| Purpose: For many years, the pathological changes of peripheral nerve affect the people's living quality badly, which is a very important and complicate process in neuroscience. Recently, reseachers found that one of pathogenesis is that oxidative irritation can make free radicel increase. Even which cause the apoptosis of Schwann cells. Schwann cell is a glial cell of peripheral nerve system, which can surround the axon to form or not form myelination. It can excrete several neurotrophic factors relate to growth and regeneration of nerve system, recently, more and more people apply external factors, such as neurotrophic factor, Chinese traditional medicine,electromagnetism, to function on Schwann cell, which can cure peripheral nerve injuries.Chinese traditional medicine can function on peripheral nerve, which is a hotspot researched by investigator. Chinese traditional medicine can promote the regeneration of PNS, which is accepted by investigators. BYHWT is a noted prescription invented by Wang qingren in the Qing dynasty, which is decocted with the root of large-flowered root of membranous milkvetch, root of common peony, Chinese angelica, earthworm, Ligusticum wallichii, peach kernel. It is pharmacological-effection is invigorating qi and invigorating blood circulation that is applied for treating the cerebral apoplexy. In recent years, it is reported that this prescription is also used to treat spinal cord intaction and peripheral neuropathy. Modern medical investigation indicates that BYHWT not only dilates blood vessels, increases the flow of brain blood and improves microcirculation but also resists the oxidization of the free radical. There are a few primary studys about the antioxidant function but few about the mechanism of protection to Schwann cell impaired from oxidation.This investigation is aimed at probing into the antioxidant and protecting function of BYHWT. The oxidization-damaged Schwann cells model e induced by the peroxide hydrogen is used to investigate the pharmacological mechanismof BYWHT treating DM. we offered pharma-cological proofs for extending the clinical application and theoretical clues for supervising the clinical remedy. Method: In this subject, Sciatic nerves and median nerves of neonatal Wistar rats is cultured in liquid-DMEM containing 10% of the new-born bovine's serum and the cultivation case of 5% CO2, 37℃, saturated humidity. The Schwann cells cultured 24 hours were divided into threel groups and added different medicines in each group.1. Preparation of BYWHT: the prescription is composed of the root of membranous milkvetch, Chinese angelica, Ligusticum wallichii, peach kernel, safflower, earthworm and red spoon. The soup was decocted by water and extracted by alcohol (75% ethanol ). Crude drug content was 2g/ml, kept in 4 degrees Centigrade and filtered the fungus while using.2. Purify and identify Schwann cells: Adding Ara-c in order to get rid of fibroblast-like cells. After cultivating 3d we identified the cultured cells and calculated the purity of Schwann cells by the immunohistochemistry dyeing (S-100 antibody is used).3. Treating by drug or oxidization damage: There were three cultured groups: one group was oxidization-damaged group treated with hydrogen whose ultimate concentration was 0.5%, another was treated with peroxide hydrogen(0.5% H2O2) and BYWHT (600μg/ml), and another was control group only treated with culture liquid.4. Investigating Schwann cells activity of different groups by MTT assay.5. Measuring endocellular MDA and SOD by biochemistry assay.6. Measuring Caspase-3 by immunohistochemistry assay.7.After Fluo-3/Am fluorescence dying, collecting the pictures of contraction varity of endocelluar calcium from every experiment group and carried on quantitative analysis by confocal laser scanning microscope.8. Measuring GDNFmRNA in three groups by RT-PCR(reverse transcription PCR).Through above experiments, we set up the oxidization-damaged Schwann cells model mutated by hydrogen, and probed into the anti-oxidant protective action of the anti-oxidant medicine- BYWHT at the same time Result: Primary Schwann cells were round, suspending in the medium when inoculated at once. Most cells attached after 24 hours. The cells were comparative small with a powerful scene of three-dimension and satiation and were shuttle-shape with two slender dendrites. After culturing 24 hours, Ara-c (the contration of Ara-c in the medium was 10-5) was added into the medium and removed after 48-72 hours, we observed the purity was increased obviously under light microscope, and immunohistochemistry(S-100) dyeing showed that more than 80% of the cells presented positive reaction. 12 hours after adding H2O2, we observed the change of the cellular shape under microscope: the dendrites withered, the cells swelled into roundness, the quantity of Schwann cells reduced, some cells took off the wall and floated in the culture liquid, the cells' three-dimensional scene were bad and refraction droped, a large number of cell piece were obviously. But under the protection of BYWHT all cells were growing well with a powerful scene of three-dimension and the cells were scarcely withered and broken, but it was observed that the cell gathered obviously.In the control group, the cells' growing station was well.The measurement of different factor-treated groups is as follows: 1. Analysing antioxidant function by MTT indicates: BYWHT can obviously resist the oxidization induced by the peroxide hydrogen. Compared with the hydrogen damaged group, there is a higher cellular livability in the BYHWT treated group. Statistics meaning is positive between them (P<0.05) but it is not existing between normal-control group and BYHWT group.2. Biochemical measure indicates: Compared with the damaged group, BYHWT kept the endocellular superoxide dismutase (SOD) in high vigorous level. It was obvious that BYHWT resisted the SOD reducing caused by the hydrogen peroxide though this level was lower than the control group. Both hadstatistics meaning(P<0.05).3. Compared with the normal control group, the peroxide hydrogen caused lipide-hydrogen to pile up inside the cells, but BYHWT obviously reduced this kind of pileup(P<0.05). In the normal control group there is less endocellular lipide peroxide.4. Mesuring Caspase-3 by immunohistochemistry: It was powerful positive in the peroxide hydrogen-damaged group, slight positive in the BYHWT protected group, and there was undyed nearly in the normal control group.5. Analysing the cellular calcium with confcoal laser scanning microscope indicates: the calcium concentration increased remarkably and the fluorescence intensity was stronger in the peroxide hydrogen-damaged group. While in the BYHWT protected group and control group the fluorescence intensity was alike and both were slighter.6. Measuring the expressing degree of GDNFmRNA by RT-PCR: the expressing degree of GDNFmRNA was lower in the peroxide hydrogen-damaged group than the degree in the BYHWT -protected group(P<0.05). While both were lower than the normal control group.Conclusion: We made the peroxide hydrogen-damaged Schwann cells model with the primary cultured Schwann cells. Discovered from the investigation, BYHWT resisted the oxidization damage induced by the peroxide hydrogen visibly. The peroxide hydrogen caused that the endocellular free radical increased, and the lipid the activity of SOD reduced and the endocellular lipide-hydrogen increased. The increased free radicals resulted in the overload of the calcium through Caspase-3 pathway and caused apoptosis finally. Howerver BYHWT can reduce the content of endocellular lipid hydrogen-MDA, maintain the higher level of SOD and reduce the concentration of calcium and Caspase-3 to avoid the failure incident happening.This results show that BYHWT's action protecting the Schwann cells by resisting the oxidization damage and the antioxidant molecular mechanismthrough the signal pathway of the oxidization damage. And it analysed and confirmed the way in which BYHWT resisted the oxidization damage for the first time , provided the theoretical foundations for the antioxidant molecular mechanism of the free radical worked in the neuropathy of peripheral nerves and for the investigations of the medicine's antioxidation. |
PDF Full Text Request