牛磺酸对大鼠高血压预防作用及其机理的研究
摘要
牛磺酸是心血管组织中含量很高的游离氨基酸之一,但其对动物高血压的预防作用机制尚不清楚。本文通过五个试验探讨了牛磺酸对大鼠高血压的预防作用及其机制。
试验一通过添加不同水平的牛磺酸和N-硝基-L-精氨酸甲酯(N-nitro-L-argininemethylester,L-NAME,NO合酶抑制剂),探讨了牛磺酸对L-NAME致高血压发生的预防作用。将100只雄性Wistar大鼠随机分为五组,对照组饮用自来水,模型Ⅰ组大鼠饮水中添加75mg/100ml L-NAME,模型Ⅱ组在饮水中添加75mg/100ml L-NAME的同时添加1%的β-丙氨酸,预防Ⅰ、Ⅱ组饮水中添加75mg/100ml L-NAME的同时分别添加1%、2%的牛磺酸。每两天测定大鼠尾动脉收缩压。结果证明:模型Ⅰ、Ⅱ组大鼠血压在造模第一周时明显升高,在以后的三周里血压持续升高,模型Ⅱ组大鼠血压略高于模型Ⅰ组差异不显著。预防Ⅰ组血压上升幅度较模型组小,预防Ⅱ组血压较对照组差异不显著。结果表明:用L-NAME处理大鼠建立大鼠高血压模型,牛磺酸可降低L-NAME所致血压升高的幅度。
试验二选取试验一中的各组试验动物,分别在第三周和第四周末处死,测定血清钙离子(Ca~(2+))、钠离子(Na~+)、肿瘤坏死因子(Tumor necrosis factor,TNF-α)、胰岛素样生长因子-Ⅰ(insulin-like growth factor,IGF-Ⅰ)的含量,血浆肾素活性(Plasma reninactivity,PRA)、肾上腺髓质素(Adrenomedullin,ADM)、降钙素基因相关肽(Calcitonin generelated peptide,CGRP)、醛固酮(aldosterone,ALD)的含量,心肌环一磷酸腺苷(Cyclic3',5'-adenosine monophosphate,cAMP)、环一磷酸鸟苷(cyclic 3',5'-guanosinemonophosphate,cGMP)含量。结果证明:模型组大鼠血中TNF-α、ALD含量和PRA活性与对照组相比显著升高(p＜0.05),预防组与模型组相比下降幅度显著(p＜0.05),预防Ⅱ组下降幅度强于预防Ⅰ组;模型组大鼠CGRP、cGMP含量与对照组相比显著下降(p＜0.05),两个预防组与模型组相比上升幅度显著(p＜0.05),预防Ⅱ组升高强于预防Ⅰ组;ADM、IGF-I、Ca~(2+)、Na~+、cAMP含量变化均不显著(p＞0.05)。结果表明:用L-NAME处理使大鼠血压升高,预防性应用牛磺酸可通过抑制肾素-血管紧张素-醛固酮系统(renin-angiotensin-aldosterone system,RAAS)活性,阻止细胞因子PRA、TNF-α、ALD的分泌,升高血中CGRP和心肌cGMP含量等机制来抑制血压的升高。
试验三分离试验一中各组大鼠腹主动脉,将其放在固定液中保存,修整大小,做常规石蜡切片,并用苏木素-伊红(HE)法染色,在普通光镜下观察各试验组大鼠腹主动脉VSMC增殖情况。结果证明:模型组大鼠腹主动脉中层VSMC大量增殖,血管壁明显增厚;预防Ⅰ组VSMC数量较模型组少;预防Ⅱ组VSMC较对照组仅有少量增殖。结果表明:牛磺酸可明显改善VSMC增殖,且2%牛磺酸抑制VSMC增殖的效果好于1%牛磺酸,这可能是牛磺酸预防大鼠高血压发生的另一机制。
试验四通过体外培养股动脉VSMC细胞,并在培养液中添加不同浓度的牛磺酸(0.1μg/mL、1μg/mL、10μg/mL、100μg/mL、500μg/mL、1000μg/mL)和AngⅡ(1×10~(-7)moL/L),采用MTT比色法测定细胞活力,初步探讨了牛磺酸对体外培养VSMC细胞增殖的作用及其机制。结果证明:培养液中添加适量的牛磺酸能抑制VSMC细胞的增殖。结果表明:牛磺酸也可通过抑制AngⅡ所致的VSMC异常增殖,从而预防高血压的发生。
试验五选取试验一中的各组试验动物在四周后处死,取各组大鼠心肌、肺脏和肾上腺组织,提取总RNA,采用SYBRⅠ实时荧光定量RT-PCR技术测定牛磺酸对心肌肾上腺素能受体β2(beta2-adrenergic receptor,β2-AR)mRNA、腺苷酸环化酶(Adenylyl cyclase,AC5)mRNA、心肌和肺脏血管紧张素转换酶(angiotensin-converting-enzyme,ACE)mRNA、肾上腺血管紧张素原(angiotensinogen,AGT)mRNA、肾上腺血管紧张素Ⅱ受体(renin-angiotensinⅡ-receptor,ATR)的ATR1a及ATR2型的mRNA表达水平影响。结果证明:与模型组相比,牛磺酸可明显降低心肌和肺脏中ACE mRNA的表达水平,可显著降低肾上腺AGT和肾上腺ATR1a mRNA的表达,可显著提高ATR2 mRNA的表达,而牛磺酸对β2-AR和(AC_5)没有明显影响。结果表明:牛磺酸可能是通过降低心肌和肺脏中ACE、肾上腺AGT与肾上腺ATR1a mRNA表达水平及提高肾上腺ATR2 mRNA表达水平,直接或间接地减少AngⅡ生成,抑制ATR1a产生和增加ATR2产生而起到预防高血压的作用。
Taurine was found to be one of the most abundant free amino acids in cardiovascular tissues of animals,but its preventive effects on hypertension is still obscure.In the present study,the effects of taurine on hypertension was investigated.
In experiment 1,male rats were given tap water containing taurine and L-NAME(inhibitor of NOS),then the preventive effect and mechanism of taurine on hypertensive induced by L-NAME were investigated.100 male wistar rats were randomly divided into five groups. The rats in the control group were given normal diet and tap water.The rats in the model groupⅠwere given normal diet and tap water containing 75mg/100ml of L-NAME,model groupⅡwere given normal diet and tap water containing 75mg/100ml of L-NAME and 1%β-Ala.Prevention groupⅠwere given normal diet and tap water containing 75mg/100ml of L-NAME and 1%taurine.Prevention groupⅡwere given normal diet and tap water containing 75mg/100ml of L-NAME and 2%taurine.Blood pressure was detected using tail-cuff method every two days.The results showed that 1 week later,blood pressure in the model group increased and the increase lasted in the following three weeks.There is no significant difference between the modelⅠandⅡgroups,and the blood pressure of model groupⅡis a little higher than that of the model groupⅠ.Blood pressure in prevention groupⅠwas lower compared with the model groups and there is no significant difference between prevention groupⅡand the control group.The results indicated that the model of hypertensive rats was established by L-NAME which could induce hypertension.Taurine could inhibit the elevation of blood pressure.
In experiment 2,experimental animals were selected from experimentⅠ.The rats were killed respectively at the end of the third and the fourth weeks.The contents of serum calciumion,sodiumion,TNF-α,IGF-Ⅰ,plasma PRA,ADM,CGRP,ALD and the contents of cAMP,cGMP in cardiac muscle were detected.The results showed that the contents of TNF-α, ALD and PRA increased significantly compared with the control group(p＜0.05),the prevention groups significantly decreased in model groups(p＜0.05),the effect of prevention groupⅡis better than that of the prevention groupⅠ,the contents of CGRP and cGMP significantly decreased compared with the control group(p＜0.05),the prevention groups significantly increased the in model groups(p＜0.05),the effect of prevention groupⅡis better than that of prevention groupⅠ,but there was significant differences in the level of ADM, IGF-Ⅰ,Ca~(2+),Na~+ ana cAMP(p＞0.05).Conclusion:the preventive mechanisms of taurine on
hypertension may due to the following reasons:it could restrain RAAS activity,inhibit the secretion of cell factor PRA,TNF-αand ALD secretion,increase the plasma level of CGRP and the level of cGMP in ardiac muscle.
In experiment 3,abodominal aortas selected from experimentⅠwere fixed in phosphate buffered formalin solutions and were embeded in paraffin wax.Sections were cut and stained with haematoxylin and eosin(HE).Proliferations of vascular smooth muscle cells(VSMC) were observed under optical microscope.The results showed:VSMC in the model groups significantly proliferated,the vessel wall thickened significantly.The number of VSMC in prevention groupⅠwas less than that of the model groups.The VSMC in prevention groupⅡproliferated a little.The results indicated that taurine could inhibit the proliferation of VSMC obviously and the effect of 2%taurine is better than that of 1%tauine,which can be regarded as another preventive mechanisms of taurine on hypertension.
In experiment 4,MTT method was used to study the effects of taurine on the proliferation of VSMC.In this experiment,taurine(0.1μg/mL,1μg/mL,10μg/mL,100μg/mL,500μg/mL,1 000μg/mL) and AngⅡ(1×10~(-7) moL/L) were addes in the culture fluid of VSMC in arteria femoralis.The results showed that taurine could significantly inhibit the proliferation of VSMC,which indicated that taurine could inhibite the proliferation of VSMC induced by AngⅡ.
In experiment 5,rats selected from experimentⅠwere killed at the end of the fourth week. At the same time,cardiac muscle,lung and adrenal gland were taken out promptly,then total RNA were extracted.The influence of taurine on the expression of receptors ofβ2-AR mRNA, AC5 mRNA,cAMP mRNA in cardiac muscle,ACE mRNA in cardiac muscle and lung,AGT mRNA,ATR1a mRNA and ATR2 mRNA in adrenal gland were detected by real time PCR. The results showed that taurine could decrease the expression level of ACE mRNA in cardiac muscle and lung,AGT mRNA,ATR1a mRNA,and increase the expression level of ATR2 mRNA.There were no significant differences between the control group and the taurine groups.The effects of taurine on the expression level ofβ2-receptor mRNA and AC5 mRNA were not significant.So it could be concluded that taurine may prevent hypertension by way of decreasing the expression level of ACE mRNA in cardiac muscle and lung,AGT mRNA and ATR1a mRNA in adrenal gland,increasing the expression level of ATR2 mRNA in adrenal gland,thus it could inhibit the production of AngⅡdirectly or indirectly,and inhibit the production of ATR1a,promote the production of ATR2.
试验一通过添加不同水平的牛磺酸和N-硝基-L-精氨酸甲酯(N-nitro-L-argininemethylester,L-NAME,NO合酶抑制剂),探讨了牛磺酸对L-NAME致高血压发生的预防作用。将100只雄性Wistar大鼠随机分为五组,对照组饮用自来水,模型Ⅰ组大鼠饮水中添加75mg/100ml L-NAME,模型Ⅱ组在饮水中添加75mg/100ml L-NAME的同时添加1%的β-丙氨酸,预防Ⅰ、Ⅱ组饮水中添加75mg/100ml L-NAME的同时分别添加1%、2%的牛磺酸。每两天测定大鼠尾动脉收缩压。结果证明:模型Ⅰ、Ⅱ组大鼠血压在造模第一周时明显升高,在以后的三周里血压持续升高,模型Ⅱ组大鼠血压略高于模型Ⅰ组差异不显著。预防Ⅰ组血压上升幅度较模型组小,预防Ⅱ组血压较对照组差异不显著。结果表明:用L-NAME处理大鼠建立大鼠高血压模型,牛磺酸可降低L-NAME所致血压升高的幅度。
试验二选取试验一中的各组试验动物,分别在第三周和第四周末处死,测定血清钙离子(Ca~(2+))、钠离子(Na~+)、肿瘤坏死因子(Tumor necrosis factor,TNF-α)、胰岛素样生长因子-Ⅰ(insulin-like growth factor,IGF-Ⅰ)的含量,血浆肾素活性(Plasma reninactivity,PRA)、肾上腺髓质素(Adrenomedullin,ADM)、降钙素基因相关肽(Calcitonin generelated peptide,CGRP)、醛固酮(aldosterone,ALD)的含量,心肌环一磷酸腺苷(Cyclic3',5'-adenosine monophosphate,cAMP)、环一磷酸鸟苷(cyclic 3',5'-guanosinemonophosphate,cGMP)含量。结果证明:模型组大鼠血中TNF-α、ALD含量和PRA活性与对照组相比显著升高(p＜0.05),预防组与模型组相比下降幅度显著(p＜0.05),预防Ⅱ组下降幅度强于预防Ⅰ组;模型组大鼠CGRP、cGMP含量与对照组相比显著下降(p＜0.05),两个预防组与模型组相比上升幅度显著(p＜0.05),预防Ⅱ组升高强于预防Ⅰ组;ADM、IGF-I、Ca~(2+)、Na~+、cAMP含量变化均不显著(p＞0.05)。结果表明:用L-NAME处理使大鼠血压升高,预防性应用牛磺酸可通过抑制肾素-血管紧张素-醛固酮系统(renin-angiotensin-aldosterone system,RAAS)活性,阻止细胞因子PRA、TNF-α、ALD的分泌,升高血中CGRP和心肌cGMP含量等机制来抑制血压的升高。
试验三分离试验一中各组大鼠腹主动脉,将其放在固定液中保存,修整大小,做常规石蜡切片,并用苏木素-伊红(HE)法染色,在普通光镜下观察各试验组大鼠腹主动脉VSMC增殖情况。结果证明:模型组大鼠腹主动脉中层VSMC大量增殖,血管壁明显增厚;预防Ⅰ组VSMC数量较模型组少;预防Ⅱ组VSMC较对照组仅有少量增殖。结果表明:牛磺酸可明显改善VSMC增殖,且2%牛磺酸抑制VSMC增殖的效果好于1%牛磺酸,这可能是牛磺酸预防大鼠高血压发生的另一机制。
试验四通过体外培养股动脉VSMC细胞,并在培养液中添加不同浓度的牛磺酸(0.1μg/mL、1μg/mL、10μg/mL、100μg/mL、500μg/mL、1000μg/mL)和AngⅡ(1×10~(-7)moL/L),采用MTT比色法测定细胞活力,初步探讨了牛磺酸对体外培养VSMC细胞增殖的作用及其机制。结果证明:培养液中添加适量的牛磺酸能抑制VSMC细胞的增殖。结果表明:牛磺酸也可通过抑制AngⅡ所致的VSMC异常增殖,从而预防高血压的发生。
试验五选取试验一中的各组试验动物在四周后处死,取各组大鼠心肌、肺脏和肾上腺组织,提取总RNA,采用SYBRⅠ实时荧光定量RT-PCR技术测定牛磺酸对心肌肾上腺素能受体β2(beta2-adrenergic receptor,β2-AR)mRNA、腺苷酸环化酶(Adenylyl cyclase,AC5)mRNA、心肌和肺脏血管紧张素转换酶(angiotensin-converting-enzyme,ACE)mRNA、肾上腺血管紧张素原(angiotensinogen,AGT)mRNA、肾上腺血管紧张素Ⅱ受体(renin-angiotensinⅡ-receptor,ATR)的ATR1a及ATR2型的mRNA表达水平影响。结果证明:与模型组相比,牛磺酸可明显降低心肌和肺脏中ACE mRNA的表达水平,可显著降低肾上腺AGT和肾上腺ATR1a mRNA的表达,可显著提高ATR2 mRNA的表达,而牛磺酸对β2-AR和(AC_5)没有明显影响。结果表明:牛磺酸可能是通过降低心肌和肺脏中ACE、肾上腺AGT与肾上腺ATR1a mRNA表达水平及提高肾上腺ATR2 mRNA表达水平,直接或间接地减少AngⅡ生成,抑制ATR1a产生和增加ATR2产生而起到预防高血压的作用。
Taurine was found to be one of the most abundant free amino acids in cardiovascular tissues of animals,but its preventive effects on hypertension is still obscure.In the present study,the effects of taurine on hypertension was investigated.
In experiment 1,male rats were given tap water containing taurine and L-NAME(inhibitor of NOS),then the preventive effect and mechanism of taurine on hypertensive induced by L-NAME were investigated.100 male wistar rats were randomly divided into five groups. The rats in the control group were given normal diet and tap water.The rats in the model groupⅠwere given normal diet and tap water containing 75mg/100ml of L-NAME,model groupⅡwere given normal diet and tap water containing 75mg/100ml of L-NAME and 1%β-Ala.Prevention groupⅠwere given normal diet and tap water containing 75mg/100ml of L-NAME and 1%taurine.Prevention groupⅡwere given normal diet and tap water containing 75mg/100ml of L-NAME and 2%taurine.Blood pressure was detected using tail-cuff method every two days.The results showed that 1 week later,blood pressure in the model group increased and the increase lasted in the following three weeks.There is no significant difference between the modelⅠandⅡgroups,and the blood pressure of model groupⅡis a little higher than that of the model groupⅠ.Blood pressure in prevention groupⅠwas lower compared with the model groups and there is no significant difference between prevention groupⅡand the control group.The results indicated that the model of hypertensive rats was established by L-NAME which could induce hypertension.Taurine could inhibit the elevation of blood pressure.
In experiment 2,experimental animals were selected from experimentⅠ.The rats were killed respectively at the end of the third and the fourth weeks.The contents of serum calciumion,sodiumion,TNF-α,IGF-Ⅰ,plasma PRA,ADM,CGRP,ALD and the contents of cAMP,cGMP in cardiac muscle were detected.The results showed that the contents of TNF-α, ALD and PRA increased significantly compared with the control group(p＜0.05),the prevention groups significantly decreased in model groups(p＜0.05),the effect of prevention groupⅡis better than that of the prevention groupⅠ,the contents of CGRP and cGMP significantly decreased compared with the control group(p＜0.05),the prevention groups significantly increased the in model groups(p＜0.05),the effect of prevention groupⅡis better than that of prevention groupⅠ,but there was significant differences in the level of ADM, IGF-Ⅰ,Ca~(2+),Na~+ ana cAMP(p＞0.05).Conclusion:the preventive mechanisms of taurine on
hypertension may due to the following reasons:it could restrain RAAS activity,inhibit the secretion of cell factor PRA,TNF-αand ALD secretion,increase the plasma level of CGRP and the level of cGMP in ardiac muscle.
In experiment 3,abodominal aortas selected from experimentⅠwere fixed in phosphate buffered formalin solutions and were embeded in paraffin wax.Sections were cut and stained with haematoxylin and eosin(HE).Proliferations of vascular smooth muscle cells(VSMC) were observed under optical microscope.The results showed:VSMC in the model groups significantly proliferated,the vessel wall thickened significantly.The number of VSMC in prevention groupⅠwas less than that of the model groups.The VSMC in prevention groupⅡproliferated a little.The results indicated that taurine could inhibit the proliferation of VSMC obviously and the effect of 2%taurine is better than that of 1%tauine,which can be regarded as another preventive mechanisms of taurine on hypertension.
In experiment 4,MTT method was used to study the effects of taurine on the proliferation of VSMC.In this experiment,taurine(0.1μg/mL,1μg/mL,10μg/mL,100μg/mL,500μg/mL,1 000μg/mL) and AngⅡ(1×10~(-7) moL/L) were addes in the culture fluid of VSMC in arteria femoralis.The results showed that taurine could significantly inhibit the proliferation of VSMC,which indicated that taurine could inhibite the proliferation of VSMC induced by AngⅡ.
In experiment 5,rats selected from experimentⅠwere killed at the end of the fourth week. At the same time,cardiac muscle,lung and adrenal gland were taken out promptly,then total RNA were extracted.The influence of taurine on the expression of receptors ofβ2-AR mRNA, AC5 mRNA,cAMP mRNA in cardiac muscle,ACE mRNA in cardiac muscle and lung,AGT mRNA,ATR1a mRNA and ATR2 mRNA in adrenal gland were detected by real time PCR. The results showed that taurine could decrease the expression level of ACE mRNA in cardiac muscle and lung,AGT mRNA,ATR1a mRNA,and increase the expression level of ATR2 mRNA.There were no significant differences between the control group and the taurine groups.The effects of taurine on the expression level ofβ2-receptor mRNA and AC5 mRNA were not significant.So it could be concluded that taurine may prevent hypertension by way of decreasing the expression level of ACE mRNA in cardiac muscle and lung,AGT mRNA and ATR1a mRNA in adrenal gland,increasing the expression level of ATR2 mRNA in adrenal gland,thus it could inhibit the production of AngⅡdirectly or indirectly,and inhibit the production of ATR1a,promote the production of ATR2.
引文
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