激光针灸及灸法的生物物理机理探索
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摘要
针灸包括针灸理论、腧穴、针灸技术以及相关器具,在形成、应用和发展的过程中,具有鲜明的中华民族文化与地域特征,是基于中华民族文化和科学传统产生的宝贵遗产。在20世纪70年代,有研究者开始提出肥大细胞与经络现象有关系的假说,该假说认为针刺使较敏感的肥大细胞受刺激或损伤,从而释放出活性物质,这些物质进一步激发了局部的经络感传现象。随后越来越多的报道发现穴位处的肥大细胞数量的确较旁开组织要多,而且肥大细胞作为一种免疫细胞,在收到刺激后会释放组胺,对人体起着重要的调节作用。随着现代科学技术的进步,针灸也在不断地发展,例如激光针灸就是一个很好的例子。然而其许多治疗机理仍然是个迷,大大的限制了针灸方法的传播和应用。我们实验室已有的研究结果表明,肥大细胞在激光刺激下会产生脱颗粒释放组胺现象,然而肥大细胞内部的信号传递过程仍然不得而知；灸法的机理研究还很少,而关于灸法热作用的研究就更是缺乏。基于这些情况,本文利用扫描共聚焦显微镜和荧光光谱方法,研究了激光照射以及不同的热刺激作用对RBL-2H3肥大细胞胞内钙离子浓度及其脱颗粒释放组胺的影响,并对作用机制进行了探讨,具体结果如下：
首先,为了观察激光波长对肥大细胞脱颗粒释放组胺的影响,采用波长、功率、照射时间分别为406nm--0.081mW--25min\530nm--0.086mW--25min\632nm—0.423mW--5min三组,在吸收能量在同一数量级的情况下,406nm释放组胺量最高,632nm其次,530nm最低,与细胞色素C氧化酶的吸收光谱吻合,说明细胞色素C氧化酶就是激光针灸过程中的靶点。细胞色素C氧化酶接受光子后会产生活性氧,改变线粒体的外膜通透性；对细胞内的细胞色素C进行荧光标记,发现在激光照射后细胞色素C浓度增加；对细胞质pH值进行监测,对30个细胞pH值荧光进行统计分析得到结果表明激光刺激后,肥大细胞细胞质确实出现碱性化；根据已有文献报道,细胞质碱性化将导致细胞膜上的TRPV通道开启,并且使外钙内流,钙离子作为细胞内最重要的第二信使之一,钙离子浓度升高将导致肥大细胞脱颗粒。Bcl-2可以有效地抑制线粒体膜通透性的改变,当对上述的实验采用Bcl-2预先处理,发现激光照射后,肥大细胞质中细胞色素C浓度和pH值没有变化,细胞的组胺释放量也没有增加,说明了在肥大细胞受激光刺激释放组胺的实验过程中,信号起始于线粒体内,当对这一信号源进行阻断,后面的所有现象都不会发生。因此我们就通过实验发现了激光刺激肥大细胞释放组胺整个细胞内的信号传递过程。
其次,在RBL-2H3肥大细胞热刺激研究中,采用自制加热样品池来满足对细胞加热同时观察的条件；用程序控温仪测量细胞环境溶液的温度,以保证温度测量的正确性。对细胞线性加热刺激,通过多次的实验统计分析,发现采取含钙离子的HEPES缓冲液作为细胞加热环境溶液时,细胞受热刺激,内部钙离子在27℃后开始逐渐增加,在每一个TRPV开启温度点,都发现细胞内部的钙离子浓度迅速的增加；在60℃时,细胞内钙离子浓度为初始浓度2-3倍；当采用含有钌红(RR,阻断TRPV通道试剂)的HEPES缓冲液时,发现细胞内部钙离子浓度随温度升高缓慢上升,但是在各个TRPV开启温度点没有明显变化,最高浓度约为初始浓度的1.5倍；而在完全无外界钙离子的生理盐水条件下,仍然发现细胞内部钙离子浓度随温度升高缓慢上升,但是在各个TRPV开启温度点没有明显变化,最高浓度约为初始浓度的1.5倍,与加RR阻断剂结果相似,证明细胞内的钙库在温度刺激导致钙离子浓度升高现象中起着重要的作用,也证明TRPV是细胞膜上主要响应温度刺激的离子通道。随后对组胺浓度测试发现组胺的释放与温度呈量效关系。对细胞直接加入65℃的HEPES缓冲液刺激,发现细胞内部的钙离子浓度也迅速增加,增幅到达3倍,说明加热速率并不是导致细胞内部钙离子浓度升高的主要因素；细胞在65℃左右开始破裂,细胞内钙离子浓度下降,暗示灸法中最高温度阈值不需要超过65℃。
因此,激光刺激作用于肥大细胞,通过线粒体启动细胞内的信号传递过程,线粒体膜通透性增强,细胞色素C释放到细胞质中,细胞质碱性化,导致细胞膜TRPV通道开启,胞外钙离子内流,引起胞内钙离子浓度的升高,进而导致肥大细胞脱颗粒,释放组胺等生物活性物质,调节机体的功能；而在加热物理刺激导致细胞膜上的TRPV通道开启时,也导致细胞内的钙库释放钙离子,细胞质钙离子浓度的升高,通过这样的方式来调节机体的功能。
Acupuncture and moxibustion are the most important methods in Traditional Chinese Medicine. The formation, application and development of acupuncture and moxibustion are distinct affected by characteristics of Chinese culture and Chinese regional characteristics, they are valuable legacies based on the traditional Chinese culture and science. In the1970s, researchers put up hypothesis that the mast cells are important for meridian phenomenon, the hypothesis believed acupuncture lead to stimulation or damage of the sensitive mast cells, which then would release active substances, these substances further stimulated local channel transmission[1]. Then more and more reports found the number of mast cells in acupoint is indeed larger then in the place near acupoint. Mast cells, as a kind of immune cells, will release histamine when receiving stimulation, and histamine plays an important role in regulating human body. Acupuncture is in constant development as the modern science and technology develops, laser acupuncture is a good example for it. Yet many of its therapeutic mechanism remain a mystery, which limits the spread and application of acupuncture and moxibustion methods. The existing research results of our laboratory show that laser stimulation of mast cells could lead to the degranulation of mast cell which contains histamine, however, mast cells internal signal transmission process remains unknown; the mechanism of moxibustion is also rarely studied, and much more insufficient in the heat action of moxibustion. Based on these conditions, this paper using scanning confocal microscope and fluorescence spectrum method, intracellular calcium ion concentration and degranulation with histamine influence in laser irradiated and different thermal stimulated RBL-2H3mast cells were studied, the mechanism was discussed, and the specific results are as follows:
First, in order to observe the laser wavelength influence to mast cell degranulation, the wavelength, the power, irradiation times are respectively406nm--0.081mW--25min\530nm--0.086mW--25min\632nm--0.423mW--5min, with the same order of magnitude in absorbing energy,406nm laser released histamine with the highest quantity, while632nm was second highest,530nm was minimum, which are in accordance with the absorption spectra of cytochrome C oxidase, suggesting cytochromes C oxidase is the target in the process of laser acupuncture. Cytochromes C oxidase absorbs photons and later generate ROS, which would change the mitochondrial membrane permeability; Marked the Cytochromes C with florescence and it's found that the concentration increased after laser irradiation. Subsequently cytoplasm pH was monitored, the statistical analysis of30cells fluorescence showed that cytoplasm alkaline appeared in mast cells. According to the existing reports, alkalization will result in the opening of TRPV channels in the membrane, and the influx of calcium outside. Calcium as one of the most important second messenger, its raise will lead to the degranulation of mast cells. Bcl-2Can effectively suppress the mitochondrial membrane permeability changes, When the above-mentioned experimental pretreated with Bcl-2, it's found that cytoplasm cytochromes C concentration and pH value did not change after laser irradiation, Cell histamine release didn't increase in mast cells, which suggesting signal begins from the mitochondria, when it's blocked, all the other phenomenon is not going to happen. So we proved the whole cell signaling process in the laser stimulated mast cells from our experiments.
Secondly, in the in thermal stimulation research of RBL-2H3mast cells, the self-made heating pool was used, and made sure the temperature was correctly monitored when heated. Thermometer measurement while controlling the temperature though cell environment solution so as to ensure the correctness of temperature measurement. Linear heating of cells, through statistics analysis of many experiment results, it's found that the HEPES containing calcium ions as the environment solution, the cell calcium ion raised from27℃temperature, whenever in TRPV open temperature point, it's found intracellular calcium ion concentration increased rapidly, in60℃, the intracellular calcium ion concentration was2～3times of initial concentration; When using HEPES buffer contained ruthenium red (RR) blocking the TRPV channels, it's found the calcium ion concentration increased with temperature rise, but a all the TRPV opening temperature point, the calcium concentration did not change significantly, the highest concentration was approximately1.5times of initial concentration; And in physiological saline conditions completely without outside calcium, we still found inside calcium ion concentration increased with temperature rise, but in all the TRPV opening temperature point they did not change significantly, too. the highest concentration was approximately1.5times of initial concentration, which was similar to the condition with RR blockers, which proofed intracellular calcium store plays an important role in temperature stimulation, the membrane TRPV is also proved to be the main response channels in temperature stimulation leading to the raise of intracelluler calcium. Then histamine concentration test in cells heated showed a quantity-efficiency relation. Cells directly added with the65℃HEPES buffer were found that the intracellular calcium ion concentration also increased rapidly, it's three times the concentration of initial state which manifested that heating rate maybe not the cause for calcium ion concentration raising in mast cells stimulated by heat; It's found that when heated to65℃, the calcium concentration decreasd in mast cells, which suggested that65℃should be high enough for moxibustion.
The wavelength effect of laser irradiations on histamine release suggests that the cytochrome c oxidase is a photoacceptor which initiates a photoreaction resulting in the histamine release in RBL-2H3cells. The fact of the sequent cytochrome c release from mitochondrial to cytoplasm in irradiated cells supports this photoacceptor suggestion, since the excited cytochrome c oxidase would produce the ROS leading to a permeability increment of mitochondrial membranes. The effects of Bcl-2on suppressing the cytosolic alkalinization,[Ca2+]i increment and histamine release demonstrate the dominate role of mitochondrial signaling. Therefore, the whole cellular response course for laser irradiations can be concluded as follows:the cytochrome c oxidase is the target of incident photons and then the induced signals from mitochondrial to cytoplasm cause the cytosolic alkalinization which opens the TRPV channel leading to a [Ca2+]i increment and consequently resulting in an enhanced histamine release. And TRPV channels on the membrane opened by heating stimulation, which also lead to the raise of intracellular calcium ions, calcium store calcium ion should also play a very important role in the calcium concentration raise in the cytoplasm, through which to regulate the function of organism.
首先,为了观察激光波长对肥大细胞脱颗粒释放组胺的影响,采用波长、功率、照射时间分别为406nm--0.081mW--25min\530nm--0.086mW--25min\632nm—0.423mW--5min三组,在吸收能量在同一数量级的情况下,406nm释放组胺量最高,632nm其次,530nm最低,与细胞色素C氧化酶的吸收光谱吻合,说明细胞色素C氧化酶就是激光针灸过程中的靶点。细胞色素C氧化酶接受光子后会产生活性氧,改变线粒体的外膜通透性；对细胞内的细胞色素C进行荧光标记,发现在激光照射后细胞色素C浓度增加；对细胞质pH值进行监测,对30个细胞pH值荧光进行统计分析得到结果表明激光刺激后,肥大细胞细胞质确实出现碱性化；根据已有文献报道,细胞质碱性化将导致细胞膜上的TRPV通道开启,并且使外钙内流,钙离子作为细胞内最重要的第二信使之一,钙离子浓度升高将导致肥大细胞脱颗粒。Bcl-2可以有效地抑制线粒体膜通透性的改变,当对上述的实验采用Bcl-2预先处理,发现激光照射后,肥大细胞质中细胞色素C浓度和pH值没有变化,细胞的组胺释放量也没有增加,说明了在肥大细胞受激光刺激释放组胺的实验过程中,信号起始于线粒体内,当对这一信号源进行阻断,后面的所有现象都不会发生。因此我们就通过实验发现了激光刺激肥大细胞释放组胺整个细胞内的信号传递过程。
其次,在RBL-2H3肥大细胞热刺激研究中,采用自制加热样品池来满足对细胞加热同时观察的条件；用程序控温仪测量细胞环境溶液的温度,以保证温度测量的正确性。对细胞线性加热刺激,通过多次的实验统计分析,发现采取含钙离子的HEPES缓冲液作为细胞加热环境溶液时,细胞受热刺激,内部钙离子在27℃后开始逐渐增加,在每一个TRPV开启温度点,都发现细胞内部的钙离子浓度迅速的增加；在60℃时,细胞内钙离子浓度为初始浓度2-3倍；当采用含有钌红(RR,阻断TRPV通道试剂)的HEPES缓冲液时,发现细胞内部钙离子浓度随温度升高缓慢上升,但是在各个TRPV开启温度点没有明显变化,最高浓度约为初始浓度的1.5倍；而在完全无外界钙离子的生理盐水条件下,仍然发现细胞内部钙离子浓度随温度升高缓慢上升,但是在各个TRPV开启温度点没有明显变化,最高浓度约为初始浓度的1.5倍,与加RR阻断剂结果相似,证明细胞内的钙库在温度刺激导致钙离子浓度升高现象中起着重要的作用,也证明TRPV是细胞膜上主要响应温度刺激的离子通道。随后对组胺浓度测试发现组胺的释放与温度呈量效关系。对细胞直接加入65℃的HEPES缓冲液刺激,发现细胞内部的钙离子浓度也迅速增加,增幅到达3倍,说明加热速率并不是导致细胞内部钙离子浓度升高的主要因素；细胞在65℃左右开始破裂,细胞内钙离子浓度下降,暗示灸法中最高温度阈值不需要超过65℃。
因此,激光刺激作用于肥大细胞,通过线粒体启动细胞内的信号传递过程,线粒体膜通透性增强,细胞色素C释放到细胞质中,细胞质碱性化,导致细胞膜TRPV通道开启,胞外钙离子内流,引起胞内钙离子浓度的升高,进而导致肥大细胞脱颗粒,释放组胺等生物活性物质,调节机体的功能；而在加热物理刺激导致细胞膜上的TRPV通道开启时,也导致细胞内的钙库释放钙离子,细胞质钙离子浓度的升高,通过这样的方式来调节机体的功能。
Acupuncture and moxibustion are the most important methods in Traditional Chinese Medicine. The formation, application and development of acupuncture and moxibustion are distinct affected by characteristics of Chinese culture and Chinese regional characteristics, they are valuable legacies based on the traditional Chinese culture and science. In the1970s, researchers put up hypothesis that the mast cells are important for meridian phenomenon, the hypothesis believed acupuncture lead to stimulation or damage of the sensitive mast cells, which then would release active substances, these substances further stimulated local channel transmission[1]. Then more and more reports found the number of mast cells in acupoint is indeed larger then in the place near acupoint. Mast cells, as a kind of immune cells, will release histamine when receiving stimulation, and histamine plays an important role in regulating human body. Acupuncture is in constant development as the modern science and technology develops, laser acupuncture is a good example for it. Yet many of its therapeutic mechanism remain a mystery, which limits the spread and application of acupuncture and moxibustion methods. The existing research results of our laboratory show that laser stimulation of mast cells could lead to the degranulation of mast cell which contains histamine, however, mast cells internal signal transmission process remains unknown; the mechanism of moxibustion is also rarely studied, and much more insufficient in the heat action of moxibustion. Based on these conditions, this paper using scanning confocal microscope and fluorescence spectrum method, intracellular calcium ion concentration and degranulation with histamine influence in laser irradiated and different thermal stimulated RBL-2H3mast cells were studied, the mechanism was discussed, and the specific results are as follows:
First, in order to observe the laser wavelength influence to mast cell degranulation, the wavelength, the power, irradiation times are respectively406nm--0.081mW--25min\530nm--0.086mW--25min\632nm--0.423mW--5min, with the same order of magnitude in absorbing energy,406nm laser released histamine with the highest quantity, while632nm was second highest,530nm was minimum, which are in accordance with the absorption spectra of cytochrome C oxidase, suggesting cytochromes C oxidase is the target in the process of laser acupuncture. Cytochromes C oxidase absorbs photons and later generate ROS, which would change the mitochondrial membrane permeability; Marked the Cytochromes C with florescence and it's found that the concentration increased after laser irradiation. Subsequently cytoplasm pH was monitored, the statistical analysis of30cells fluorescence showed that cytoplasm alkaline appeared in mast cells. According to the existing reports, alkalization will result in the opening of TRPV channels in the membrane, and the influx of calcium outside. Calcium as one of the most important second messenger, its raise will lead to the degranulation of mast cells. Bcl-2Can effectively suppress the mitochondrial membrane permeability changes, When the above-mentioned experimental pretreated with Bcl-2, it's found that cytoplasm cytochromes C concentration and pH value did not change after laser irradiation, Cell histamine release didn't increase in mast cells, which suggesting signal begins from the mitochondria, when it's blocked, all the other phenomenon is not going to happen. So we proved the whole cell signaling process in the laser stimulated mast cells from our experiments.
Secondly, in the in thermal stimulation research of RBL-2H3mast cells, the self-made heating pool was used, and made sure the temperature was correctly monitored when heated. Thermometer measurement while controlling the temperature though cell environment solution so as to ensure the correctness of temperature measurement. Linear heating of cells, through statistics analysis of many experiment results, it's found that the HEPES containing calcium ions as the environment solution, the cell calcium ion raised from27℃temperature, whenever in TRPV open temperature point, it's found intracellular calcium ion concentration increased rapidly, in60℃, the intracellular calcium ion concentration was2～3times of initial concentration; When using HEPES buffer contained ruthenium red (RR) blocking the TRPV channels, it's found the calcium ion concentration increased with temperature rise, but a all the TRPV opening temperature point, the calcium concentration did not change significantly, the highest concentration was approximately1.5times of initial concentration; And in physiological saline conditions completely without outside calcium, we still found inside calcium ion concentration increased with temperature rise, but in all the TRPV opening temperature point they did not change significantly, too. the highest concentration was approximately1.5times of initial concentration, which was similar to the condition with RR blockers, which proofed intracellular calcium store plays an important role in temperature stimulation, the membrane TRPV is also proved to be the main response channels in temperature stimulation leading to the raise of intracelluler calcium. Then histamine concentration test in cells heated showed a quantity-efficiency relation. Cells directly added with the65℃HEPES buffer were found that the intracellular calcium ion concentration also increased rapidly, it's three times the concentration of initial state which manifested that heating rate maybe not the cause for calcium ion concentration raising in mast cells stimulated by heat; It's found that when heated to65℃, the calcium concentration decreasd in mast cells, which suggested that65℃should be high enough for moxibustion.
The wavelength effect of laser irradiations on histamine release suggests that the cytochrome c oxidase is a photoacceptor which initiates a photoreaction resulting in the histamine release in RBL-2H3cells. The fact of the sequent cytochrome c release from mitochondrial to cytoplasm in irradiated cells supports this photoacceptor suggestion, since the excited cytochrome c oxidase would produce the ROS leading to a permeability increment of mitochondrial membranes. The effects of Bcl-2on suppressing the cytosolic alkalinization,[Ca2+]i increment and histamine release demonstrate the dominate role of mitochondrial signaling. Therefore, the whole cellular response course for laser irradiations can be concluded as follows:the cytochrome c oxidase is the target of incident photons and then the induced signals from mitochondrial to cytoplasm cause the cytosolic alkalinization which opens the TRPV channel leading to a [Ca2+]i increment and consequently resulting in an enhanced histamine release. And TRPV channels on the membrane opened by heating stimulation, which also lead to the raise of intracellular calcium ions, calcium store calcium ion should also play a very important role in the calcium concentration raise in the cytoplasm, through which to regulate the function of organism.
引文
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