The Study Of Didangtang On RBC Clearance By Microglia After Intracerebral Hemorrhage Based On Poxuehuayu Method

Objective:To explore the Study of Didangtang on RBC clearance by Microglia after intracerebral hemorrhage based on Poxuehuayu method.Method : 1.Establish a co-culture model of microglia and RBC to simulate the conditions of ICH in vitro.Microglia were labeled with PKH26 as orange-yellow fluorescence,microglia nucleus with H33342 as blue fluorescence,RBC with CFSE as green fluorescence,and co-culture of microglia with RBC and cell morphological changes were observed in real time by high content living cell analyzer.2.Using the Flow cytometer to screen suitable the percentage of co-culture of microglia with RBC,Calcium ion concentration in vivo and cell surface area of RBC at different ages were measured to identify the aging degree of erythrocyte.Normally,microglia and RBC were co-cultured for3,6 and 9 hours to calculate the number of microglia that phagocytized RBC and to verify the phagocytic ability of microglia.3.Flow cytometer was used to detect the change of microglia's phagocytosis to RBC under the intervention of Poxuehuayu decoction.Result:1.The co-culture model of microglia and RBC was established successfully,and the phenomenon of microglia devour RBC was observed.After co-culture for 1 hour,microglia extended to RBC;2-4 hours,a large number of RBC aggregated to microglia;5hours later,microglia absorbed and devour RBC.2.Microglia and Red blood cells co-culture,compared with common culture ratio of RBC:microglia=20:1,when common culture ratio of RBC:microglia=40:1,micro-g-lia devour more RBC(P<0.05);Compared with the age of RBC for 3d and 20 d,the surface area of RBC at 40 days,the concentration of intracellular calcium ions increased,which verified the degree of aging(P<0.05),and the number of RBC with age of 40 d was selected From the results,compared with co-culture for 3 hours,a total of 6h and 9h were co-cultured,the number of microglia increased of devouring RBC,and the total culture of 9h was more significant(P< 0.05).3.The detection of 52.5 ?g/mL to 500 ?g/mL by MTT method had little effect on the survival rate of microglia and had no toxic effect.4.Microglia were pre-stimulated by 100ug/mL Didangtang Decoction for 24 hours,the phagocytosis ability of microglia was enhanced at 6 and 9 hours of co-culture,9h when more significant(P<0.05).Conclusion:The co-culture model of microglia and RBC is established,and the abilityof microglia to phagocytize RBC is enhanced under the intervention of Didangtang decoction.