An Experiment Study Of Bushen Huoxue Therapy On The Influence Of High Glucose Level And Ages Intervened Hypoxia Signal Pathway Mediated By Hif-1α Of Retinal Myller Cells#

Objective:To investigate the protection of the traditional chinese medicine(TCM) named Bu-Shen-Huo-Xue(BSHX) decoction on purified retinal Muller cells (RMCs) in high glucose conditions, high dose of advanced glycationend products(AGEs) conditions and low dose of AGEs conditions,and to explore the mechanism. Further more, to establish the certain foundation of therapy BSHX used in treating the diabetic retinopathy (DR).Methods:1. Cultured RMCs by way of modified enzyme-digestion method of from7day old SD rats.2. Purified RMCs were cultured in high(terminal concentration:50mmol/L) glucose conditions(terminal concentration:50mmol/L). low (terminal concentration:50mg/L)dose of advanced glycationend products(AGEs)conditions、 high (terminal concentration:100mg/L)dose of AGEs conditions and normal control conditions,BSHX medicine-containing serum was added to culture medium. The experiment is assigned into normal control group、TCM intervening group、high glucose group、high glucose group intervened with TCM、low dose of AGEs group、low dose of AGEs intervened with TCM、high dose of AGEs group、high dose of AGEs group intervened with TCM.3. Measured the extracellular lactate dehydrogenase (LDH) leakage、hypoxia-inducible factor-1α(HIF-1α) expression vascular endothelial growth factor (VEGF) expression and proline hydroxylase domain1、2、3(PHD1、2、3) activity of the co-culture cells by enzyme-linked immunosorbent assay (ELISA); in addition, tested the HIF-1αmRNA and VEGFmRNA expression by reverse transcription polymerase chain reaction (RT-PCR), All data was analyzed with SPSS13.0.Results:1. In alternative groups, the expression of LDH and HIF-1α in the high dose of AGEs group is the highest, it was significantly increased (P<0.05) compared with the normal control group and high glucose group; the expression of HIF-1αmRNA in the high glucose group、low dose of AGEs group and high dose of AGEs group was significantly increased (P<0.05); the expression of HIF-1αmRNA in the low dose of AGEs group is the highest, it was significantly increased (P<0.05) compared with the high glucose group and the high dose of AGEs group; the expression of LDH、HIF-1α and HIF-1αmRNA was obviously decreased by TCM intervention under different added conditions of this experiment.2. Both the expression of VEGF and VEGFmRNA in the high glucose group、 low dose of AGEs group and high dose of AGEs group was significantly increased (P<0.05)compared with the normal control group; both the expression of VEGF and VEGFmRNA was obviously decreased by TCM intervention under different added conditions of this experiment.3. Compared with the normal control group, the PHD1activity obviously reduced (P<0.05) in the high glucose group, low dose of AGEs group and high dose of AGEs group; the PHD2activity obviously increased(T<0.05) in the low and high dose of AGEs group, but the PHD3activity was no significant difference. The PHD1activity in the high dose of AGEs group was significantly reduced (P<0.05) compared with low dose of AGEs group, BSHX medicine-containing serum could raise the activity of PHD1in high glucose conditions, reduces the PHD2activity in all different conditions.4. BSHX medicine-containing serum had no significant effect on the leakage of LD^the expression of HIF-1α and HIF-1αmRNA、the expression of VEGF and VEGFmRNA and the activity of PHD1, PHD2、PHD3、under normal control conditions.Conclusion:1. In this experiment, under the short-term and simple high glucose intervention conditions, the membrane permeability of purified RMCs and the expression of HIF-1α have no obvious change,but it caused a significant increased of HIF-1αmRNA、VEGF、VEGFmRNA and obviously decreased of PHD1activity in the hypoxia signal pathway mediated by HIF-1α of purified RMCs.2. In this experiment, AGEs intervention (terminal concentration:100mg/L) not only caused the damage of purified RMCs、the expression of HIF-1α increased, but also both the terminal concentration of50mg/L and100mg/L AGEs intervention caused the expression of HIF-1αmRNA、VEGF and VEGFmRNA upregulated, obviously decreased of PHD1activity, increased of PHD2activity.3. No significant effect on the activity of PHD3under different conditions include high glucose and AGEs of this experiment.4. In this experiment, under the intervention of AGEs (terminal concentration:50mg/L) and high glucose intervention conditions, the increased expression of HIF-1α of purified RMCs mainly related to the up-regulated level of HIF-1αmRNA. Under the intervention of AGEs (terminal concentration:100mg/L), the increased expression of HIF-1α of purified RMCs not only related to the up-regulated level of HIF-1αmRNA, but also maybe involved in the level after HIF-1α protein expressed.5.BSHX medicine-containing serum could decrease RMCs’ membrane permeability in the high glucose and AGEs conditions, accordingly it may be one of medicine intervention routes of bushenhuoxuefa could prevent and cure diabetic retinopathy.6. BSHX medicine-containing serum could inhibited the expression of HIF-1α、 HIF-1αmRNA、VEGF、VEGFmRNA、reduced the activity of PHD2and enhanced the activity of PHD1in the hypoxia signal pathway mediated by HIF-1α of purified RMCs. Accordingly it may be another one of medicine intervention routes of bushenhuoxuefa could prevent and cure diabetic retinopathy.7. BSHX medicine-containing serum had no significant effect on the hypoxia signal pathway mediated by HIF-la of purified RMCs under normal control conditions.