从“脉浊”探讨导痰汤干预颈动脉硬化时内皮细胞粘附分子1-表达的作用机制
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摘要
本研究在传统中医理论的基础上,以中医“脉浊”概念为切入点,结合现代医学研究成果,对导痰汤干预颈动脉硬化的机制进行了诠释。
研究认为,脉为奇恒之腑,具有相对的独立性和整体性,结合现代对动脉粥样硬化的研究进展,可以发现：①动脉粥样硬化的发病部位在脉；②多种原因导致的大量以低密度脂蛋白为主的脂质颗粒沉积于动脉内皮下是动脉粥样硬化的关键环节,这些沉积于脉管内的脂质颗粒由于具有重浊、黏滞、浑秽的特性,因此应属浊邪侵袭；③动脉粥样硬化的病理表现完整地体现了浊邪留滞、袭扰脉管的过程。因此,依据中医理论,动脉粥样硬化系浊邪内留血脉所致,应统称为“脉浊”。脉浊发病过程中,存在“正虚”与“邪损”两方面因素相互影响与转化,其病位在于“脉”,本虚标实是脉浊的病机关键。颈动脉硬化作为脉浊的一个具体体现形式,其发病率随着人口老龄化进程的加快,而不断上升。
中医理论认为,在脉浊发病过程中,痰浊内阻是一个重要病理基础和致病因素。而导痰汤作为临床常用化痰名方,能够燥湿化痰,行气开郁。但是导痰汤等中医化痰治疗在干预颈动脉硬化过程中的具体作用渠道,目前尚缺乏大量深入的基础研究。
由于内皮细胞损伤和内皮细胞粘附分子-1(in tercellular adhesion molecular-1, ICAM-1)的活化是动脉硬化的起始环节。ICAM-1的表达与丝裂原活化蛋白激酶家族中的JNK和p38通路与ICAM-1表达之间密切相关。另外, p53、p21等抑癌基因是细胞衰老的关键效应物,在颈动脉硬化等老年性疾病中,p53/p21通路可以直接诱导内皮细胞ICAM-1过表达。
因此,本研究在“脉浊”理论指导下,使用导痰汤干预体外、体内模型的基础上,分析了颈动脉硬化时导痰汤对影响ICAM-1表达的相关基因的干预情况,发现：
一、导痰汤通过JNK、p38信号通路而干预ICAM-1的表达
动物实验中,对照组颈动脉血管内膜完整,中膜为梭形平滑肌细胞,血管壁有少量ICAM-1、JNK和p38阳性表达细胞。模型组有粥样斑块形成,内膜明显增厚,内膜下有大量泡沫细胞堆积,血管壁泡沫细胞、平滑肌细胞和内皮细胞的胞浆中有ICAM-1、JNK和p38强阳性表达,明显高于对照组(p<0.01)。导痰汤大、中、小剂量组内膜也有轻度的增生,内膜下仅有少量的泡沫细胞形成,程度轻于模型组,导痰汤组细胞内ICAM-1、JNK和p38阳性表达显著低于模型组,且中、大剂量组对ICAM-1和JNK的抑制均明显强于小剂量组(p<0.01),大剂量组对p38的抑制明显强于小剂量组(p<0.01)。直线相关分析结果表明,JNK与ICAM-1水平呈显著正相关(p<0.01)。p38与ICAM-1水平呈显著正相关(p<0.01)。
体外实验发现,TNF-α诱导后ICAM-1、JNK活性和p38活性均显著高于正常对照组和导痰汤对照组(p<0.01)。使用导痰汤含药血清或SB203580处理后,ICAM-1表达显著下降(p<0.05)。使用10%,20%导痰汤含药血清或SP600125处理后,JNK活性显著下降(p<0.01)。使用20%导痰汤含药血清或SB203580处理后,p-p38表达显著下降(p<0.05)。直线相关分析结果表明,JNK活性及p38活性与ICAM-1mRNA水平呈显著正相关(p<0.01)。
二、导痰汤通过p53、p21干预ICAM-1的表达正常对照组血管壁有少量ICAM-1、p53和p21阳性表达细胞。模型组血管壁内皮细胞的胞浆中有ICAM-1、p53和p21强阳性表达,显著高于对照组(p<0.01)。导痰汤治疗组细胞内ICAM-1、p53和p21阳性表达显著低于模型组(p<0.01),且大剂量组的导痰汤对ICAM-1、p53和p21的抑制均显著强于小剂量组(p<0.01)。直线相关分析结果表明,p53和p21均与ICAM-1水平呈显著正相关(p<0.01)。
TNF-α诱导组ICAM-1、p53与p21蛋白显著高于正常对照组和空白血清对照组(p<0.01)。使用导痰汤含药血清或PFT-α处理后,1CAM-1、p53与p21表达显著下降(p<0.05),且随导痰汤剂量的增加,抑制逐渐增强。直线相关分析表明, p53、p21与ICAM-1mRNA及蛋白均水平呈显著正相关(p<0.05)。
To investigate the mechanism of action of endothelial cell adhesion molecule-1expression in Dao Tan Tang intervention with carotid atherosclerosis from the "artery turbid"
This study on the basis of the theory of traditional Chinese medicine, the concept of the Chinese medicine "arterys turbid", as the starting point of modern medical research, on the the Dao Tan soup intervention mechanism for the interpretation of carotid atherosclerosis.
Artery is one of extraordinary fu-visceras, with relative independence and integrity."Arterys turbid" is caused by both "weakened body resistance" and "evil damage", and two factors affect each other and the transformation of their disease is "artery" bit, the vacuity arterys cloud pathogenesis key. The carotid atherosclerosis embodied in the form as artery turbid of a specific incidence with the accelerated process of population aging and rising.
TCM theory holds that phlegm resistance is an important pathological basis of carotid atherosclerosis and pathogenic factors. Dao Tan Decoction (DTD), as a prescription to the commonly used clinical phlegm, could treat phlegm dampness. DTD, which could used in the phlegm treatment, also has a specific role in the process of carotid atherosclerosis, yet lacking of a large number of in-depth fundamental research.
The activation of endothelial cell injury and endothelial cell adhesion molecule-1(intercel lular adhesion molecular-1, ICAM-1) are the star t ing part s of atherosclerosis. Expression of ICAM-1and mitogen-activated protein kinase family, JNK and p38pathway and ICAM-1expression between closely related. In addition, p53, p21and other tumor suppressor genes are key effectors of cellular senescence; p53/p21pathway can be directly induced ICAM-1over-expression of the endothel ial cells in carotid atheroscleros is and other age-related dis eases.
Therefore, this study was under the guidance of the theory of the artery turbidity. DTD intervention would be used in vitro, in vivo models based on analysis ICAM-1expression in the carotid atherosclerosis. It was found:
I. DTD interfere with the expression of ICAM-1via JNK, p38signaling pathway In animal experiments, the control group carotid intimal integrity, in the film spindle smooth muscle cells, blood vessel wall with a small amount of ICAM-1, JNK and p38positive cells. Plaque formation in model group intimal obvious thickening, subintimal accumulation of a large number of foam cells in the cytoplasm of the vascular wall foam cells, smooth muscle cells and endothelial cells, ICAM-1, JNK and p38strongly positive expression significantly higher (p<0.01). DTD, and mild in the low-dose group, endometrial hyperplasia, subintimal only a small amount of foam cell formation, to a lesser degree in the model group, DTD group of cells within the expression of ICAM-1, JNK, and p38positive expression significantly lower than the model group, see, in the high-dose group of ICAM-1and JNK inhibition was significantly stronger than the low-dose group (p<0.01), the high-dose group p38inhibition was stronger in the small dose group (p<0.01). The correlation analysis showed that, JNK, and ICAM-1levels were significantly positively correlated (p<0.01). p38and ICAM-1levels were significantly positively correlated (p<0.01).
In vitro experiments, It was found that TNF-α-induced expression of ICAM-1, JNK activity and p38activity were significantly higher than the normal control group and DTD the control group (p<0.01). DTD containing serum or SB203580treatment, ICAM-1express ion was significant ly decreased (p<0.05). In10%,20%DTD containing serum or SP600125treatment, JNK activity was significantly decreased (p<0.01).20%DTD containing serum or SB203580treatment, p-p38expression was significantly decreased (p<0.05). The correlation analysis showed that JNK activity and p38act ivi ty and ICAM-1mRNA level was significantly positively related (p<001).
II. DTD interfere with the expression of ICAM-1via p53, p21pathway
There is a small amount of ICAM-1, p53and p21positive cells, in vessel wall of the normal control group. Strong positive expression of ICAM-1, p53and p21, significantly higher than that in the control group (p<0.01), were shown in the cytoplasm of the endothelial cells of the vessel wall of the model group. DTD treatment group cell express ion of ICAM-1of p53and p21positive express ion significant ly with less than the model group (p<0.01), and the high-dose group DTD of ICAM-1, p53and p21in suppress ion were s igni ficant with strong in small dose group (p<0.01). The correlation analysis showed that p53, p2l and ICAM-1levels were significantly positively related (p<0.01).
In TNF-α induced group ICAM-1, p53and p21protein were significantly higher than the normal control group and blank serum control group (p<0.01). DTD containing serum or PFT-α treatment, TCAM-1, p53and p21expression was significantly decreased (p<0.05), and inhibit gradually increased with the increase in the dose of Dao Tan Tang. The linear correlation analysis showed that p53, p21and ICAM-1mRNA and protein levels were significantly positively correlated (p<0.05).
研究认为,脉为奇恒之腑,具有相对的独立性和整体性,结合现代对动脉粥样硬化的研究进展,可以发现：①动脉粥样硬化的发病部位在脉；②多种原因导致的大量以低密度脂蛋白为主的脂质颗粒沉积于动脉内皮下是动脉粥样硬化的关键环节,这些沉积于脉管内的脂质颗粒由于具有重浊、黏滞、浑秽的特性,因此应属浊邪侵袭；③动脉粥样硬化的病理表现完整地体现了浊邪留滞、袭扰脉管的过程。因此,依据中医理论,动脉粥样硬化系浊邪内留血脉所致,应统称为“脉浊”。脉浊发病过程中,存在“正虚”与“邪损”两方面因素相互影响与转化,其病位在于“脉”,本虚标实是脉浊的病机关键。颈动脉硬化作为脉浊的一个具体体现形式,其发病率随着人口老龄化进程的加快,而不断上升。
中医理论认为,在脉浊发病过程中,痰浊内阻是一个重要病理基础和致病因素。而导痰汤作为临床常用化痰名方,能够燥湿化痰,行气开郁。但是导痰汤等中医化痰治疗在干预颈动脉硬化过程中的具体作用渠道,目前尚缺乏大量深入的基础研究。
由于内皮细胞损伤和内皮细胞粘附分子-1(in tercellular adhesion molecular-1, ICAM-1)的活化是动脉硬化的起始环节。ICAM-1的表达与丝裂原活化蛋白激酶家族中的JNK和p38通路与ICAM-1表达之间密切相关。另外, p53、p21等抑癌基因是细胞衰老的关键效应物,在颈动脉硬化等老年性疾病中,p53/p21通路可以直接诱导内皮细胞ICAM-1过表达。
因此,本研究在“脉浊”理论指导下,使用导痰汤干预体外、体内模型的基础上,分析了颈动脉硬化时导痰汤对影响ICAM-1表达的相关基因的干预情况,发现：
一、导痰汤通过JNK、p38信号通路而干预ICAM-1的表达
动物实验中,对照组颈动脉血管内膜完整,中膜为梭形平滑肌细胞,血管壁有少量ICAM-1、JNK和p38阳性表达细胞。模型组有粥样斑块形成,内膜明显增厚,内膜下有大量泡沫细胞堆积,血管壁泡沫细胞、平滑肌细胞和内皮细胞的胞浆中有ICAM-1、JNK和p38强阳性表达,明显高于对照组(p<0.01)。导痰汤大、中、小剂量组内膜也有轻度的增生,内膜下仅有少量的泡沫细胞形成,程度轻于模型组,导痰汤组细胞内ICAM-1、JNK和p38阳性表达显著低于模型组,且中、大剂量组对ICAM-1和JNK的抑制均明显强于小剂量组(p<0.01),大剂量组对p38的抑制明显强于小剂量组(p<0.01)。直线相关分析结果表明,JNK与ICAM-1水平呈显著正相关(p<0.01)。p38与ICAM-1水平呈显著正相关(p<0.01)。
体外实验发现,TNF-α诱导后ICAM-1、JNK活性和p38活性均显著高于正常对照组和导痰汤对照组(p<0.01)。使用导痰汤含药血清或SB203580处理后,ICAM-1表达显著下降(p<0.05)。使用10%,20%导痰汤含药血清或SP600125处理后,JNK活性显著下降(p<0.01)。使用20%导痰汤含药血清或SB203580处理后,p-p38表达显著下降(p<0.05)。直线相关分析结果表明,JNK活性及p38活性与ICAM-1mRNA水平呈显著正相关(p<0.01)。
二、导痰汤通过p53、p21干预ICAM-1的表达正常对照组血管壁有少量ICAM-1、p53和p21阳性表达细胞。模型组血管壁内皮细胞的胞浆中有ICAM-1、p53和p21强阳性表达,显著高于对照组(p<0.01)。导痰汤治疗组细胞内ICAM-1、p53和p21阳性表达显著低于模型组(p<0.01),且大剂量组的导痰汤对ICAM-1、p53和p21的抑制均显著强于小剂量组(p<0.01)。直线相关分析结果表明,p53和p21均与ICAM-1水平呈显著正相关(p<0.01)。
TNF-α诱导组ICAM-1、p53与p21蛋白显著高于正常对照组和空白血清对照组(p<0.01)。使用导痰汤含药血清或PFT-α处理后,1CAM-1、p53与p21表达显著下降(p<0.05),且随导痰汤剂量的增加,抑制逐渐增强。直线相关分析表明, p53、p21与ICAM-1mRNA及蛋白均水平呈显著正相关(p<0.05)。
To investigate the mechanism of action of endothelial cell adhesion molecule-1expression in Dao Tan Tang intervention with carotid atherosclerosis from the "artery turbid"
This study on the basis of the theory of traditional Chinese medicine, the concept of the Chinese medicine "arterys turbid", as the starting point of modern medical research, on the the Dao Tan soup intervention mechanism for the interpretation of carotid atherosclerosis.
Artery is one of extraordinary fu-visceras, with relative independence and integrity."Arterys turbid" is caused by both "weakened body resistance" and "evil damage", and two factors affect each other and the transformation of their disease is "artery" bit, the vacuity arterys cloud pathogenesis key. The carotid atherosclerosis embodied in the form as artery turbid of a specific incidence with the accelerated process of population aging and rising.
TCM theory holds that phlegm resistance is an important pathological basis of carotid atherosclerosis and pathogenic factors. Dao Tan Decoction (DTD), as a prescription to the commonly used clinical phlegm, could treat phlegm dampness. DTD, which could used in the phlegm treatment, also has a specific role in the process of carotid atherosclerosis, yet lacking of a large number of in-depth fundamental research.
The activation of endothelial cell injury and endothelial cell adhesion molecule-1(intercel lular adhesion molecular-1, ICAM-1) are the star t ing part s of atherosclerosis. Expression of ICAM-1and mitogen-activated protein kinase family, JNK and p38pathway and ICAM-1expression between closely related. In addition, p53, p21and other tumor suppressor genes are key effectors of cellular senescence; p53/p21pathway can be directly induced ICAM-1over-expression of the endothel ial cells in carotid atheroscleros is and other age-related dis eases.
Therefore, this study was under the guidance of the theory of the artery turbidity. DTD intervention would be used in vitro, in vivo models based on analysis ICAM-1expression in the carotid atherosclerosis. It was found:
I. DTD interfere with the expression of ICAM-1via JNK, p38signaling pathway In animal experiments, the control group carotid intimal integrity, in the film spindle smooth muscle cells, blood vessel wall with a small amount of ICAM-1, JNK and p38positive cells. Plaque formation in model group intimal obvious thickening, subintimal accumulation of a large number of foam cells in the cytoplasm of the vascular wall foam cells, smooth muscle cells and endothelial cells, ICAM-1, JNK and p38strongly positive expression significantly higher (p<0.01). DTD, and mild in the low-dose group, endometrial hyperplasia, subintimal only a small amount of foam cell formation, to a lesser degree in the model group, DTD group of cells within the expression of ICAM-1, JNK, and p38positive expression significantly lower than the model group, see, in the high-dose group of ICAM-1and JNK inhibition was significantly stronger than the low-dose group (p<0.01), the high-dose group p38inhibition was stronger in the small dose group (p<0.01). The correlation analysis showed that, JNK, and ICAM-1levels were significantly positively correlated (p<0.01). p38and ICAM-1levels were significantly positively correlated (p<0.01).
In vitro experiments, It was found that TNF-α-induced expression of ICAM-1, JNK activity and p38activity were significantly higher than the normal control group and DTD the control group (p<0.01). DTD containing serum or SB203580treatment, ICAM-1express ion was significant ly decreased (p<0.05). In10%,20%DTD containing serum or SP600125treatment, JNK activity was significantly decreased (p<0.01).20%DTD containing serum or SB203580treatment, p-p38expression was significantly decreased (p<0.05). The correlation analysis showed that JNK activity and p38act ivi ty and ICAM-1mRNA level was significantly positively related (p<001).
II. DTD interfere with the expression of ICAM-1via p53, p21pathway
There is a small amount of ICAM-1, p53and p21positive cells, in vessel wall of the normal control group. Strong positive expression of ICAM-1, p53and p21, significantly higher than that in the control group (p<0.01), were shown in the cytoplasm of the endothelial cells of the vessel wall of the model group. DTD treatment group cell express ion of ICAM-1of p53and p21positive express ion significant ly with less than the model group (p<0.01), and the high-dose group DTD of ICAM-1, p53and p21in suppress ion were s igni ficant with strong in small dose group (p<0.01). The correlation analysis showed that p53, p2l and ICAM-1levels were significantly positively related (p<0.01).
In TNF-α induced group ICAM-1, p53and p21protein were significantly higher than the normal control group and blank serum control group (p<0.01). DTD containing serum or PFT-α treatment, TCAM-1, p53and p21expression was significantly decreased (p<0.05), and inhibit gradually increased with the increase in the dose of Dao Tan Tang. The linear correlation analysis showed that p53, p21and ICAM-1mRNA and protein levels were significantly positively correlated (p<0.05).
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