| AIM:To investigate the effect of salidroside (Sal) on T lymphocyte, peritoneal macrophage and microglia cell behaviors of normal mouse in vitro, and the protective effect on cerebral ischemia-reperfusion model mice by salidroside preconditioning as well as to explore the related mechanisms preliminarily.Methods:1. To investigate the effect of Sal on the behaviors of the mouse T lymphocytes in vitro, in order to elucidate the immunoregulation effect of Sal. The toxicity of Sal to T lymphocytes was detected by MTT.3,3'-Dihexyloxacarbocyanine iodide (DIOC6(3)) which could put green colour on living cells but not dead cells together with FCM was used to test the influence of Sal on the activity of T lymphocytes. T lymphocytes were activated by concanavalin (Con A). The expression of CD69, which is early activation marker, was measured by FCM combined with two fluoresceins conjugated monoclonal antibodies. The impact of Sal on proliferation of T lymphocytes in response to Con A was detected by FCM combined with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining for 72 h.2',7'-Dichlorofluorescin diacetate (H2DCFDA)staining together with FCM was used to analyze the changes of reactive oxygen species (ROS) in un-activated T lymphocytes.2. To study the effect of Sal on peritoneal macrophages behaviors of mouse in vitro. The toxicity of Sal to peritoneal macrophages was detected by MTT. Peritoneal macrophages were stimulated with lipopolysaccharide (LPS) and interferon-y (IFN-y), the proliferation of macrophages was measured by MTT. The effect of Sal on the apoptosis of Sytox(?) Green-labelled peritoneal macrophages, which induced by cycloheximide(CHX), ionomycin (ION), ION and deferoxamine (DFO) as well as ION and thapsigargin (TG) respectively,was detected by Fluorescence enzyme-labelled meter. FCM were used to detect the effect of Sal on phagocytosis of peritoneal macrophages. H2DCFDA staining was used to analyze the changes of ROS in peritoneal macrophages which measured by Fluorescence enzyme-labelled meter, and the changes of ROS in bone marrow cells checked by FCM. Griess Gragent was used to detect the role of Sal in production of nitric oxide (NO) in peritoneal macrophages activated by LPS and IFN-y.3. To investigate the effect of Sal on microglia cells behaviors of mouse in vitro. DiOC6(3) staining combined with FCM was not only used to test the influence of Sal on the activity of microglia cells, but also used to check the influence of Sal on the changes of the mitochondrial membrane potential of activated microglia cells induced by LPS and glutamate respectively. H2DCFDA staining combined with FCM was used to analyze the changes of ROS in microglia cells induced by LPS. FCM was used to measure the role of Sal on the endoplasmic reticulum(ER) stress-induced apoptosis of microglia cells with ER Tracker(?) Red staining, which induced by dithiothreitol (DTT) and hydrogen peroxide (H2O2), separately. Fluorescence enzyme-labelled meter was used to measure the effects of Sal on the apoptosis of Sytox(?) Green-labelled microglia cells, which induced by ION, H2O2, ION and DFO as well as ION and TG, respectively.4. To investigate protective effects of on cerebral ischemia-reperfusion mouse by salidroside preconditioning in vivo. Cerebral ischemia-reperfusion model mouse was preconditioned by Sal, which was injected into the lateral cerebral ventricle via stereotaxic technique. The concentration of Sal in vivo was determined by the decrease of neurologic deficit score. The effect of Sal preconditioning on the percentage of infarct area of cerebral ischemia-reperfusion model mouse was measured by TTC staining. The effect of Sal preconditioning on the index of mouse spleen, thymus gland and weight of cerebral ischemia-reperfusion model mouse were checked by weighed. And the effect of Sal preconditioning on the life-span of cerebral ischemia-reperfusion model mouse was calculated via breeding the model mouse, which pretreated and un-pretreated with Sal separately, under the same condition for long time.RESULT:1. The studies in vitro showed that, Sal had no any toxic-side effect on T lymphocytes. Sal could enhance the activity of T lymphocytes, promote the activation and proliferation of T lymphocytes stimulated by ConA, and reduce the production of ROS of un-activated T lymphocytes. 2. The studies in vitro showed that, Sal had no any toxic-side effect on peritoneal macrophages. Sal could promote the proliferation of peritoneal macrophages activated by LPS and IFN-y, and inhibit apoptosis of peritoneal macrophages induced by CHX, ION ION and DFO as well as ION and TG respectively. Sal could promot the phagocytosis of peritoneal macrophages. And Sal could reduce the production of ROS in both of activated peritoneal macrophages and bone marrow cells. Sal could increase the NO of production in peritoneal macrophages induced by LPS and IFN-γ.3. Sal could enhance the activity of microglia cells in vitro, and Sal could also inhibit the decrease of mitochondrial membrane potential of microglia cells induced by LPS and glutamate respectively. The result of H2DCFDA staining showed that Sal could reduce the production of ROS in activated microglia cells induced by LPS. Sal could inhibit the ER stress-induced apoptosis of microglia cell which induced by DTT and H2O2, respectively. And also could inhibit the apoptosis of micoglia cells which induced by ION, H2O2, ION and DFO as well as ION and TG, respectively.4. The studies in vivo showed that, Sal preconditioning could reduce the infarct area of cerebral ischemia-reperfusion mouse obviously. Sal preconditioning could also inhibit the decrease of cerebral ischemia-reperfusion mouse's index of spleen, thymus gland and weight. Sal preconditioning could also prolong life time of cerebral ischemia-reperfusion mouse.Conclusion:Sal can regulate the behaviors of mouse T lymphocytes, peritoneal macrophages and microglia cells in certain concentration range, moreover, it can protect cerebral ischemia-reperfusion model mouse by salidroside preconditioning. |
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