Study On The Effects And Bioactive Compounds Of Danggui-shaoyao-san Based On Plasmin Mediated Sodium Retention And Edema In Nephrotic Syndrome

Aims:Nephrotic syndrome is a common clinical kidney disease.Large proteinuria and edema are the main indicators for diagnosing nephrotic syndrome.Edema affects the quality of life of patients seriously.Sodium and water retention is the main factor of edema.Renin-angiotensin-aldosterone system blockers and diuretics are often used clinically to treat edema in nephrotic syndrome,but the efficacy is inaccurate,and multiple diuretics are resistant.Danggui-Shaoyao-San(DSS)composed of three kinds of blood-nourishing blood-activating drugs(Angelicae Sinensis Radix,Paeoniae Radix Alba,Chuanxiong Rhizoma)and three kinds of spleen-promoting and water-enhancing drugs(Atractylodis Macrocephalae Rhizoma,Poria,Alismatis Rhizoma),as a representative prescription of classic treatment of invigorating blood circulation and promoting diuresis,has definite curative effect when applied to nephrotic syndrome.But the effect and mechanisms on sodium retention of DSS are not clear yet.Based on the clinical efficacy and predictive mechanism,this study revealed the effect and mechanism of DSS in the treatment of sodium retention in nephrotic syndrome and clarified the key targets and effective material basis of sodium retention in nephrotic syndrome.Material and methods:In this study,we investigated the animal model of nephrotic syndrome,and selected NS models to evaluate the pharmacologic effect and key targets of DSS.Rat model of nephrotic syndrome induced with puromycin aminonucleoside was established.We disassembled DSS into YXHX and JPLS groups according to both the "promoting blood circulation and diuresis" guidance of Traditional Chinese Medicine theory and the efficacies of herbs.The positive drug,amiloride hydrochloride,was a double inhibitor of urokinase type plasminogen activator(uPA)and epithelial sodium channel.The effects and potential pathways on urinary sodium excretion,body weight,urinary protein,urinary plasmin(gen)content,and plasmin activity of DSS,YXHX,and JPLS extracts were dynamically observed.The inhibition of DSS and its decomposed prescriptions on potential targets was verified by inhibition experiments in vitro.Based on clear targets,we established an affinity ultrafiltration method and evaluated method reliability.Affinity ultrafiltration mass spectrometry spectrometry(AUF-MS)was used to rapidly screen and qualitatively analyze the uPA/plasmin affinity compounds from DSS extract.Additionally,uPA/plasmin inhibition assays and molecular docking were used to verify the activity and affinity mechanisms of the potential bioactive compounds and cleared the bioactive compounds of DSS in improving sodium retention of nephrotic syndrome.Result:The models of adriamycin and puromycin aminonucleoside nephrotic syndrome were established,both of models were reflected the clinical characteristics of nephrotic syndrome.In this study,we focused on the dynamic changes of urinary sodium excretion and edema indexes.The urinary sodium excretion of puromycin aminonucleoside model rats decreased significantly from 5 to 10 days after modeling(p<0.01),the ascites increased significantly on the 12th day(p<0.01),and the total body fluid and extracellular fluid increased significantly(p<0.01).However,puromycin aminonucleoside decreased urinary sodium excretion more significantly and stably than adriamycin model.Therefore,puromycin aminonucleoside nephropathy model was selected in this study.We evaluated the effect of DSS on improving the retention of sodium and water in nephrotic syndrome and its potential targets in vivo.Based on the puromycin aminonucleoside rat model,the rats were treated with DSS,YXHX and JPLS for 12 days.The results showed that DSS,YXHX and JPLS could correct the abnormal blood indexes and renal dysfunction,promote the excretion of sodium and improve the state of sodium retention.DSS,YXHX and JPLS can promote urinary sodium excretion and relieve sodium retention in nephrotic rats in different degrees(p<0.05 orp<0.01)and the effect of sodium excretion in YXHX group is the best,followed DSS group.However,DSS and disassembled group had no effect on urinary plasminogen excretion,DSS and YXHX group significantly decreased urinary plasmin activity(p<0.05 or p<0.01).And the increase of urinary sodium excretion in DSS and YXHX group was synchronized with the decrease of urinary plasmin activity.Therefore,DSS and YXHX group may play an important role in improving sodium retention through interfering in plasmin production and inhibiting its activity.In vitro enzyme activity experiments showed that the extract of DSS and YXHX group had dual inhibitory effects on urokinase type plasminogen activator(uPA)and plasmin.uPA and plasmin are the targets of DSS to improve the sodium retention in nephrotic syndrome.An affinity ultrafiltration screening method targets on urokinase plasminogen activator and plasmin was successfully established.The reliability of the method was verified by positive and negative drugs.Based on this method,urokinase plasminogen activator and plasmin affinity compounds in the extract of DSS were screened.Gallic acid,methyl gallate,paeoniflorin and pentagalloylglucose are moderate or high affinity substances of uPA or plasmin.Enzyme activity inhibition experiment was used to verify the activity of affinity substances,and molecular docking was used to verify the binding between affinity substances and enzymes.Pentagalloylglucose has dual inhibitory effects on plasminogen activator and plasmin.Conclusion:The puromycin aminonucleoside rat model established in this study is suitable for dynamic observation of sodium retention in nephrotic syndrome.DSS and YXHX group promoted urinary sodium excretion and alleviated ascites by interfering with plasmin production and plasmin activity but did not change the excretion of urinary protein and plasmin.uPA and plasmin are the targets of DSS to improve the sodium retention in nephrotic syndrome.With this dual target,affinity ultrafiltration technology is used to screen the effective substance basis of DSS.Pentagalloylglucose is the effective material basis for improving the retention of sodium in nephrotic syndrome.