Study On The Protective Effect And Mechanism Of Bushen Huoxue Formula On Retinal Müller Cells Based On The Coupling Of AQP4/Kir4.1

Background:Ddiabetic macular edema is one of the main causes of moderate and severe visual acuity decline in patients with diabetes.The abnormal water drainage pathway mediated by aquaporin 4（AQP4）and inward rectifier potassium channel（Kir4.1）in Müller cells is one of the necessary conditions for early diabetic retinal edema and DME.However,there are few studies on the mechanism of Traditional Chinese Medicine compound improving DR-related water excretion pathway.Previous studies have shown that Bushen Huoxue Formula can improve retinal aqueous drainage in diabetic animal models,but the mechanism of this formula against DR and its complications has not been fully understood in vitro based on AQP4/Kir4.1 coupling on Müller cell.Objective:To investigate the molecular mechanism of Bushen Huoxue Formula in the treatment of DME by using network pharmacology,summarizing and analyzing the therapeutic targets of Bushen Huoxue Formula in previous studies,and to clarify the overall mechanism of this formula in the prevention and treatment of DR.Further,we explore the effect of Bushen Huoxue Formula on Müller cells cultured in vitro under hypoxic and AGEs interventions,aiming to comprehensively elucidate the possible mechanisms of Bushen Huoxue Formula on the prevention and treatment of DR.Methods:Network pharmacology was applied to analyze the potential drug ingredients,key targets,and molecular pathways of Bushen Huoxue Formula in treating diabetic macular edema（DME）.The primary retinal Müller cells of neonatal 7-day SD rats were isolated and cultured by a modified enzyme digestion method,then identified by HE staining and immunohistochemistry（GFAP staining and GS staining）.Müller cells were treated with Na₂S₂O₄ and different concentrations of AGEs to prepare the hypoxia and AGEs cell models respectively.The cell models were divided into control group（Ctr）,Traditional Chinese Medicine group（M）,hypoxia group（Hy）,hypoxia intervened by Medicine group（Hy+M）,low and high dose of AGEs groups（l AGEs）,（h AGEs）,and different doses of AGEs intervened by medicine group（l AGEs+M）,（h AGEs+M）.The drug-containing serum of Bushen Huoxue Formula and the serum of control group were prepared by the method of serum pharmacology of traditional Chinese medicine to intervene different groups of cell models.The expression of AQP4and Kir4.1 protein was detected and quantified by immunofluorescence double staining at 24h,48h and 72h.Elisa assay was used to detect the expression of AQP4,Kir4.1,VEGF,PEDF and IL-1βin Müller cells at different time points.Results:（1）A total of 425 drug targets,1797 DME disease targets and 168potential targets of Bushen Huoxue Formula acting on DME were screened by network pharmacological analysis.Among them,tanshinone IIB,β-sitosterol,kaempferol,luteolin and puerarin are the key compounds of Bushen Huoxue Formula on DME,while AKT1,TNF,IL6,INS,TP53,VEGFA,IL-1β,CASP3,PTGS2,EGFR,PPARG,MMP9,STAT3 and HIF1A are the key targets of Bushen Huoxue Formula on DME.Lipid metabolism related pathway,HIF-1 signal pathway,AGE-RAGE signal pathway,PI3K-AKT signal pathway and TNF signal pathway in diabetic complications are the key signal pathways of Bushen Huoxue Formula acting on DME.（2）Previous studies have shown that hypoxia and AGEs can induce the decrease of the expression of tight junction-related proteins Occludin and ZO-1 in retinal i BRB,and promote the expression of inflammation-related factors IL-1β,ICAM-1 and the imbalance between VEGF and PEDF,resulting in i BRB leakage.Hypoxia,high glucose combined with hypoxia and glucose fluctuation can increase the expression of Glu,Gly and LDH,decrease the expression of GS in ganglion cells and Müller cells,damage the membrane stability of ganglion cells and Müller cells,and increase neuro-excitotoxicity.Hypoxia,high glucose,AGEs and TGF-β2 can increase LDH expression,decrease Glu uptake,decrease GS activity,up-regulate HIF-1αand VEGF,and further damage the structure and function of Müller cells.Bushen Huoxue Formula can reverse the expression of various factors under the above pathological conditions and protect i BRB,ganglion cells and Müller cells to a certain extent.（3）Immunofluorescence showed that under hypoxic conditions,the fluorescence intensity of AQP4 in Hy was significantly higher than that in Ctr and M at 24h,48h and72h（p<0.05）,Hy+M could significantly reduce the fluorescence expression of AQP4at 24h and 48h under hypoxia,and showed an increasing tendency at 72h（p>0.05）.The immunofluorescence intensity of Kir4.1 in Hy was significantly lower than that in Ctr and M,and the fluorescence expression of Kir4.1 in Hy+M was significantly higher than that in Hy at 48h.Compared with Ctr,AQP4 fluorescence intensity in M decreased at 24h and 48h and increased at 72h.There was a significant difference between groups at 48h（p<0.05）.Kir4.1 decreased at 24h and 48h and increased at 72h,and there was a significant difference between groups at 72h（p<0.05）.（4）Elisa assay showed that AQP4,VEGF,IL-1βsignificantly increased and Kir4.1,PEDF significantly decreased in Hy compared with Ctr and M at the same time point（p<0.05）.Hy+M significantly reduced AQP4,VEGF,IL-1βexpression and increased Kir4.1 and PEDF expression under hypoxia（p<0.05）.Compared with Ctr,AQP4 in M was significantly decreased,Kir4.1 and PEDF were significantly increased at different time points（p<0.05）.VEGF expression showed a tendency to decrease at 72h,and IL-1βdecreased at all time points（p>0.05）.Compared at different time points within the same group,Hy and Hy+M all differed significantly with significant time effect,while Ctr and M did not change significantly with 24h time interval.The specific value of AQP4/Kir4.1 and VEGF/PEDF increased significantly with time prolongation.（5）Under AGEs intervention,immunofluorescence showed that the fluorescence intensity and expression of AQP4 in different doses of AGEs（l AGEs and h AGEs）were significantly higher than those in Ctr and M at all time points（p<0.05）.The intervention groups of drug-containing serum（l AGEs+M and h AGEs+M）could reduce the AQP4fluorescence expression at each time point under the conditions of AGEs at each concentration.Kir4.1 fluorescence intensity of each AGEs intervention group was significantly reduced at 48h and 72h（p<0.05）.The drug-containing serum of different concentrations of AGEs could reduce the fluorescence expression of AQP4 in the group with corresponding concentration of AGEs and and increase that of Kir4.1 at 24h and48h.At 72h,Kir4.1 increased in l AGEs+M compared to l AGEs,and Kir4.1fluorescence intensity decreased in h AGEs+M compared to h AGEs.（6）Elisa assay showed that AQP4,VEGF and IL-1βwere significantly increased and Kir4.1 and PEDF were significantly decreased in l AGEs and h AGEs compared with the Ctr and M at the same time point between the groups（p<0.05）.The drug-containing groups could significantly reverse this alteration.Compared with l AGEs,the expression of AQP4,VEGF and IL-1βin h AGEs significantly increased while the expression of Kir4.1 and PEDF significantly decreased.Compared with Ctr,AQP4 in M was significantly lower,Kir4.1 and PEDF were significantly higher,and VEGF was significantly lower at 24h and 48h（p<0.05）,with a trend of lowering at 72h,and IL-1βwas lower at all time points（p>0.05）.The expression of each factor in Ctr and M was more stable within the same group,and there was obvious time effect in l AGEs,h AGEs,l AGEs+M and h AGEs+M.The expression of AQP4,VEGF and IL-1βincreased significantly with prolongation of time,while the expression of Kir4.1 and PEDF decreased significantly.Under the intervention of AGEs,the ratio of AQP4/Kir4.1 and VEGF/PEDF increased significantly with the extension of culture time,and the change of h AGEs was more significant than that of l AGEs.（7）At all time points,the effect of h AGEs on AQP4 and VEGF expression was stronger than that of Hy.The effect of l AGEs on Kir4.1 and PEDF expression was stronger than that of Hy.The effect of h AGEs on IL-1βexpression was not significantly different from that of Hy.Conclusion:（1）Network pharmacology study revealed that the treatment of DME with Bushen Huoxue Formula involves a wide range of physio-pathological processes.KEGG analysis showed that Bushen Huoxue Formula treats DME mainly through signaling pathways such as lipid metabolism-related pathway,HIF-1 signaling pathway,and AGE-RAGE signaling pathway in diabetic complications,indicating that hypoxia and AGEs are important pathological factors in the pathogenesis of DME.（2）The concentration of AGEs was positively correlated with the degree of Müller cell injury.Reducing the concentration of AGEs is one of the important ways to protect Müller cells.（3）Hypoxia and AGEs can promote AQP4,VEGF,and IL-1βexpression,reduce Kir4.1 and PEDF expression,disrupt AQP4/Kir4.1 coupling and the relative balance of VEGF/PEDF in Müller cells in vitro,and upregulate the proinflammatory factor IL-1βto damage Müller cell function with significant temporal effects.Early intervention is of great importance to ameliorate Müller cell function.（4）Bushen Huoxue Formula can restore AQP4/Kir4.1 coupling and VEGF/PEDF balance of Müller cells under hypoxia and different concentrations of AGEs to a certein extent,and reduce expression of proinflammatory factor IL-1β,which may be an important mechanism of Bushen Huoxue Formula in the prevention and treatment of DME by protecting Müller cells.（5）The effect of AGEs on the expression of AQP4,Kir4.1,VEGF and PEDF was stronger than that of hypoxia,and the effects on IL-1βwere not significantly different between the two.In the prevention and treatment of DR,we should take into account the initiating factor AGEs and secondary hypoxic pathological changes to better prevent and cure DR.（6）Bushen Huoxue Formula may comprehensively regulate retinal neurovascular unit（NVU）function by reducing ganglion cytotoxicity,mitigating i BRB leakage and protecting Müller cells to prevent and treat DR and DME.