| In recent years, there have been a great deal of valuable researches on the splenasthenic syndrome in the area of traditional Chinese medicine. Spleen deficiency refers not only to the functional disorder of digestive systems and immunological function, but also the related changes in brain neurotransmitters, neurotrophic factors, signal transduction, gene expression. What's more, spleen deficiency can lead to the changes in the structure of cerebral cortex, hippocampus and other organizations by affecting nerve cell differentiation and proliferation, and changing the ability of organism's remodeling, thereby affecting brain functions. There is a lot of clinical and experimental foundation to confirm that acupuncture can cure the splenasthenic syndrome. Therefore, for the damage to brain functions caused by splenasthenic syndrome, efforts are made in our researches to the recovery of brain functions through curing this syndrome or improving the splenic deficiency condition. Based on the assumption and literature study, we chose the differentiation and proliferation of nerve cell as cut-in point. From the aspects of literature and experiment study, we can approach the possible mechanism of electro-acupuncture on protecting nerve cell from damaging caused by splenasthenic syndrome.1. Objective:To observe the differentiation and proliferation of nerve stem cell and expression of bFGF protein and mRNA in subventricular zone(SVZ)and Denatate Gyrus(DG) in spleen deficiency Rats, and approach the effects of electro-acupuncturing Zusanli (ST36) and Sanyinjiao (SP6) on the differentiation and proliferation of nerve stem cell and expression of bFGF protein and mRNA. By observing nerve stem cell, we hope to find the possible mechanism of electro-acupuncture on protecting nerve cell from damaging caused by splenasthenic syndrome, and provide theoretical and experimental evidence for electro-acupuncture to treat nerve cell damage caused by splenasthenic syndrome.2. Methods:A total of 192 healthy, male, Sprague Dawley rats, weighing (55±5) g and aged 4 weeks, from SPF-grade laboratory were taken into the study. Before the test, they were raised to adapt to the surroundings. According to the random digits table, they were divided into control group, model group and electro-acupuncture treated group, each group contained 64 rats. Then each group was averagely divided into 7 days group,14 days group and 28 days group at random. There were 16 rats in each subset group. Spleen deficiency was induced via intragastric administration of rhubarb,intraperitoneal injection of reserpine connected with feeding irregular diet for 14 days. We observed the change of appearance and weight in the spleen deficiency rats. Rats in electro-acupuncture treated group were treated with electro-acupuncturing Zusanli and Sanyinjiao points after modeling. As for electric acupuncture, distant-dense wave was chosen. The frequency was15Hz for dense wave and intensity was 6 to15 V. The treatment lasted for twenty minutes, and was done once every day until the rats got killed. On the other hand, rats in model group were fixed on the operation board without any treatment for twenty minutes after modeling.2.1 We select 2 rats from each subset group randomly. All together 24 rats were killed at the design time, and their brains were taken after fixed by perfusion. Then we observed the pathomorphism of subventricular zone (SVZ) and Denatate Gyrus(DG) in normal group, model group and electro-acupuncture treated group by light microscope and electronmicroscope.2.2 We select 6 rats from each subset group randomly. All together 72 rats were injected Brdu peritoneal 2 days before the design time that they were killed, and their brains were taken after fixed by perfusion. Then we observed the cell marked by Brdu,Brdu/Nestin,Brdu/GFAP and Brdu/NSE in SVZ and DG in normal group, model group and electro-acupuncture treated group by immunohistochemistry. At last, we counted positive cells at light microscope of 400X, analyzed the image by analytical system of Image-Pro Plus software from USA, and compared the difference between groups by Stata 10.0 software.2.3 We select 8 rats from each subset group randomly. All together 96 rats were killed at the design time, and their brains were taken immediately. Immunohistochemistry and PCR reaction was performed to detect the protein and mRNA expression of bFGF in SVZ and DG. At last, we counted positive cells at light microscope of 200X, analyzed the image by analytical system of Image-Pro Plus software from USA, and compared the difference between groups by Stata 10.0 software.3. Results:3.1 Compared to the normal group, rats in the model group were obviously emaciated, poor hair quality, and rolling up together, lassitude, closing eyes, fatigue, reacting slowly, defecating frequently.3.2 Compared to the normal group at light microscope, the outline of neuron was blurred, the cell body was swelling, the interspace around cells was widen, and nuclear fragmentation, cavity in kytoplasm, besides, the zonal layer of cells was diminished in model group. But in electro-acupuncture treated group, the outline of neuron was clear, the cell nucleus was eumorphism, the zonal layer of cells was well-distributed, and the glial cells were infiltrating. At electron microscope, some structures of the pericaryon and neuropil were destroyed in model group, especially the structure of mitochondrion and the terminal of synapse. But in electro-acupuncture group, degree of injury in nerve cells is lower and most of the cell organs still exist.3.3 The same conclusion could be seen in the same period either in subventricular zone or Denatate Gyrus. The number of positive cells of Brdu,Brdu/Nestin,Brdu/GFAP,Brdu/NSE were coincident in different periods in the normal control group (P>0.05). The number of Brdu cells in model group was less than that in the normal group in the 7th day and the 14th day (P<0.05), but it recovered to the normal level in the 28th day. The number of Brdu cells in electro-acupuncture treated group was almost the same as that in the normal group in the 7th day, and it was extremely high in the 14th day, but it decreased in the 28th day and 49th day, however, the level was still higher than that in model group and normal group in this period (P<0.05). In model group, there was little Brdu/Nestin cells in the 7th day, while the number increased and reached to a normal level in the 14th day (P>0.05), then it achieved the peak level in the 28th day (P<0.05). In electro-acupuncture treated group, the number of Brdu/Nestin cells was just the same as that in normal group in the 7th day. And it reached to a peak level in the 14th day. In the 28th day and 49th day, the number declined but it was still higher than that in model group and normal group in this period (P<0.05). In model group, the number of Brdu/GFAP cells was less than that in normal group in the 7th day and the 14th day (P<0.05). But it reached to the normal level in the 28th day (P>0.05). The number of Brdu/GFAP cells in electro-acupuncture treated group was just the same as that in normal group in the 7th day (P>0.05). And it reached to a peak level in the 14th day. Then it delined to the basal line after the 28th day. The number of Brdu/NSE cells in model group was less than that in normal group in the 7th day and the 14th day (P<0.05), but it recovered to the normal level in the 28th day (P>0.05). In electro-acupuncture treated group, the number was just the same as that in normal group in the 7th day (P>0.05). And it increased in the 14th day and reached to a peak level in the 28th day, then delinced to the basic line gradually.3.4 The expression of bFGF protein and mRNA are coincident in the same period either in subventricular zone or Denatate Gyrus. In model group, the expression was low in the 7th day after model treated period. However, it increased quickly in the 14th day and the level was higher than that in normal group (P<0.05) It stayed for some time and delined to the basal line in the 49th day. The expression of bFGF protein and mRNA in electro-acupuncture treated group was different. It generally increased in the 7th day, and the level was higher than that in model group and in normal group (P<0.01). Then it slightly descended in the 14th day; however, the level was still higher than that in normal group in this period. The high level lasted to the 49th day.4. Conclusions:In this empirical study, we set up a spleen deficiency rat model by combinding with intragastric administration of rhubarb, intraperitoneal injection of reserpine and fed on irregular die. In order to observe the damage of nerve cell caused by splenasthenic syndrome, and provide theoretical and experimental evidence for electro-acupuncture protecting nerve cell, we observed the growth, differentiation and proliferation of nerve stem cell by histomorphology and immunohistochemistry. The results were as follows:The splenasthenic syndrome may result in the changes of pericaryon and synapses, and degrade the proliferation and differentiation potency of nerve stem cells. The electro-acupuncturing Zusanli and Sanyinjiao points treatment can inhibit the damage and protect the nerve cells. It maintains the normal structure of cells and synapse. Besides, it can enhance the proliferation potency of nerve stem cell in the spenasthenic syndrome, and encourage nerve stem cells differentiating to astrocyte and nerve cell in the Subventricular Zone of the Lateral Ventricle and dentate gyrus of hippocampus. The protection of electro-acupuncture may be relevant to maintaining a high level of bFGF and mRNA expression。In a word, we could consider that the encephalodysplasia due to the splenasthenic syndrome may be cured by electro-acupuncture. Besides, the elecrto-acupuncture can induce the cell proliferation and differentiation, so as to repair the nerve cells which are impaired by splenic asthenia.
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