The Regulatory Effects Of EPIc On Macrophages In Mouse And Protective Effects On Cerebral Ischemia-reperfusion Mouse

ObjectiveStudy the regulatory effects of EPIc on macrophages in mouse and the protective effects on cerebral ischemia-reperfusion mouse to explore the related mechanisms preliminarily.Methods1. Research about the regulatory effects of EPIc on peritoneal macrophages in mouse:The toxicity of EPIc to peritoneal macrophages was detected by MTT and CCK-8 test. Microscopic photographs were used to check the influence of EPIc on long term survival of peritoneal macrophages. Fluorescence enzyme-labelled meter was used to measure the effects of EPIc on apoptosis of SytoxGreen labelled macrophages induced by CHX, CTX and H2O2. The effects of EPIc on mitochondria (labelled by MitoTrackerred and Calcein-AM together) and lysosomes (labelled by LysoTrackerred and Fluo-4AM together) changes of peritoneal macrophages were detected by Laser scanning confocal microscope (LSCM). The effects of EPIc on adhesion of peritoneal macrophages which were labelled by DIOC6(3) were examined by LSCM. Griess Gragent was used to detect the role of EPIc in NO production of peritoneal macrophages and the mouse IL-1βproduction was measured by mouse IL-1βelisa kit together with enzyme-labelled meter. IL-6, IL-10, MCP-1, IFN-y and TNF production was analyzed by Cytometric Bead Array (CBA) method together with FCM. LSCM and FCM were used to detect the effect of EPIc on phagocytosis of peritoneal macrophages.2. Research about the regulatory effects of EPIc on microglia cells (macrophges located in brain) from mouse:X-Celligence RTCA was used to detect the effects of EPIc on microglia cells survival index and apoptosis of microglia induced by H2O2. Apoptosis of microglia cells induced by intracellular [Ca2+]i overload was measured by X-Celligence RTCA and LSCM. DIOC6(3) which could put green colour on living cells but not dead cells together with LSCM was used to test the influence of EPIc on activation and Nano-tubes (NTs) of microglia cells. LSCM was also used to detect the effect of EPIc on translocation of NF-κB.3. Research about the protective effects of EPIc on cerebral ischemia-reperfusion mouse:Apoptosis of neurocytes induced by intracellular [Ca2+]i overload was measured by LSCM. TTC staining was used to detect brain infarct areas of cerebral ischemia-reperfusion mouse. Dry/wet specific gravity method was used to test brain water content (bwc) of cerebral ischemia-reperfusion mouse. Cerebral ischemia-reperfusion mouse treated with EPIc and untreated with EPIc were both bred under the same condition for long enough, then the life-span was calculated.Results1. The regulatory effects of EPIc on peritoneal macrophages in mouse:Both MTT and CCK-8 methods demonstrated that EPIc had no any toxic-side effect. The inhibitory effect of EPIc on apoptosis of peritoneal macrophages induced by CHX, CTX and H2O2 also has been proved by Sytox red staining together with Fluorescence enzyme-labelled meter. And then the role of EPIc to prevent the disintegration of mitochondria and lysosomes of peritoneal macrophages was confirmed by related florescence staining under LSCM. Immediately the positive effect on adhesion of peritoneal macrophages has been found. Griess Gragent and mouse IL-1βElisa kit respectively detected the production of NO and IL-1β, and the data showed that EPIc promoted NO and IL-1βproduction of "rest" peritoneal macrophages but inhibited NO and IL-1βproduction of peritoneal macrophages stimulated by LPS. The detection of CBA showed that EPIc promoted IL-6 and MCP-1 production of "rest" peritoneal macrophages, but inhibited IL-6, IL-10, MCP-1, IFN-y and TNF production of peritoneal macrophages stimulated by LPS. Both LSCM and FCM data showed the promoted effect of EPIc on phagocytosis of peritoneal macrophages.2. The regulatory effects of EPIc on microglia cells (macrophges located in brain) from mouse:The data of microglia cells suvival index by X-Celligence RTC A showed that EPIc had no any toxic-side effect on microglia cells, moreover demonstrated that EPIc had inhibitory effect on apoptosis of microglia cells stimulated by H2O2. Both X-Celligence RTCA and LSCM proved that the average death time of microglia cells induced by intracellular [Ca2+]i overload had been dalayed by EPIc. The active action of EPIc towards activation and NTs production of microglia cells was attested by DIOC6(3) staining together with LSCM. The data about NF-κB showed that EPIc promoted translocation of NF-κB of "rest" microglia cells but inhibited translocation of NF-κB of microglia cells stimulated by LPS, that is to say, EPIc had dual directional regulatory effect on translocation of NF-κB of microglia cells.3. The protective effects of EPIc on cerebral ischemia-reperfusion mouse:EPIc dalayed the time of apoptosis of neurocytes induced by intracellular [Ca2+]i overload. The infarct size of cerebral ischemia-reperfusion mouse treated with EPIc was obviously smaller than cerebral ischemia-reperfusion mouse untreated with EPIc. EPIc also alleviate brain water content of cerebral ischemia-reperfusion mouse and prolong life time of schemia-reperfusion mouse.