Biophysical Mechanism Exploration Of Laser Acupuncture And Moxibustion

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.