Study On The Effect Of Liang Xue Hua Yu Decoction On Apoptosis And Phagocytosis In Cultured Human Retinal Pigment Epithelium Cells

ObjectiveTo observe the effect of Liang Xue Hua Yu decoction to blue light-induced apoptosis in cultured retinal pigment epithelial(RPE) cells of human.And also to investigate the effect of Liang Xue Hua Yu decoction to the phagocytosis of cultured human RPE cells in vitro.And thereby to reveal the mechanism of Liang Xue Hua Yu decoction in curing age-elated macular degeneration(AMD) by cytobiology.And also show the evidence of Liang Xue Hua Yu decoction as a effective therapy for AMD.Methods1.The culture and indentification of RPE cells:The human RPE cells were separated and depurated by the method of trypsin digestion,the cells were cultured in vitro,then to observe the process of growth.And they were identified by the way of immunohistochemisty.2.Screening of suitable concentration of Liang Xue Hua Yu decoction for the following experiment:To observe the effects of different concentration of Liang Xue Hua Yu decoction and the blood serum which contain different concentration of different dose Liang Xue Hua Yu decoction to the RPE cells.In order to choose the suitable concentrations of Liang Xue Hua Yu decoction to go on the experiment.3.To set the photodamage model of RPE cells in vitro:To induce the apoptosis of RPE cells by the blue light which the wave length is between 470～500nm and the intensity between(2000±500) LUX.The RPE cells were exposure to it for 12 hours.4.To observe the effect of Liang Xue Hua Yu decoction to blue light-induced apoptosis in cultured RPE cells:All the groups of RPE cells were exposed to blue light with the same intensity,same time of illumination,same finish time of the culture.The protection of Liang Xue Hua Yu decoction to the bule light induced apoptosis in RPE cells was observed by Annexin V fluorescein isothiocyanate (FITC)/propidiumiodide(PI) flowcytometry.5.To investigate the effect of the Liang Xue Hua Yu decoction to the phagocytosis of cultured RPE cells:The rod outer segments(ROS) were labeled by FITC.Human RPE cells were divided into different groups,and to feed them with different concentration of liang xue hua yu decoction respectively.All the groups of RPE cells were incubated with same quantity of FITC labled ROS.And then were rinsed at the same incubation time to terminate the phagocytosis.At the same time cell was dyed by DAPI.Samples were photoed by a fluorescent microscope.Images were analysis by Lecia Q-Win image analysis software.The average quantity of the ingested ROS was analyzed by the quantification of FITC-fluorescence emission.Results1.The culture and indentification of RPE cells:The primary RPE cells adhere to the bottom of culture dish in 2～3 days.In about 1 week the cells began to appear to colony.After about 10 days the cells on the bottom of the culture dish appear to confluence.The primary RPE cells were irregularity and multi-angles,melanin pigment was full fill the cytoplasm.But with the transfer of culture went on,the melanin pigment in cytoplasm decreased gradually until totally vanished.There were no melanin pigment in the fifth generation of RPE cells.And it looks like fibroblasts.The identification results showed that the test of CK and S100 were all positive. 2.Screening of suitable concentration of Liang Xue Hua Yu decoction for the experiment:Liang Xue Hua Yu decoction that of 2mg/ml,200ug/ml,20ug/ml,2ug/ml.And all the blood serum groups that contain Liang Xue Hua Yu decoction of all the three doses(high,middle and low)with all the concentrations(40%,20%,10%,5%) had no evident influence to the RPE cells.3.To observe the effect of Liang Xue Hua Yu decoction to blue light-induced apoptosis in cultured RPE cells:the human RPE cells was exposure to bule light,and the main influence to the cells was apoptosis.The quantity of apoptosis cell of all the groups which was cultured with Liang Xue Hua Yu decoction were less.The protection of Liang Xue Hua Yu decoction was superior to VitC.The quantity of apoptosis cell of all the blood serum groups which contain Liang Xue Hua Yu decoction of all the three doses(high,middle and low)with all the concentrations(40%,20%,10%,5%) were less.The protection of blood serum which contain Liang Xue Hua Yu decoction of the following groups(40%,20%,10%,5%of high dose,the 40%,20%,10%of middle dose,the 40%,20%of low dose)were superior to VitC.4.To investigate the effect of the Liang Xue Hua Yu decoction to the phagocytosis of cultured RPE cells:the study showed that the phagocytosis of ROS by RPE cells was cofirmed,and the phagocytosis of the cells feeded with 2mg/ml and 200ug/ml Liang Xue Hua Yu decoction were more than the group feed with no Liang Xue Hua Yu decoction.The phagocytosis of the cells feeded with the blood serum which contain Liang Xue Hua Yu decoction of 40%high dose,40%middle dose and 40%low dose were more than the group feed with no Liang Xue Hua Yu decoction.Conclusion1.Trypsin digestion is a effective method for the culture of RPE cells in vitro.To culture the RPE cells in vitro successfully is the basis of RPE cells related experiments.2.The blue light which the wave length is between 470～500nm and the intensity of(2000±500) LUX,and the RPE cells was exposure to it for 12 hours could definitely induce the RPE cells to apoptosis.In our study the protection of Liang Xue Hua Yu decoction of suitable concentration to the blue light induced RPE cells apoptosis is confirmed.3.In our study Liang Xue Hua Yu decoction of suitable concentration could enhance the phagocytosis ofRPE ccells4.The protection of Liang Xue Hua Yu decoction to the blue light induced RPE cells apoptosis,and Liang Xue Hua Yu decoction enhance the phagocytosis of RPE cells may be one of the mechanism of Liang Xue Hua Yu decoction as one of a effective therapy to AMD in clinical.And it showed the evidence of Liang Xue Hua Yu decoction as a effective way of therapy for wet AMD.