杜仲-刺蒺藜对老龄自发性高血压大鼠肠道微生物组的影响
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摘要
目的观察杜仲-刺蒺藜对自发性高血压大鼠(SHR)的血压及肠道微生物组的影响。方法选用18月龄雄性SHR 28只,将其随机分为对照组(SHR)和杜仲-刺蒺藜干预高、中、低剂量组(H、M、L)共4组,灌胃给药8周,每周测体质量、血压。8周后,酶联免疫吸附试验(ELISA)法检测血清氨基末端脑钠尿肽前体(NT-proBNP)、神经肽Y、C反应蛋白(CRP)、血管紧张素Ⅱ(AngⅡ)、去甲肾上腺素(NE)水平;实时荧光定量聚合酶链反应(qRT-PCR)法检测胸主动脉IκB激酶(IKK)α、IKK-β、核转录因子κB抑制蛋白(IκB)α、核转录因子(NF)-κB p65的mRNA表达水平;无菌原则取大鼠应激粪便样本,16S V4区扩增子测序与分析;采用气相色谱法检测粪便样本中短链脂肪酸(SCFA)的含量。结果高、中剂量杜仲-刺蒺藜干预后,大鼠血压明显低于SHR对照组[收缩压/舒张压:(170.3±10.7)/(114.0±6.6)、(171.6±10.4)/(112.4±7.3)比(203.8±11.4)/(136.8±8.0)mm Hg,均P<0.05];与对照组比较,杜仲-刺蒺藜中剂量组血清NT-proBNP[(321.5±31.4)比(471.7±61.9) ng/L]、神经肽Y[(2001.9±26.6)比(3826.7±68.4)ng/L]、CRP[(42.4±13.0)比(68.5±9.5)μg/mL]、AngⅡ[(481.9±21.4)比(591.6±88.7)ng/L]水平显著降低(均P<0.05),杜仲-刺蒺藜高剂量组血清神经肽Y水平[(2977.6±64.5)比(3826.7±68.4)ng/L,P<0.05]显著降低。高、中剂量杜仲-刺蒺藜显著改善胸主动脉中IκBα、IKKβ、NF-κB p65、IKKα的mRNA表达水平(均P<0.05)。药物干预后大鼠肠道微生物的组成、α多样性及β多样性发生改变,Anaeroplasma为杜仲-刺蒺藜中剂量组在属水平上的特征菌属。高、中剂量杜仲-刺蒺藜显著升高大鼠粪便乙酸、丙酸水平;低剂量杜仲-刺蒺藜显著升高大鼠粪便丁酸、异丁酸、戊酸、异戊酸水平(均P<0.05);Anaeroplasma丰度与CRP浓度(r=-0.85)、NF-κB p65水平(r=-0.78)相关(均P<0.05)。结论杜仲-刺蒺藜可降低老龄SHR血压、降低炎症水平,其降压机制与肠道菌群的组成和多样性的改变相关。
Objective To observe the effects of the bark of eucommia(Eucommia ulmoides Oliv.) and tribulus terrestris(Tribulus terrestris L.)(ET) on blood pressure and intestinal microbiota in spontaneously hypertensive rats(SHR). Methods A total of 28 18-month-old male SHR were randomly divided into control group, ET high, medium and low dose group. Rats in four groups were gavaged for 8 weeks. Blood pressure and body weight were measured weekly. Eight weeks later, serum level of N-terminal pro brain natriuretic peptide(NT-proBNP), neuropeptide Y(NPY), C reactive protein(CRP), angiotensin Ⅱ(AngⅡ) and norepinephrine(NE) were measured by enzyme-linked immunosorbent assay(ELISA), and the expression levels of IKK-α, IKK-β, IκBα and NF-κB p65 were detected by real-time fluorescence quantitative PCR(qRT-PCR). Stool samples were taken from rats under the aseptic operation principle, and the amplicon information of 16 S V4 region was collected and analyzed. The content of short chain fatty acids(SCFA) in stool samples was detected by gas chromatography. Results The blood pressure of rats in ET high and medium dose group were significantly lower than that of SHR in control group [systolic blood pressure/diastolic blood pressure:(170.3±10.7)/(114.0±6.6),(171.6±10.4)/(112.4±7.3) vs(203.8±11.4)/(136.8±8.0)mm Hg, P<0.05]. Compared with the control group, medium dose of ET significantly decreased the levels of NT-proBNP, NPY, CRP and AngⅡ [NT-proBNP:(321.5±31.4) vs(471.7±61.9)ng/L; NPY:(2001.9±26.6) vs(3826.7±68.4)ng/L, CRP:(42.4±13.0) vs(68.5±9.5)mg/L, AngⅡ:(481.9±21.4) vs(591.6±88.7)ng/L, P<0.05], high dose of ET significantly decreased NPY level [(2977.6±64.5) vs(3826.7±68.4)ng/L, P<0.05]. High and medium dose of ET can significantly change the expression of IκBα, IKKβ, NF-κB p65 and IKKα in thoracic aorta. The composition, alpha diversity and beta diversity of intestinal microbiota were significantly different after ET treatment. Anaeroplasma was characteristic microbiota of ET medium dose group at genus level. High and medium dose ET significantly increased the levels of acetic acid and propionic acid in stool samples, while low dose ET significantly increased the levels of butyric acid, isobutyric acid, valeric acid and isovaleric acid. The abundance of anaeroplasma was linearly correlated with CRP concentration(r=-0.85) and NF-κB p65 level(r=-0.78). Conclusion ET effectively reduced blood pressure and inflammation level in elderly SHR. The mechanism of antihypertension effect may be related to the changes of composition and diversity of intestinal microbiota.
Objective To observe the effects of the bark of eucommia(Eucommia ulmoides Oliv.) and tribulus terrestris(Tribulus terrestris L.)(ET) on blood pressure and intestinal microbiota in spontaneously hypertensive rats(SHR). Methods A total of 28 18-month-old male SHR were randomly divided into control group, ET high, medium and low dose group. Rats in four groups were gavaged for 8 weeks. Blood pressure and body weight were measured weekly. Eight weeks later, serum level of N-terminal pro brain natriuretic peptide(NT-proBNP), neuropeptide Y(NPY), C reactive protein(CRP), angiotensin Ⅱ(AngⅡ) and norepinephrine(NE) were measured by enzyme-linked immunosorbent assay(ELISA), and the expression levels of IKK-α, IKK-β, IκBα and NF-κB p65 were detected by real-time fluorescence quantitative PCR(qRT-PCR). Stool samples were taken from rats under the aseptic operation principle, and the amplicon information of 16 S V4 region was collected and analyzed. The content of short chain fatty acids(SCFA) in stool samples was detected by gas chromatography. Results The blood pressure of rats in ET high and medium dose group were significantly lower than that of SHR in control group [systolic blood pressure/diastolic blood pressure:(170.3±10.7)/(114.0±6.6),(171.6±10.4)/(112.4±7.3) vs(203.8±11.4)/(136.8±8.0)mm Hg, P<0.05]. Compared with the control group, medium dose of ET significantly decreased the levels of NT-proBNP, NPY, CRP and AngⅡ [NT-proBNP:(321.5±31.4) vs(471.7±61.9)ng/L; NPY:(2001.9±26.6) vs(3826.7±68.4)ng/L, CRP:(42.4±13.0) vs(68.5±9.5)mg/L, AngⅡ:(481.9±21.4) vs(591.6±88.7)ng/L, P<0.05], high dose of ET significantly decreased NPY level [(2977.6±64.5) vs(3826.7±68.4)ng/L, P<0.05]. High and medium dose of ET can significantly change the expression of IκBα, IKKβ, NF-κB p65 and IKKα in thoracic aorta. The composition, alpha diversity and beta diversity of intestinal microbiota were significantly different after ET treatment. Anaeroplasma was characteristic microbiota of ET medium dose group at genus level. High and medium dose ET significantly increased the levels of acetic acid and propionic acid in stool samples, while low dose ET significantly increased the levels of butyric acid, isobutyric acid, valeric acid and isovaleric acid. The abundance of anaeroplasma was linearly correlated with CRP concentration(r=-0.85) and NF-κB p65 level(r=-0.78). Conclusion ET effectively reduced blood pressure and inflammation level in elderly SHR. The mechanism of antihypertension effect may be related to the changes of composition and diversity of intestinal microbiota.
引文
[1] 亓英姿,姜月华,杨传华,等.肠道菌群与高血压的研究概况[J].中华高血压杂志,2018,26(5):412-416.
[2] 徐乐,郭子宏.肠道微生物与高血压的关系及其机制研究进展[J].中华高血压杂志,2018,26(3):214-218,200.
[3] 周丹,黄雨晴,余雪菊,等.肠道菌群与心血管病关系的研究进展[J].中华高血压杂志,2018,26(5):417-423.
[4] 冯晗,周宏灏,欧阳冬生.杜仲的化学成分及药理作用研究进展[J].中国临床药理学与治疗学,2015,20(6):713-720.
[5] 姜凌宇,姜月华,郭金昊,等.杜仲治疗高血压研究进展[J].山东中医杂志,2017,20(3):249-252.
[6] 郭金昊,姜月华,杨传华,等.刺蒺藜对老年自发性高血压大鼠胸主动脉血管重塑的影响[J].中医杂志,2016,57(11):957-961.
[7] 陈兴娟.补肾和脉降浊法治疗老年高血压肾损害经验总结[J].长春中医药大学学报,2012,28(1):77-78.
[8] 孟宪卿.肥胖性高血压大鼠肾脏损伤的生物学特征及刺蒺藜干预的机制研究[D].济南:山东中医药大学,2016.
[9] Yamamoto K,Hamada H,Shinkai H,et al.The KDEL receptor modulates the endoplasmic reticulum stress response through mitogen-activated protein kinase signaling cascades[J].J Biol Chem,2003,278 (36):34525-34532.
[10] 曾桥,韦承伯.杜仲叶药理作用及临床应用研究进展[J].药学研究,2018,37(8):482-486,489.
[11] 陈启洪,李晓飞,段灿灿,等.网络药理学探讨杜仲主要活性成分及药理作用机制[J].中药材,2018,41(2):419-426.
[12] 项丽玲,温亚娟,苗明三.杜仲叶的化学、药理及临床应用分析[J].中医学报,2017,32(1):99-102.
[13] 姜月华.刺蒺藜通过下调水孔蛋白2的表达改善肥胖相关性肾病大鼠水代谢紊乱的机制研究/中国中西医结合学会肾脏疾病专业委员会.2016年中国中西医结合学会肾脏疾病专业委员会学术年会论文摘要汇编[C].中国中西医结合学会肾脏疾病专业委员会,2016:1.
[14] Blacher J,Staessen JA,Girerd X,et al.Pulse pressure not mean pressure determines cardiovascular risk in older hypertensive patients[J].Arch Intern Med,2000,160(8):1085-1089.
[15] Pickard JM,Zeng MY,Caruso R,et al.Gut microbiota:role in pathogen colonization,immune responses,and inflammatory disease[J].Immunol Rev,2017,279(1):70-89.
[16] Tang WH,Kitai T,Hazen SL.Gut microbiota in cardiovascular health and disease[J].Circ Res,2017,120(7):1183-1196.
[17] 丁慧芳,薛萍,张宁.腹盆腔放线菌病误诊为卵巢癌[J].临床误诊误治,2017,30(4):14-15.
[18] 刘勇刚,施立煌,张子杨,等.牙周炎患者病原菌感染状况及RANKL/OPG变化[J].检验医学,2018,33(12):1123-1126.
[19] Adnan S,Nelson JW,Ajami NJ,et al.Alterations in the gut microbiota can elicit hypertension in rats[J].Physiol Genomics,2017,49(2):96-104.
[20] Nagel R,Peters RJ.Investigating the phylogenetic range of gibberellin biosynthesis in bacteria[J].Mol Plant Microbe Interact,2017,30(4):343-349.
[21] Guzow-Krzemińska B,Gasior T,Szalewska-Pa?asz A.Phylogenetic relationship of the stringent response-related genes of marine bacteria[J].Acta Biochim Pol,2015,62(4):773-783.
[22] Houghton D,Stewart CJ,Stamp C.Impact of age-related mitochondrial dysfunction and exercise on intestinal microbiota composition[J].J Gerontol A Biol Sci Med Sci,2018,73(5):571-578.
[23] Liang Y,Zhang Y,Deng Y,et al.Chaihu-Shugan-San decoction modulates intestinal microbe dysbiosis and alleviates chronic metabolic inflammation in NAFLD rats via the NLRP3 inflammasome pathway[J].Evid Based Complement Alternat Med,2018,2018:9390786.
[24] Andoh A.Physiological role of gut microbiota for maintaining human health[J].Digestion,2016,93(3):176-181.
[25] Hu J,Lin S,Zheng B,et al.Short-chain fatty acids in control of energy metabolism[J].Crit Rev Food Sci Nutr,2018,58(8):1243-1249.
[26] Hung TV,Suzuki T.Short-chain fatty acids suppress inflammatory reactions in Caco-2 cells and mouse colons[J].J Agric Food Chem,2018,66(1):108-117.
[27] 蒙丹丽,梁列新,宋怀宇.短链脂肪酸在肠道中的生理作用[J].中国临床新医学,2018,11(2):198-202.
[28] Liu C,Kellems RE,Xia Y.Inflammation,autoimmunity,and hypertension:the essential role of tissue transglutaminase[J].Am J Hypertens,2017,30(8):756-764.
[29] 赵晓亚,江振作,王跃飞.人的粪便、血浆、唾液、呼出气体中短链脂肪酸的分析[J].中国微生态学杂志,2015,27(2):130-134.
[30] Bloemen JG,Venema K,Van De Poll MC,et al.Short chain fatty acids exchange across the gut and liver in humans measured at surgery[J].Clin Nutr,2009,28(6):657-661.
[31] Van Eijk HM,Bloemen JG,Dejong CH.Application of liquid chromatography-mass spectrometry to measure short chain fatty acids in blood[J].J Chromatogr B,2009,877(8/9):719-724.
[2] 徐乐,郭子宏.肠道微生物与高血压的关系及其机制研究进展[J].中华高血压杂志,2018,26(3):214-218,200.
[3] 周丹,黄雨晴,余雪菊,等.肠道菌群与心血管病关系的研究进展[J].中华高血压杂志,2018,26(5):417-423.
[4] 冯晗,周宏灏,欧阳冬生.杜仲的化学成分及药理作用研究进展[J].中国临床药理学与治疗学,2015,20(6):713-720.
[5] 姜凌宇,姜月华,郭金昊,等.杜仲治疗高血压研究进展[J].山东中医杂志,2017,20(3):249-252.
[6] 郭金昊,姜月华,杨传华,等.刺蒺藜对老年自发性高血压大鼠胸主动脉血管重塑的影响[J].中医杂志,2016,57(11):957-961.
[7] 陈兴娟.补肾和脉降浊法治疗老年高血压肾损害经验总结[J].长春中医药大学学报,2012,28(1):77-78.
[8] 孟宪卿.肥胖性高血压大鼠肾脏损伤的生物学特征及刺蒺藜干预的机制研究[D].济南:山东中医药大学,2016.
[9] Yamamoto K,Hamada H,Shinkai H,et al.The KDEL receptor modulates the endoplasmic reticulum stress response through mitogen-activated protein kinase signaling cascades[J].J Biol Chem,2003,278 (36):34525-34532.
[10] 曾桥,韦承伯.杜仲叶药理作用及临床应用研究进展[J].药学研究,2018,37(8):482-486,489.
[11] 陈启洪,李晓飞,段灿灿,等.网络药理学探讨杜仲主要活性成分及药理作用机制[J].中药材,2018,41(2):419-426.
[12] 项丽玲,温亚娟,苗明三.杜仲叶的化学、药理及临床应用分析[J].中医学报,2017,32(1):99-102.
[13] 姜月华.刺蒺藜通过下调水孔蛋白2的表达改善肥胖相关性肾病大鼠水代谢紊乱的机制研究/中国中西医结合学会肾脏疾病专业委员会.2016年中国中西医结合学会肾脏疾病专业委员会学术年会论文摘要汇编[C].中国中西医结合学会肾脏疾病专业委员会,2016:1.
[14] Blacher J,Staessen JA,Girerd X,et al.Pulse pressure not mean pressure determines cardiovascular risk in older hypertensive patients[J].Arch Intern Med,2000,160(8):1085-1089.
[15] Pickard JM,Zeng MY,Caruso R,et al.Gut microbiota:role in pathogen colonization,immune responses,and inflammatory disease[J].Immunol Rev,2017,279(1):70-89.
[16] Tang WH,Kitai T,Hazen SL.Gut microbiota in cardiovascular health and disease[J].Circ Res,2017,120(7):1183-1196.
[17] 丁慧芳,薛萍,张宁.腹盆腔放线菌病误诊为卵巢癌[J].临床误诊误治,2017,30(4):14-15.
[18] 刘勇刚,施立煌,张子杨,等.牙周炎患者病原菌感染状况及RANKL/OPG变化[J].检验医学,2018,33(12):1123-1126.
[19] Adnan S,Nelson JW,Ajami NJ,et al.Alterations in the gut microbiota can elicit hypertension in rats[J].Physiol Genomics,2017,49(2):96-104.
[20] Nagel R,Peters RJ.Investigating the phylogenetic range of gibberellin biosynthesis in bacteria[J].Mol Plant Microbe Interact,2017,30(4):343-349.
[21] Guzow-Krzemińska B,Gasior T,Szalewska-Pa?asz A.Phylogenetic relationship of the stringent response-related genes of marine bacteria[J].Acta Biochim Pol,2015,62(4):773-783.
[22] Houghton D,Stewart CJ,Stamp C.Impact of age-related mitochondrial dysfunction and exercise on intestinal microbiota composition[J].J Gerontol A Biol Sci Med Sci,2018,73(5):571-578.
[23] Liang Y,Zhang Y,Deng Y,et al.Chaihu-Shugan-San decoction modulates intestinal microbe dysbiosis and alleviates chronic metabolic inflammation in NAFLD rats via the NLRP3 inflammasome pathway[J].Evid Based Complement Alternat Med,2018,2018:9390786.
[24] Andoh A.Physiological role of gut microbiota for maintaining human health[J].Digestion,2016,93(3):176-181.
[25] Hu J,Lin S,Zheng B,et al.Short-chain fatty acids in control of energy metabolism[J].Crit Rev Food Sci Nutr,2018,58(8):1243-1249.
[26] Hung TV,Suzuki T.Short-chain fatty acids suppress inflammatory reactions in Caco-2 cells and mouse colons[J].J Agric Food Chem,2018,66(1):108-117.
[27] 蒙丹丽,梁列新,宋怀宇.短链脂肪酸在肠道中的生理作用[J].中国临床新医学,2018,11(2):198-202.
[28] Liu C,Kellems RE,Xia Y.Inflammation,autoimmunity,and hypertension:the essential role of tissue transglutaminase[J].Am J Hypertens,2017,30(8):756-764.
[29] 赵晓亚,江振作,王跃飞.人的粪便、血浆、唾液、呼出气体中短链脂肪酸的分析[J].中国微生态学杂志,2015,27(2):130-134.
[30] Bloemen JG,Venema K,Van De Poll MC,et al.Short chain fatty acids exchange across the gut and liver in humans measured at surgery[J].Clin Nutr,2009,28(6):657-661.
[31] Van Eijk HM,Bloemen JG,Dejong CH.Application of liquid chromatography-mass spectrometry to measure short chain fatty acids in blood[J].J Chromatogr B,2009,877(8/9):719-724.