| BackgroundZige lyophilized powder, a novel drug developed by Xu (China), was characterized as a neuroprotective agent against cerebral ischemia. Its prescription and preparation had applied for national invention patents, research of pharmacodynamics was supported by Science and Technology Development of Chongqing(CSTC,2010AC5011), and its protection for "Brain Neurovascular unit" was supported by postgraduate technology innovation project of Southwest University, its preparation technology, quality standard, stability were supported by the ministry of education teachers scientific research funds.As the third largest killer disease, Stroke is seriously hazard to human health. So far, there is no way to recover nerve function after stroke clinical. To rebuild the damaged neural network, finding new alternative fittings, such as new nerve cells, new blood vessels, new synapses and new electrical excitability connection is necessary. Stem cells because of their proliferation and differentiation potential, are alternative good sources of lost cells after stroke. Endothelial progenitor cells (endothelial progenitor cells, EPCs) from the bone marrow of another type of adult stem cells, plays an important role in the treatment of vascular regeneration. In vitro autologous transplantation of amplification EPCs may be a novel and effective strategy for the treatment of cerebral ischemia, So intensive researches should be done to further elucidate the effects of Zige lyophilized powder on endothelial progenitor cells of cerebral ischemic rat and endothelial cell proliferation. Objective(1) To investigate the effects of Zige lyophilized powder on HUVECs proliferation under normal and hypoxia/reoxygenation environment and to explore its possible protection mechanism against damage;(2) To research the effects of Zige lyophilized powder on EPCs proliferation under normal and hypoxia/reoxygenation injury environment;(3) The focal cerebral ischemia model was induced by electrocoagulation of the right middle cerebral artery in rats as described previously with minor modifications to study the protective effect of Zige lyophilized powder on nerve function in focal cerebral ischemic rats, especially whether its role of promoting the proliferation of EPCs to provide the basis for its new drug innovation.Methods(1) At the cellular level, HUVECs were cultured, we studyed the effects of Zige lyophilized powder on HUVECs proliferation under normal environment using MTT colorimetric method. Krebs liquid was adopted to establish the hypoxia/reoxygenation injury model. After intervention of Zige lyophilized powder, MTT colorimetric method was used to determine cell activity, the xanthine oxidase method was adopted to detect the activity of superoxide dismutase (SOD) in cells, the thibabituric acid developing method was adopted to determine the content of malondialdehyde (MDA), the nitrate reductase method was used to detect the content of nitric oxide (NO), the2,4-dinitrophenylhydrazine developing method was adopted to the content of extracellular lactate dehydrogenase (LDH). Additionally, the levels of apoptotic related proteins Bcl-2, Bax and Caspase-3were measured by Western blotting analysis.(2) EPCs was successfully isolated and purified by density gradient centrifugation and adherent culture combinated dedicated EGM-2medium. Morphological identification, cell surface molecule markers and phagocytosis function were detected to identificate EPCs; MTT colorimetric method was used to observe the effects of Zige lyophilized powder on EPCs proliferation under normal and hypoxia/reoxygenation injury environment in vitro study.(3) The focal cerebral ischemia model was induced by electrocoagulation of the right middle cerebral artery in rats. Neurological behavioral Longa score, balance beam score and infarct area were evaluated to determine the experimental animal models. Experimental study was done to detect the effects of Zige lyophilized powder on EPCs proliferation in peripheral blood of rats after cerebral ischemic injury in vivo by flow cytometry.Results1. The experimental study in vitro of Zige lyophilized powder on HUVEC proliferation under normal environment12.5μg·mL-1,24.5μg·mL-1Zige lyophilized powder can significantly promote EPCs proliferation under normoxic environment (P<0.01). Hypoxia(H)/Reoxygenation(R) caused significant viability loss and an release of LDH increase compared to the normoxic control,12.5μg·mL-1Zige lyophilized powder enhanced cell viability(P<0.05). Also,49.0μg·mL-1,24.5μg·mL-1,12.5μg·mL-1Zige lyophilized powder prevented the decline of SOD activity during H/R(P<0.05). Furthermore,24.5μg·mL-1,12.5μg·mL-1Zige lyophilized powder significantly inhibited the increase of MDA and NO content. Additionally, apoptotic related protein levels were measured by Western blotting analysis. Bcl-2protein level increased significantly in the24.5μg·mL-1,12.5μg·mL-1Zige lyophilized powder group.49.0μg·mL-1,24.5μg·mL-1,12.5μg·mL-1Zige lyophilized powder significantly decreased Bax level and up-regulated Bcl-2/Bax ratio. Caspase activation and apoptosis were monitored in the reoxygenated cells.49.0μg·mL-1,24.5μg·mL-1Zige lyophilized powder decreased Caspase-3expression(P<0.01).2. The experimental study in vitro of Zige lyophilized powder on EPCs proliferation from the bone marrowWe isolated and purified EPCs. Morphological identification, cell surface molecule markers and phagocytosis function were detected to identificate EPCs.24.5μg·mL-1Zige lyophilized powder can significantly promote EPCs proliferation under normoxic environment (P<0.05).98.0μg·mL-1,49.0μg·mL-1Zige lyophilized powder can significantly promote EPCs proliferation under Hypoxia/Reoxygenation injury environment (P<0.05, P<0.01).3. The experimental study in vitro of Zige lyophilized powder on EPCs proliferation from the bone marrowCompared with the sham group, model group and drug groups will appear with forelimb curl, the balance beam not smooth walking, forelimbs or hind legs slipping symptoms. Neurological behavioral Longa score in the drug groups reduced significantly compared with the fourth and seventh ischemic model and the balance beam walking score in the drug groups reduced significantly compared with the first and seventh ischemic model.65.4mg’kg"1Zige lyophilized powder can promote the number of EPCs in peripheral blood of cerebral ischemic rats.Conclusion1. Zige lyophilized powder could promote the proliferation of HUVECs under normoxic environment, the results indicate that Zige lyophilized powder prevents damage H/R induced to HUVECs, the cytoprotection of Zige lyophilized powder appears to be associated with the enhancement of the ability anti-oxidation and anti-apoptosis.2. Zige lyophilized powder could promote the proliferation of EPCs from the bone marrow in vitro and prevents damage H/R induced to EPCs.3. Zige lyophilized powder could promote EPCs to mobilize from the bone marrow into the peripheral blood and has a protective effect on nerve function of cerebral ischemia injury in rats... |
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