二至丸影响自然衰老大鼠肝脏AQP9表达的潜在物质基础与分子机制

Potential Substance Basis and Molecular Mechanism of Erzhi Pill(二至丸)Affecting the Expression of AQP9 in Aging Rats

目的:利用分子对接与生物学验证方法探析滋阴补益肝肾方二至丸调控水通道蛋白9(Aquaporin9,AQP9)表达,延缓衰老的潜在物质基础与分子机制。方法:运用AutoDock Vina将二至丸活性成分与AQP9进行分子对接;利用ELISA法测定肝脏SOD活力、MDA含量;应用qPCR法测定肝脏端粒长度;采用WB法检测二至丸干预自然衰老大鼠的肝脏AQP9蛋白表达情况。结果:二至丸活性成分木犀草素、槲皮素、山柰酚、紫杉叶素、圣草酚、刺槐素、紫铆素、红车轴草素、蟛蜞菊内酯、β-谷甾醇、去甲基蟛蜞菊内酯、3’-O-甲酚均与AQP9分子结合能<-5.0 kcal/mol,配体与受体可自发结合。与幼年组相比,模型对照组SOD活力降低,MDA含量升高,肝脏端粒长度明显降低;与幼年、青年对照组比较,模型对照组肝脏AQP9表达下调(P<0.05);与模型对照组比较,二至丸3.17 g/kg组MDA含量明显降低,二至丸0.79、1.59、3.17 g/kg组肝脏AQP9表达明显上调(P<0.05)。结论:二至丸活性成分能与AQP9自发结合,可能为二至丸调控AQP9表达的物质基础,二至丸可能通过影响肝脏AQP9表达调控物质代谢,发挥延缓衰老作用。

Objective:To explore the potential substance basis and molecular mechanism of Erzhi Pill(二至丸)for ziyin(滋阴)and tonifying liver and kidney in affecting the expression of aquaporin 9(AQP9)in aging rats and delaying aging by Autodock Vina and biological assays.Methods:The active components of Erzhi Pill and AQP9 were docked by Autodock Vina.The superoxide dismutase(SOD)activity and malondialdehyde(MDA)content in liver were measured by enzyme-linked immuno sorbent assay(ELISA).The telomere length was measured by quantitative polymerase chain reaction(qPCR).The expression of AQP9 in aging rats intervened by Erzhi Pill was detected by Western blot.Results:The binding energy between AQP9 and luteolin,quercetin,kaempferol,taxifolin,eriodictyol,acacetin,butin,pratensein,wedelolactone,beta-sitosterol,demethylwedelolactone,and 3'-O-methylorobol was less than-5.0 kcal/mol,indicating free binding of ligands to receptors.Compared with the infant control group,SOD activity was decreased,MDA content was increased,and liver telomere length was significantly reduced in model group.Compared with the infant and young control groups,the expression of AQP9 in model control group was reduced(P<0.05).Compared with the model group,the MDA content in Erzhi Pill 3.17 g/kg was significantly decreased,and the expression of AQP9 in Erzhi Pill 0.79 g/kg,1.59 g/kg,and 3.17 g/kg groups was increased(P<0.05).Conclusion:The active components of Erzhi Pill bound to AQP9 spontaneously,which might be the substance basis of regulating the expression of AQP9.Erzhi Pill might regulate substance metabolism by affecting the expression of AQP9 in liver,thus delaying aging.