An Experiment Study Of Bushen Huoxue Therapy On The Influence Of Membrance Stability Of RGCs And Müller Cells In The High Glucose Level Conditions

Objective: To investigate protective effect of bushen huoxue therapy on RGCs and retina Muller cells cultured in vitro under high glucose conditions, in addition, the mechanism of protective action was also preliminary studied.Methods:1. The retinal cell suspension was made of retinal from 1 day old SD rats, and the cell suspension was screened by separately anti-rat signal-regulated protein (CD172G) monoclonal antibody and anti-rat Thy-1.1 monoclonal antibody, and finally got purified RGCs; RGCs were inoculated in retinal Muller cells culture medium so that the co-culture model was established, which including both RGCs and retinal Muller cells.2. Then the RGCs and retinal Muller cells were co-cultured in stable high glucose condition,fluctuation of glucose condition and normal condition separately, all of the groups were intervened by blood serum including the medicine of Bushen Huoxue Chinese medicine. The experiment was grouped into normal control group, traditional Chinese medicine intervening group, stable high glucose group, stable high glucose and traditional Chinese medicine intervening group, glucose fluctuation group, glucose fluctuation and traditional Chinese medicine intervening group.3.The glutamine synthetase (GS) activity and extracellular lactate dehydrogenase (LDH) leakage of the co-culture cells were measured by enzyme-linked immunosorbent assay (ELISA). All data was analyzed with SPSS 17.0.1.At 24hrs and 48hrs, the LDH leakage of co-cultured cells in the stable high glucose group,glucose fluctuation group was significantly increased (P<0.05) compared with the normal control group.The LDH leakage of co-cultured cells in the stable high glucose group was significantly increased at 72hrs(P<0.05) compared with its 48 hrs. At 24hrs, the LDH leakage of co-cultured cells in glucose fluctuation group was significantly increased (P<0.05) compared with stable high glucose group. At 24hrs and 72hrs,the LDH leakage of co-cultured cells in the stable high glucose and traditional Chinese medicine intervening group was significantly reduced (P<0.05) compared with stable high glucose group. At 24hrs, the LDH leakage of co-cultured cells in the glucose fluctuation and traditional Chinese medicine intervening group was significantly reduced (P<0.05) compared with the glucose fluctuation group. The LDH leakage of co-cultured cells in the glucose fluctuation and traditional Chinese medicine intervening group was significantly increased at 72hrs (P<0.05) compared with its 48hrs.2.At 48hrs and 72hrs, the GS activity of glucose fluctuation group was significantly decreased compared with the normal control group (P<0.05); the GS activity of the stable high glucose group and the glucose fluctuation group was obviously increased at 48hrs (P<0.05) compared with its 24hrs. At 72hrs, the GS activity of stable high glucose group was still significantly increased (P<0.05), but that of glucose fluctuation group was obvious decreased (P<0.05), separately comparing with their 48hrs; the GS activity of glucose fluctuation group was obvious decreased (P<0.05) comparing with stable high glucose group of same time period.At 48hrs the GS activity of stable high glucose and traditional Chinese medicine intervening group was significantly increased comparing with 24hrs (P <0.05); at 24hrs and 48hrs the GS activity of glucose fluctuation and traditional Chinese medicine intervening group was significantly increased (P<0.05) comparing with glucose fluctuation in the same time period.1.In the either stable high glucose or glucose fluctuation conditions, cell viability of RGCs and retina Muller cell of co-cultured is decreased, the stability of the cell membrane is lower, Bushen Huoxue medicine-containing serum could strengthen the viability of RGCs and retina Muller cell and decrease cells' membrane permeability.2.In the glucose fluctuation condition, the GS activity of co-cultured retina Muller cell is obvious dropped, Bushen Huoxue medicine-containing serum could increase the GS activity of retina Muller cell in glucose fluctuation condition, heighten the elimination of Glu to protective function of RGCs and the retina Muller cell, accordingly it may be one of medicine intervention routes of bushenhuoxuefa could prevent and cure diabetic retinopathy.3. Bushen Huoxue medicine-containing serum could enhance the GS activity of retina Muller cell in the glucose fluctuation condition, its one of the important mechanism may be that serum could enhance the viability of RGCs and retina Muller cell, accordingly it may be another one of medicine intervention routes of bushenhuoxuefa could prevent and cure diabetic retinopathy.