【作者】 杨强；

【导师】 王文健； 何燕铭；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2012， 博士

【摘要】 目的1.建立高盐高脂饮食诱导的盐敏感性高血压大鼠模型,并观察模型大鼠是否存在肾素-血管紧张素系统激活,以及是否伴有胰岛素抵抗等状态；2.研究补肾中药对盐敏感性高血压模型大鼠血压以及肾素-血管紧张素系统和肾功能等指标的影响,了解其是否能降低模型大鼠血压、抑制肾素-血管紧张素系统激活以及对肾功能是否有改善作用；3.研究补肾中药对盐敏感性高血压模型大鼠伴有的胰岛素抵抗、促炎、促凝状态等的影响,观察中药对这些病理状态是否有改善作用；4.通过研究补肾中药对肾素-血管紧张素系统与胰岛素抵抗等环节的影响,探讨中药可能的作用机制,为盐敏感性高血压的治疗探索可行途径,阐明“咸入肾”理论的科学内涵。方法1.高盐高脂饮食诱导的盐敏感性高血压大鼠模型的建立。清洁级SD大鼠30只,雄性,6周龄。适应性饲养10天,随机分为4组：正常饲料组(CON)5只,高盐组(HS)5只,高脂组(HF)5只,高盐高脂组(HSF)15只。各组大鼠自由饮水,分别对应给予普通饲料、高盐饲料、高脂饲料和高盐高脂饲料。每周用尾套法测血压,2周后,隔夜空腹12小时,用毛细管眼眶后静脉丛采血,测血糖、胰岛素、血脂、肾素活性、血管紧张素Ⅱ、血管紧张素Ⅰ转化酶,计算HOMA-IR、ISI;代谢笼收集24小时尿液,测尿钠、尿钾、尿醛固酮、尿微量白蛋白,计算大鼠24h尿微量白蛋白排泄率。2.研究补肾中药对盐敏感性高血压模型大鼠血压、肾素-血管紧张素系统和肾功能等的干预作用。动物分组：在饮食干预2周后,将高盐高脂组(HSF)大鼠随机分为3组,每组5只大鼠：HSF组(不予药物干预)、缬沙坦组(VAL)和六味地黄组(LW),连同原来的正常饲料组,共为4组。缬沙坦剂量为13.33mg/kg/d(体重),六味地黄组剂量为8.13g/kg/d(体重)。缬沙坦、六味地黄方每天灌胃1次,持续6周。实验过程中,正常饲料组大鼠予普通饲料,其余各组大鼠均继续给予高盐高脂饲料。每周测体重、用尾套法测量血压。药物干预后的第6周,隔夜空腹12小时后采血,测血液中肾素活性、血管紧张素Ⅱ、血管紧张素Ⅰ转化酶；代谢笼收集24小时尿液,测尿量、尿钠、尿钾、尿醛固酮、尿微量白蛋白,计算大鼠24小时尿钠、尿钾排泄量以及24小时尿微量白蛋白排泄率等。取肾脏组织-80℃冻存,RT-PCR检测Renin、AngⅡ、AT1R,并做石蜡包埋切片,HE染色,进行组织病理学形态观察。3.研究补肾中药对盐敏感性高血压模型大鼠伴有的胰岛素抵抗及促炎、促凝状态等的作用。动物分组和造模方法同前。饮食干预8周(药物治疗6周)后,隔夜空腹12小时后采血,生化法检测血液中血糖、血脂四项(TC、TG、HDL-c、LDL-c)等；ELISA法检测血液中胰岛素、瘦素(LEP)、脂联素(ADP)、组织纤溶酶原激活物(t-PA)、纤溶酶原激活物抑制剂1(PAI-1)、C反应蛋白(CRP)、肿瘤坏子因子-α(TNF-α)、白介素-6(IL-6)、游离脂肪酸(FFA)等,计算HOMA-IR和ISI。结果1.高盐高脂饮食诱导的盐敏感性高血压大鼠模型的建立。与正常饲料组大鼠相比,各组大鼠在高盐或／和高脂饮食干预2周后,血压显著升高,其中高盐高脂组血压升高最多(119.4±4.7vs.94.0±2.2mniHg, p<0.05);经过饮食干预2周后,高盐高脂组血AngⅡ显著降低(66.39±3.62vs.82.16±14.68ng/L,p<0.05),其它循环RAS组分(PRA、ACE)变化不大。在尿电解质方面,高盐高脂组24小时尿钠排泄量显著升高(1.92±0.48vs.0.75±0.18mmol/L,p<0.05),24小时尿钾排泄量显著升高(1.49±0.23vs.0.98±0.28mmol/L,p<0.05),尿钾／尿钠比值较正常饲料组显著降低(0.80±0.16vs.1.35±0.49,p<0.05)。高盐高脂组尿微量白蛋白排泄率显著升高(22.16±4.01vs.6.66±1.17ug/L,p<0.05)。在胰岛素敏感性方面,与正常饲料组大鼠相比,各组大鼠在高盐或／和高脂饮食干预2周后,空腹血糖显著升高,其中高盐高脂组升高最明显(3.66±0.39vs.2.60±0.52mmol/L,p<0.05);空腹血胰岛素水平与血糖水平的变化趋势基本相同,同样是高盐高脂组升高最明显(3.83±0.29vs.2.99±0.23mIU/L,p<0.05);高盐高脂组HOMA-IR显著升高(p=0.000),ISI显著降低(p=0.001)。上述血压、循环RAS活性、肾脏功能和胰岛素敏感性等方面指标的结果表明：高盐高脂饮食诱导盐敏感性高血压大鼠模型成功,可用于本课题的实验研究。2.研究中药对盐敏感性高血压模型大鼠血压以及肾素-血管紧张素系统和肾功能相关指标的影响。经过1周治疗后,与高盐高脂组相比,缬沙坦组(VAL)血压就有显著降低(89.2±4.8vs.117.6±9.3mmHg,p<0.05),在治疗第2周,六味地黄组(LW)显示出降压效果(105.2±8.8vs.118.6±5.3mmHg,p<0.05);治疗6周后,LW显著降低血压至正常水平(103.0±8.1vs.118.4±6.3mmHg,p<0.05)。在循环RAS方面,饮食干预2周,与正常饲料组大鼠相比,高盐高脂组AngⅡ显著降低(66.39±3.62vs.82.16±14.68nG/L,p<0.05),至饮食干预第8周,高盐高脂组AngⅡ较正常饲料组有所下降,但无统计学意义；经过6周药物治疗,LW及VAL均能显著升高AngⅡ浓度。而其它RAS组分如血管紧张素转换酶(ACE)、肾素活性(PRA)各组间无显著差异。饮食干预2周,与正常饲料组大鼠相比,高盐高脂组尿醛固酮(UALD)显著上升(135.02±9.93vs.101.27±13.30ng/L,p<0.05),经过6周药物治疗,LW与VAL均可显著降低UALD至正常水平。在肾脏局部RAS方面,经过8周的高盐高脂饮食干预,与正常饲料组大鼠相比,高盐高脂组大鼠肾脏局部AngⅡ、肾素、血管紧张素Ⅱ受体Ⅰ型(AT1R) mRNA的表达水平都显著升高。经过6周的药物治疗,与高盐高脂组相比,LW能显著抑制肾脏RAS的激活状态,与VAL效果相当。在肾功能方面,经过8周的高盐高脂饮食干预,与正常饲料组大鼠相比,高盐高脂组大鼠24小时尿钠排泄量(p=0.007)、尿钾排泄量显著增多(p=0.017),而尿钾／尿钠比值偏低,提示在高钠摄入的情况下,高盐高脂组大鼠仍存在钠潴留现象；经过6周的药物治疗,与高盐高脂组大鼠相比,LW能促进尿钠的排出及减少尿钾流失,疗效与VAL相当。尿微量白蛋白(MAU)检测结果表明,经过8周的高盐高脂饮食干预,与正常饲料组大鼠相比,高盐高脂组大鼠MAU显著升高(p=0.001)；经过6周的药物治疗,与高盐高脂组相比,LW能显著降低MAU水平(p=0.010),与VAL疗效相当(p=0.008)。。肾脏组织HE染色结果表明,高盐高脂组大鼠可见肾小球入球动脉轻度玻璃样变性,其余各组肾小球入球动脉、小动脉和肾单元未见显著改变。血肌酐、尿素氮各组间无显著差异,说明我们采用的造模方法是造成以血压升高为主伴有轻度肾脏损害的动物模型,与临床上大部分SSHT患者的病变程度相似。3.研究补肾中药对盐敏感性高血压模型大鼠伴有的胰岛素抵抗及其相关机制的作用。在糖脂代谢方面,至饮食干预第8周时,高盐高脂组大鼠血糖和血胰岛素水平进一步升高,与正常饲料组大鼠相比差异显著(分别为5.55±0.62mmol/L vs.2.87±0.77mmol/L, p<0.05和4.92±1.34mIU/L vs.2.79±0.26mIU/L,p<0.05),血脂方面,TC、TG与LDL-c各组之间无显著差异；而经过6周的药物治疗,与高盐高脂组大鼠相比,VAL组的血糖(p=0.000)及血胰岛素(p=0.005)浓度下降至正常,LW的血糖水平显著下降(p=0.000)；ISI及HOMA-IR结果表明,LW与VAL均能改善胰岛素敏感性及降低IR程度(p<0.05)。在相关脂肪细胞因子方面,治疗6周后,高盐高脂组与正常饲料组大鼠相比,瘦素(p=0.047)、PAI-1显著升高(p=0.031),t-PA显著降低(p=0.037)；CRP. TNF-α、IL-6、ADP、FFA各组之间无显著差异。经过6周的药物治疗,与高盐高脂组相比,LW方能显著降低瘦素、PAI-1至正常水平,并升高tPA至正常水平,与VAL疗效相当。结论1.高盐高脂饮食干预可诱导出稳定可靠的盐敏感性高血压大鼠模型,该方法简便易行,较好的模拟了现代饮食习惯,适用于改善盐敏感性高血压药物筛选及机制的研究。2.六味地黄方显著降低盐敏感性高血压模型大鼠血压,疗效与缬沙坦相当；对循环RAS的抑制有良性调节作用,抑制肾局部RAS激活；对该模型大鼠的钠潴留及尿钾流失现象也有改善作用,并减少尿微量白蛋白排泄率。3.六味地黄方显著降低盐敏感性高血压模型大鼠血糖、血胰岛素水平,提高胰岛素敏感性,降低HOMA-IR;对脂肪细胞因子及纤溶功能紊乱有显著的良性调节作用。总之,高盐高脂饮食干预可诱导出稳定可靠的盐敏感性高血压大鼠模型,六味地黄方可用于盐敏感性高血压的治疗,显著降低该模型大鼠血压、抑制肾脏RAS激活及改善IR,其作用机制可能与拮抗肾脏RAS、改善肾功能、改善糖脂代谢紊乱及改善纤溶功能紊乱等有关,体现了中医药多环节、多靶点综合作用的特点,为“咸入肾”及从肾论治盐敏感性高血压提供了可靠的实验依据。更多还原

【Abstract】 Objective1. To establish rat model of high salt and high fat diet induced salt sensitive hypertension (SSHT); To observe the Renin-Angiotensin system (RAS) activity as well as insulin resistance (IR) in SSHT model rats.2. To investigate the effects of Chinese herbal medicine on reducing blood pressure, inhibiting RAS activity, improve kidney metabolism and kidney function in SSHT rats.3. To investigate the effects of Chinese herbal medicine on ameliorating IR and other aspects of SSHT rats, such as inflammatory reaction, procoagulant and etc.4. To investigate possible mechanism of Chinese herbal medicine from the point of view of RAS and IR; To pave new way to the treatment of SSHT; To scientifically elucidate the theory of "salty flavor entering the kidney".Method1. Establishing rat model of high salt and high fat diet induced salt sensitive hypertension.30male SD rat,6weeks old and acclimate for about10days before study. Randomly divided into4groups:Control group (CON, n=5); High salt group (HS, n=5); High fat group (HF, n=5); High salt and fat group (HSF, n=15). All rats are allowed access to drinking water and subjected to different dietary regimens as described below. CON-normal pellet diet; HS-high salt diet; HF-high fat diet; HSF-high salt and high fat diet. Measure blood pressure weekly by tail-cuff method. Two weeks later, rats are overnight fasting (12h) and blood was collected from retro-orbital vein, followed by glucose, insulin, blood fat, Plasma renin activity (PRA), Angiotensin Ⅱ (Ang Ⅱ), ACE (Angiotensin-converting enzyme) measurement. HOMA-IR and ISI are calculated accordingly.24h urine is collected by metabolic cages. Urine Na+and urine K+, Aldosterone (ALD), Microalbuminuria (MAU) are measured and excretion rate are calculated accordingly.2. To investigate the effects of Chinese herbal medicine on inhibiting RAS activity, improve kidney metabolism and kidney function in SSHT rats.Grouping:two weeks after diet intervention, HSF rats are randomly divided into three groups, five rats in each group and together4groups as the following:HSF group (non-diet intervention); Valsartan (VAL) group; Liu-Wei-Di-Huang-Fang (LW) group and the control groups. Doing regime is as follows:VAL,13.33mg/kg/d, i.g. QD; LW,8.13g/kg/d, i.g. QD. Both drugs are treated for6weeks. HSF diet are provided to all HSF rats during the treatment except control group with the normal diet. Body weight and blood pressure by tail-cuff method are measured weekly. Six weeks later, rats are overnight fasting (12h) and blood was collected, followed by PRA, AngⅡ, ACE measurement.24h urine are collected by metabolic cages. Urine volume, Na+, K+, ALD, MAU are measured and excretion rate of MAU, Urine Na+and K+are calculated accordingly. All rats are sacrificed at the end of study and kidney are kept at-80℃for further analysis. RT-PCR is carried for determining renin, Ang II receptor type I (AT1R), AngⅡ mRNA expression level in kidney. And HE staining was used for histopathology analysis.3. To investigate the effects of Chinese herbal medicine on ameliorating IR, inflammatory reaction and procoagulant activity.SSHT model establishment and grouping is as described above. After dietary change for8weeks (drug treated for6weeks), blood are collected after overnight fasting (12h), followed by biochemical analysis of glucose and blood fat (TC, TG, LDL-c, HDL-c). ELISA was performed to determine insulin, Leptin (LEP), Adiponectin (ADP), Tissue plasminogen activator (t-PA), Plasminogen activator inhibitor1(PAI-1), C-reactive protein (CRP), Tumor necrosis factor-a (TNF-a), Interleukin-6(IL-6) and Free fatty acid (FFA) in blood. HOMA-IR and ISI are calculated accordingly.Results1. Establish rat model of high salt and high fat induced SSHT.Compared with CON group, the blood pressure of all other groups increased significantly after2weeks of dietary change, among which HSF group shows highest blood pressure (119.4±4.7vs.94.0±2.2mmHg, p<0.05); In HSF group, AngⅡ level in the blood was significantly reduced however (66.39±3.62vs.82.16±14.68ng/L, p<0.05), there is no significant change of RAS indicator in other groups. Regarding to urine electrolyte,24h urine Na+excretion in HSF group was significantly increased.24h urine K+excretion (1.49±0.23vs.0.98±0.28mmol/L, p<0.05) in HSF group is very similar to urine Na+excretion (1.92±0.48vs.0.75±0.18mmol/L, p<0.05). The ratio of urine K+/urine Na+in HSF group is lower than CON group (0.80±0.16vs.1.35±0.49,p<0.05). UMAU excretion as another sensitive marker of early kidney injury in HSF group is also higher than CON group (22.16±4.01vs.6.66±1.17ug/L, p<0.05). In insulin sensitivity, compared with the CON group, all the other group shows obvious increase in fasting blood glucose after2weeks’ diet intervention on high salt or/and high fat and HSF group shows the highest increase (3.66±0.39vs.2.60±0.52mmol/L, p<0.05). Also HSF group shows obvious increase in HOMA-IR (p=0.000) and decreased in ISI (p=0.001). All the above data like blood pressure, RAS activity, kidney function and insulin sensitivity shows that we successfully establish rat model of SSHT induced by HSF, which can be used in the following pharmacology evaluation of Chinese herbal medicine.2. To investigate the effects of Chinese herbal medicine on reducing blood pressure, inhibiting RAS activity, improve kidney metabolism and kidney function in SSHT rats.After one week treatment, compared with HSF group, VAL shows greater decrease in blood pressure (89.2±4.8vs.117.6±9.3mmHg, p<0.05) and after two weeks treatment, LW shows effect in decreasing blood pressure (105.2±8.8vs.118.6±5.3mmHg,p<0.05); After6weeks treatment, blood pressure returned to the normal level after LW treatment (103.0±8.1vs.118.4±6.3mmHg,p<0.05).Regarding to circulating RAS, after2weeks’diet, compared with CON group, HSF group shows obvious decrease in Ang Ⅱ (66.39±3.62vs.82.16±14.68ng/L, p<0.05) and on the eighth week on diet intervention, Ang Ⅱ has come back to base line. However, there’s no significance difference in ACE, PRA and others RAS indicators. After2weeks’diet, compared with CON group, HSF group shows obvious rise in UALD (135.02±9.93vs.101.27±13.30ng/L, p<0.05) and after6weeks treatment, both of LW and VAL can bring down UALD to the base line.Regarding to local kidney RAS, after8weeks diet intervention on high salt and high fat, compared to CON group, HSF group shows obvious mRNA expression on Ang II, rennin and AT1R. After6week drug treatment, compared with HSF group, LW and VAL show the equal effect on inhibiting kidney RAS activation.Concerning kidney function, after8week diet intervention on high salt and high fat, compared with CON group,24h urine Na+(p=0.007) and urine K+(p=0.017) in HSF group is significantly increased, while the ratio urine Na+/urine K+is relatively low, which indicates the retention of Na+when having diet intervention on high Na+treatment. After6week drug treatment, compared with HSF group, LW and VAL can have equal effect on promoting excretion of urine Na+and decreasing the loss of urine K+. The MAU data shows that, after6weeks drug treatment, compared with HSF group, LW and VAL can obvious decrease MAU level. HE results show that, HSF group shows slight hyaline degeneration in glomerular afferent arteriole and there is no significant change in glomerular afferent arteriole, arterioles and each kidney unit. Another two kidney injury indexes, Serum Creatinine and Urea Nitrogen were not seen significant change among the groups and indicate our rat model is created to cause blood pressure rise and slight kidney function injuries, lesions of which is similar to the actual clinical patients.3. To investigate the effects of Chinese herbal medicine on ameliorating IR and other aspects of SSHT rats.After8weeks of diet intervention, blood glucose and insulin level are continuing rising in HSF group, which shows obvious difference compared with CON group (5.55±0.62mmol/L vs.2.87±0.77mmol/L, p<0.05and4.92±1.34mIU/L vs.2.79±0.26mIU/L, p<0.05), but no significance difference for TC、TG、LDL-c; After6weeks drug treatment, compared with HSF group, blood glucose (p=0.000) and insulin level (p=0.005) are back to base line in VAL and blood glucose (p=0.000) is significantly deceased in LW. Regarding ISI and HOMA-IR results, both of LW and VAL can improve insulin sensitivity and IR (p<0.05).Regarding adipose cell cytokine, after6weeks treatment, obvious increase in LEP (p=0.047) and PAI-1(p=0.031) and decrease in t-PA (p=0.037) in HSF group compared to the CON group; there’s no significant difference between CRP、TNF-α、 IL-6、ADP、FFA in each group. After6week drug treatment, compared with HSF group, LW and VAL have equal effect on significantly decreasing LEP and PAI-1and rise t-PA back to base line.Conclusion1. HSF diet can be used to induce reliable rat model of SSHT. It is easy to carry out and mimic modern dietary habit, which can be used to improve anti-SSHT drug as well as mechanism study.2. LW can significantly reduce blood pressure of SSHT model rats which is as good as VAL; inhibit local RAS activation vigorously; reduce urine ALD level; LW can also improve the high urine Na+excretion and urine K+loss. LW has effect on UMAU too.3. LW shows significant effect on improving IR, deducing blood glucose, insulin and HOMA-IR; It has good effect on the cytokine release and fibrinolytic function, too.In summary, HSF diet can induce reliable rat model of SSHT. LW can be used for the treatment of SSHT. It has obvious effect on the reducing blood pressure, inhibit renal RAS activation and improving IR.The underlying mechanism is probably related to RAS antagonism, improve kidney function, improve the glucose metabolism and fibrinolytic function etc, which reflects the multiple factors and target of Chinese herbal medicine. It provides reliable evidence to the treatment of SSHT from the point of view of "Salty flavor entering the kidney".更多还原