xu WD,et al.Effect of combination of Herba Sargassii and Radix Glycyrrhizae Uralensis in different proportion on the expression of thyroid-related regulatory factors Fas and Bcl-2[J].Journal of Nanjing TCM University,2010,26(6):450-452.
[16] 高天舒,齐腾澈.海藻玉壶汤及其拆方对大鼠碘缺乏致甲状腺肿的干预作用[J].中医杂志,2012,53(19):1671 -1676.Gao TS,Qi TC.Intervention effect of Haizaoyuhu De coction and Its decomposed recipes for goiter caused by iodine deficiency[J].Journal of Traditional Chinese Medicine,2012,53(19):1671-1676.
[17] 陈杰,李甘地.病理学[M].北京:人民卫生出版社,2005:35-37.Chen J,Li GD.Pathology[M].Beijing:People’s Medical Publishing House,2005:35-37.
[18]Oltvai ZN,Milliman CL,Korsmeyer SJ.Bcl-2 het erodimerizes in vivo with a conserved homolog,Bax that accelerates programmed cell death[J].Cell,1993,74(4):609-619.