Effect Of Huoxue Tongluo Lishui Recipe On Hypoxia And Antagonism Of Glutamate Toxicity In The Retinal Müller Cells Of Rats

ObjectivesTo observe the effect of Huoxue Tongluo Lishui recipe on the expression of effector molecules protein in retinal Müller cells stimulated by glutamate?Glu?and hypoxia,and to explore the mechanism of Huoxue Tongluo Lishui recipe on Müller cells induced by ischemia and hypoxia.Methods40 SD rats were randomly divided into blank group,low dosage group,high dose group and ginkgo biloba leaf group.The rats in the drug group were given the corresponding drugs,and the blank group were given the same volume of normal saline,once a day.After 7 days,the blood was collected from the abdominal aorta,and the normal rat serum and the serum containing the drug were extracted by centrifugation and filtration.Primary Müller cells from sd suckling rats were cultured and purified in vitro.After passage to p3,the cells were identified and used in the experiment.HE staining and electron microscopy were used to observe the morphological changes of the cells.Müller cells were treated with different concentrations of Glu.The optimal concentration of Glu was determined by MTT method to prepare the cytotoxic glutamate model,and the hypoxia model of Müller cells cultured with 500?mol/L CoCl₂ for 24 h was prepared.The p3 Müller cells were randomly divided into control group,model group,low dose group,high dose group and ginkgo leaf group.Cytotoxicity glutamate model and hypoxia model were used to culture for 24 h.In the two model groups,normal rat serum,low-dose Chinese medicine serum,high-dose Chinese medicine-containing serum and ginkgo biloba leaf drug-containing serum were added respectively.Determination of residual Glu concentration in cells of each group stimulated by Glu toxicity by HPLC method.The expression levels of GLAST,GS and AQP4 protein were detected by western-blot method in each group under the intervention of two kinds of models.Results1 Müller cells were successfully cultured,purified and identified.Under light microscope,Müller cells grew in clusters,and the cell bodies were larger,showing star,fusiform,polygonal and other morphological distribution.HE staining showed that the cell envelope was abundant,the cell membrane was clear,and the cell membrane was light red.The nucleus was multi-ovoid,and it was located in the middle of the cell.2 Compared with the control group,the survival rate of Müller cells decreased gradually with the increase of glutamate concentration by MTT assay.The survival rate of Müller cells reached 60%to80%at the concentration of 25 mmol/L glutamic acid concentration.The model of glutamate toxicity was successfully constructed.3 The results of HPLC showed that compared with the model group,the glutamic acid concentration of Müller cells in each dose group of Huoxue Tongluo Lishui recipe and Ginkgo biloba leaf group was lower than that of the model group.?P<0.01?.There was no significant difference among the three groups?P>0.05?.4 The results of Western blot showed that the expression of GLAST,GS protein was lower and the expression of AQP4 protein was higher than that of control group under glutamate toxicity?P<0.01?.The expression of GLAST,GS protein was higher and the expression of AQP4 protein was lower than that of model group after serum intervention in each dose group of Huoxue Tongluo Lishui recipe and ginkgo biloba leaf group.The difference was statistically significant.?P<0.05 or P<0.01?.It suggested that the drug administration was effective.Compared with ginkgo leaf group,the treatment effect of high-dose Chinese medicine group was better than that of ginkgo leaf group,the difference was statistically significant?P<0.05?.5 The expression of GLAST,GS protein was lower and the expression of AQP4 was higher than that of control group under hypoxia.The difference was statistically significant?P<0.01?.The expression of GLAST,GS was higher and the expression of AQP4 was lower than that of model group after serum intervention in each dose group of Huoxue Tongluo Lishui recipe and ginkgo biloba leaf group.The difference was statistically significant.?P<0.05 or P<0.01?.It suggested that the drug administration was effective.Compared with ginkgo leaf group,the treatment effect of high-dose Chinese medicine group was better than that of ginkgo leaf group,the difference was statistically significant?P<0.05?Conclusions1 Huoxue Tongluo Lishui recipe can promote the absorption of glutamate by Müller cells and antagonizing the excitotoxicity of glutamic acid 2 Huoxue Tongluo Lishui recipe may up-regulate the expression of GLAST,GS protein and decrease the expression of AQP4 protein in Müller cells stimulated by glutamate toxicity and hypoxia,so as to maintain the normal uptake of glutamate by Müller cells and effectively improve the condition of retinal ischemia and hypoxia.Figure28;Table4;Reference 120...