Mechanism Of Inhibitory Effect Of Anmeitang On Cortex Of Insomnic Rats Based On The Morphology And Function Of Mitochondria

ObjectiveIn this study, based on the correlation theory between heart and brain from theory of visceral manifestations, we explored the relationship between yinyang imbalance of insomnia and the imbalance of excitation and suppression of the cerebral cortex, and mainly observed the changes of the cortical mitochondria of rats at different time points which were made from insomnic models by PCPA peritoneal injection, and also observed the effect of PCPA on the structure and function of the cortical mitochondria of different month rat. Followed by an experimental basis in the previous phase, the next experiment will go on in order to study the effect mechanism of Anmeitang from mitochondrial pathway, and urther develop medicine to improve the symptom of insomnia and provide the some evidence for clinic treatment.MethodsPart I:First,3-month-old SPF SD rats, male and female accounted for half, were dealed with chlorobenzene alanine (PCPA) by intraperitoneal injection for insomnic model according to the literature, and we observed the changes of the mitochondrial morphology and function of the frontal cortical neurons on1st,2nd,3rd and4th days after intraperitoneal injection by the super live staining and electron microscopy, which can measure the number and ultrastructure of mitochondria, meanwhile we also used fluorescence microscopy to detect mitochondrial membrane potential and WST-1to determine the ATP enzyme content of mitochondria. Secondly, based on the best time of change point, different aged rats,3,6,9, and12-month-old rats, will be produced for insomnic models by the intraperitoneal injection for3days, then we will observe the changes of the number and ultrastructure of mitochondria using the the super live staining and electron microscopy, while we will use the fluorescence microscopy to detect mitochondrial membrane potential and the WST-1to determine the ATP enzyme content of the mitochondria.Part Ⅱ:Based on the best time point and the best month age of the first part experiment, the9-month-old rats produced by PCPA for insomnic models were randomly divided into normal control group, model group, antipsychotic treatment group, melatonin treatment group and Anmeitang low, medium and high dose treatment group; and we observed the changes of the number and ultrastructure of mitochondria by super live staining and electron microscopy, and we also measured the membrane potential and ATP enzyme content of mitochondria by the fluorescence microscopy and WST-1method.As follows are the methods of two parts.1, preparation of animal modelAll rats were made for the insomnic model with p-chlorophenyl alanine (PCPA) by intraperitoneal injection. In addition to the normal saline group, the other groups received intraperitoneal injection of PCPA0.3ml/100g suspension (concentration0.1g/ml).2, preparation of mitochondriaThe rats were anaesthetized with10%chloral hydrate (350mg/kg) by intraperitoneal injection at the end of the experiment, and the brains of rats were removed quickly and immediately placed in ice-cold saline, then blood was washed. We peeled the cortex and placed them into the separation medium (2ml), and cut the cortex into pieces, then quickly transferred to a glass homogenizer tube (completed within lmin), electric homogenate diluted separation medium, centrifuged, and the resulting precipitate was suspended with a separation medium into lml suspension. The samples were placed into the centrifuge tube, which were stored in the trash ice. The target would immediately complete after the extraction. Mitochondrial protein content was measured with the method of Coomassie brilliant blue (Bradford).3, mitochondrial super live staining observationThe rats were sacrificed by spinal dislocation in the foramen magnum at the end of the experiment, then we cut the medulla oblongata quickly, peeled the skull, and the brain tissue was completely exposed, and then picked up the brain tissue, isolated frontal cortex after washing the cortex with the cold saline bath conditions, fully homogenized, centrifuged, and the supernatant set aside. Janus Green B (JGB) could specifically stain the activated mitochondria into blue-green, which could help to determine the integrity of mitochondrial function. The specific steps of super-live staining we can refer to the kit instructions of Janus Green B (JGB).4, the observation of mitochondrial ultrastructureAnimals were anesthetized with1%sodium pentobarbital (0.2ml/10g) by intraperitoneal injection at the end of the experiment, then perfused, and the brain would taken out;3mm diameter specimens from frontal cortex were made, then placed into the mixed stationary liquid. Next, the specimens would be trimed into1mm×1mm×1mm size tissue blocks, and placed into the mixed stationary liquid of2.5%glutaraldehyde and1%osmium tetroxide acid, rinsed, dehydrated in acetone, Epon812embedded; Next we made the semi-thin section,which would be positioned by optical microscope, and ultrathin sections(50nm) were made for observation after the stain of uranyl acetate and lead citrate by electron microscopy. A copper mesh for each rat was observed, and each copper mesh was moved from top left to bottom right diagonal moves up and down, and five photographs randomly photographed, whose negatives would magnified to15000times.5, the measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPaseThe rats were decapitated rapidly and their brains were taken out in the ice tray at the end of the experiment, and the frontal cortexs were cut out for the test, then the residual blood outside the cerebral cortex, accurately weighed, was dried with filter paper, and the icy physiological saline was added to prepare a tissue homogenate, which would be centrifuged with the centrifugal machine. Next, the supernatant was reserved and the pellet was discarded, and then centrifuged, and the precipitate was mitochondria. All the procedure of kit was followed.6, determination of mitochondrial membrane potentialthe frontal cortex samples excised freshly0.5cm were flatten, and the cleaning solution2ml preheated to37℃was used to clean the cortical tissue and then carefully removed. The entire samples were placed into the900ul working fluid, then put into37℃incubator for20min, and then the staining fluid should be removed, and the slices were made about100um thick after the samples were cleaned once again, and then placed on a slide, cover glass, flatten gently with your fingers, and we should immediately observe the slices under a fluorescence microscope and photograph.Results Part I:1, Animal model of insomniaThe circadian rhythm of the insomnic model was normal on the first day after the injection, and no restlessness occurred;but the activity of the insomnic model increased, and the models were very sensitive to the sound and light outside on the second day after the injection; the circadian rhythm disappeared on the third day after the injection, and all models walked non-stop day and night; on the fourth day after the injection, the restlessness of all models were very obvious.2, observation of super live staining of mitochondriaThe mitochondrial morphology of the three-month rats in each group appeared different shaps, some looked like short stick, and some were nearly linear and oval-shaped, and some appeared aggregated state in high-powered optical microscope. The number of mitochondria of each model group was significantly reduced compared with normal control group, especially on the third、fourth day after the injection. The number of mitochondria of the different model group reduced comparing with the normal control group, and the mitochondrial number of In-9m-3d group was the minimum, but the mitochondrial number of In-12m-3d group rised a little. The decrease of the number of mitochondria of the different month group gradually accompanied with the the rising age, and the number of12-month group was minimum.3, observation of mitochondrial ultrastructureThe morphology of motochondria of the frontal cortex appeared slight swollen, and some cristae of mitochondrial inner membrane broke on the first day after the injection; the mitochondria swollen obviously, and many cristae broke and assembled at the edge of the inner membrane on the second day after the injection; the mitochondria swollen further, and most of the cristae broke and the whole mitochondria became a vacuole on the third day after the injection; the mitochondria nearly turned into vacuolation on the fourth day after the injection. The mitochondrial size of the frontal cortex of the normal rats,3,6month old, was small, but the size of9month old rats became significantly larger; not only the size of12month old rats was larger, but also a little cristae inside broke, which indicating that the mitochondrial apoptosis of the frontal cortex of12-month-old rat happened, and mitochondrial function started to decline. The mitochondrial size of normal control group was small, and the number of cristae per unit area was large, while the mitochondria of the corresponding model groups larger and the number of cristae per unit area was small, many cristae fractured, even some mitochondria became vacuoles. The mitochondria of nine-month-old normal rats slightly swollen, and the mitochondria of the corresponding model rats swollen seriously, and most of the cristae fractured, then serious vacuolization occured. The mitochondria of12-month-old normal rats not only swollen slightly, but some cristae broke; but the mitochondria of the corresponding model rats swollen very clearly, and almost all cristae broken and gathered to the edge of the membrane, so the clear vacuoles appeared.4, observation of mitochondrial membrane potentialCompared with membrane potentials (MP) of3-month-old normal rats, MPs of the rats in model group all declined, and the red fluorescence intensity in model group declined gradually, and the largest decrease occurred on the fourth day after the injection. The green fluorescence intensity began to increase with the day going on after the injection, and presented the tendency of shifting from low to high, which suggested that the mitochondrial amount of the frontal cortex began to change on the fourth day after the injection. The red fluorescence intensity in frontal cortex of9-month-group normal group was the strongest, which suggested that the mitochondrial membrane potential was normal and mitochondrial function was the most exuberant, with the month increased, the red fluorescence intensity of12-month-old normal rats became to decline and tan fluorescence appeared, which illustrated that mitochondrial membrane potential began to descend and mitochondrial function was subsiding. Compared to9-month-old normal rats, the red fluorescence intensity of frontal cortex of all the other model groups declined more obviously, but the green fluorescence intensity enhanced. As the rats grow older, yellow-green fluorescence turned to dark green, which showed that the membrane potential of some mitochondria initiated to go down.5, measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPaseTo3-month-old rats, the activity of Na+-K+-ATP, Ca2+-Mg2+-ATPase of each model group decreased gradually with the day going on after the injection, which was statistically significant comparing to the normal control group (p<0.05); moreover, the decrease was the most significant on the3rd,4th day after the intraperitoneal injection, but no significance exsisted between the two model groups. Meanwhile, with the increase of month age, the mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPase activity of the frontal cortex of the normal group rose first and then decreased. The enzyme activity of6-month-rat was the strongest in the normal groups, while the enzyme activity of12-month-rat was the lowest, but there was no significance between9-month-old and3-month-old normal rats. The table shows that ATP enzyme activity of the model group and normal control group was statistically significant, but there was no statistical significance between the enzyme activity 9-month-old and12-month-old rats after the injection, which indicated that the9-month-old rats were better for the insomnic model.Part II:1, observation of super live staining of mitochondriaThe mitochondrial number of the frontal cortex of the model rats decreased obviously compared with the normal rats in high-powered optical microscope, while the mitochondrial number of after administration of diazepam and melatonin increased significantly; in addition, the mitochondrial number of the frontal cortex also increased a little after two courses of low-dose Anmeitang treatment, and the mitochondrial number of the frontal cortex increased more obviously after two courses of middle-dose Anmeitang treatment, while the mitochondrial number of the frontal cortex increased the mo.st obviously after two courses of high-dose Anmeitang treatment,2, observation of mitochondrial ultrastructureThe size of the frontal cortical mitochondria of9-month-old rats swollen obviously after the injection of PCPA, and most of the cristae broke and gathered at the edge of the mitochondrial membrane even vacuolation occurred, which indicated that mitochondrial function decreased significantly; the swelling mitochondria of the frontal cortex got better after two courses of diazepam and melatonin treatment, and the mitochondrial size were relatively integrated; while the swelling reduced to a certain extent, yet larger than the normal after low-dose treatment of Anmeitang, and some cristae broke; but the mitochondrial size was next door to that of the normal mitochondria after two courses of middle-dose and high-dose treatment of Anmeitang, and the mitochondrial integrality was betrer than that of loe-dose treatment of Anmeitang, and there was almost no breakage in the cristae.3, Determination of mitochondrial membrane potentialThe mitochondrial membrane potential of the model group significantly decreased compared with the9-month-old normal rats. It could be seen that the red fluorescence obviously reduced, while the green fluorescence significantly increased.the red fluorescence of diazepam and melatonin treatment increased compared with the model group, while the red fluorescence of low, middle, high-dose treatment of Anmeitang increased gradually, indicating that the mitochondrial membrane potential began to improve little by little.4, measurement of mitochondrial Na+-K+-ATP, Ca2+-Mg2+-ATPaseas for Na+-K+-ATP, Ca2+-Mg2+-ATPase of the frontal cortex, there was statistical significance (p<0.01) between the model group and all the treatment groups, which hinted that a variety of drug was effective after two courses of treatment. However, compared with the control group, only the model group and Anmeitang low, middle-dose group were statistically significant (p<0.05), suggesting that Anmei tang low and middle dose were effective, but still not enough to return to normal level; moreover, showing the curative effect of high-dose Anmeitang treatment was close to diazepam and melatonin treatment group and the normal group; there was statistical significance (p<0.05) between diazepam treatment group and low-dose treatment group of Anmeitang, tne model group, indicating that low doses of Anmeitang effect was not significant. With the increasing doses of Anmeitang and Na+-K+-ATP content also increasing, but there was no statistical significance among the low, middle, high-dose treatment groups of Anmeitang.On the other hand, Ca2+-Mg2+-ATPase of the frontal cortex of the model group and all the treatment groups declined compared with the control group, but they were statistically significant (p<0.05); while there was statistical significance between each treatment group and the model group (p<0.01); while there was also statistical significance between the normal, diazepam treatmeng group and low, middle-dose treatment group of Anmeitang (p <0.01), but there was no statistical significance between the low-dose Anmeitang treatment and the melatonin treatment, middle, high-dose Ameitang treatment groups, which indicated that although Ca2+-Mg2+-ATPase content also increased with increasing doses of Anmeitang, as well as the statistical significance between low-dose Anmeitang treatment and high-dose Anmeitang treatment (p<0.05), there was no statistical significance between middle-dose Anmeitang treatment and high-dose Anmeitang treatment, which suggested that the dose-dependent of Anmeitang was not obvious.In summary1, the mitochondrial number and ultrastructure of the cortex of3-month-old model rats reduced significantly on the3rd day after intraperitoneal injection; and the mitochondrial membrane potential and ATP activity started to decline obviously on the2n day, and the most significantly(p<0.05) on the3rd day, but rise slightly on the fourth day.2, the most obviously changes of the mitochondrial ultrastructure, membrane potential and ATP activity of the cortex existed in the9-month-old model group, but these changes were not statistically significant compare with12-month-old model group.3, the mitochondrial number began to increase, and the mitochondrial ultrastructure and the mitochondrial membrane potential of the cortex also began to get better after the continuous treatment of Anmeitang; and the content of Na+-K+-ATPase Ca2+-Mg2+-ATPase also began to increase along with the increase of Anmeitang dose, but no significance existed in the both.Conclusion1, To3-month-old rats, the most obvious changes of the mitochondrial ultrastructure and function of the frontal cortex of the model rats after a continuous injection of3days were on3rd day.2,9-month-old rats were the best choice for the insomnic model by PCPA, and the possible insomnic mechanism is the damage of the mitochondrial morphology and function of the prefrontal cortex by the intraperitoneal injection of PCPA.3, Anmeitang can increase the number of mitochondria of the prefrontal cortex of the model rats, and improve the degeneration of the mitochondrial ultrastructure; while Anmeitang can also increase the mitochondrial membrane potential and the content of Na+-K+-ATPase、Ca2+-Mg2+-ATPase; the dose-dependent of effectivenessin is not obvious although the content of Na+-K+-ATPaseN Ca2+-Mg2+-ATPase increase along with the dose increase of Anmeitang.4, The decrease and the improvement of the morphology and function of the cortical mitochondria is possibly related with the cortical excitation and inhibition imbalance and excess of Yin and shortage of Yang.Innovation1, it is frequent for us to see the clinical reports of Chinese medicine treatment about the insomnia, but the basic research are less, and these basic researches are performed based on the theory of Western neurotransmitter of the lower brain stem and hypothalamus levels. But we firstly investigated the etiopathogenesis of the insomnic models by PCPA and the effect mechanism of Anmeitang from the highest level of the neurous system.2, we fistly discussed the relationship between the structure and function of mitochondria and the cortical excitation and inhibition imbalance, excess of Yin and shortage of Yang of insomnia. Inadequacy1, we don’t carry out more experiments about month-ages and time points of model rats because of the difficulty of the rat breeding cycle and funding issues.2, our study does not carry out the further examination at different levels of the gene, mRNA and protein expression, but we will perform the next continuous, exhaustive exploration in order to provide some references for the basis of external research.