| Part ?:Pharmacodynamic material basis of Er Zhi Fang(EZF)in anti-diabetic nephropathy1.Based on the pharmacokinetic characteristics of umbellolactone,oleanolic acid and ursolic acid in rats to explore the pharmacodynamic basis of EZF in treating diabetic nephropathyObjective:To investigate the pharmacodynamic material basis of EZF for anti diabetic nephropathy.Methods:HPLC method was established for simultaneous determination of umbelliferol,oleanolic acid and ursolic acid in serum and target tissues(kidneys)of rats fed Erzhi Fang and its extracts.The concentration-time curves were drawn and the pharmacokinetic parameters were calculated by DAS 2.0 software.Results:The regression equations of umbellolactone,oleanolic acid and ursolic acid were Y=5.047*103X-24.550(R2=0.9998),Y=6.511X+2.181(R2=0.9998),Y=5.413X-1.627(R2=0.9998),and the linear ranges were 0.5-50.0 ug/mL,and the quantitative limits(LOD)were 0.53,1.24 and 1.26 mg/kg,respectively.The recoveries of umbellolactone,oleanolic acid and ursolic acid were 98.8%,99.8%and 102.1%,respectively.The recoveries of umbellolactone,oleanolic acid and ursolic acid in plasma samples were 82.15%,82.15%and 83.73%,respectively.The concentrations of umbelliferol in liver,heart,spleen,lung and kidney were 7.32,12.30,21.19,84.95 and 111.36 mg/L,respectively,and those of oleanolic acid in liver,heart,spleen,lung and kidney were 1.10,43.70,11.70,197.60 and 40.10 mg/L,respectively.The concentrations of ursolic acid in liver,heart,spleen,lung and kidney were 37.90,307.50,61.80,92.90 and 301.30 mg/L,respectively;Tmax of oleanolic acid and ursolic acid were 0.5 h,Cmax were 111.33±10.50 and 153.72±3.35 mg/L,AUC(0-?)were 342.44±10.15 and 589.79±9.49 mg/L*h.t1/2z were 2.93±0.03 and 3.19±0.02 h respectively.The Cmax of umbelliferol was 100.30±3.81mg/L,AUC(0-?)was 316.89±15.01 mg/L*h,ti/2z was 2.87±0.37.Conclusion:According to the results of tissue distribution,umbelliferol,oleanolic acid and ursolic acid can be detected in the target tissues of rats.It is important to study the molecular mechanism of umbelliferol,oleanolic acid and ursolic acid against diabetic nephropathy.2.Based on the pharmacokinetic characteristics of Specnuezhenide and Wedelolactone in rats to explore the pharmacodynamic basis of EZF in anti-diabetic nephropathyObjective:To elucidate the pharmacodynamic basis of EZF for diabetic nephropathy(DN)on the basis of the contents of Specnuezhenide and Wedelolactone in EZF and their pharmacokinetic characteristics in rats.Methods:HPLC-MS/MS method was established for the first time to determine the contents of Specnuezhenide and Wedelolactone in EZF and rats' serum and tissues.The main pharmacokinetic parameters were calculated by DAS 2.0 program,and the concentration-time curves were drawn.Results:The regression equations of Specnuezhenide and Wedelolactone were Y=123.408+181.663 X(R2=0.9999),Y=92127.2+3822.94 X(R2=0.9987),the linear range was 25-1000 ?g/L,LOD was 2.04,2.60 ?g/L,and the recovery was 101.2%and 100.3%,respectively.The content of Specnuezhenide in Erzhi Formula ranged from 0.27 to 9.79%and Wedelolactone from 0.16 to 0.61%.The Tmax of Specnuezhenide was 1.50 h,the Tmax of Wedelolactone was 2.0 h,the Cmax of Wedelolactone was 30.24±1.65 ug/L,the Cmax of Specnuezhenide was 6.39±0.05 ug/L,the AUC of Wedelolactone was 123.30±2.68 ug/L*h,the AUC(0-?)of Specnuezhenide was 16.56±0.98 ug/L*h.Conclusions:The method was suitable for the pharmacokinetic study of Specnuezhenide and Wedelolactone in Chinese herbal compound and rats.Meanwhile,Specnuezhenide and Wedelolactone are important pharmacodynamic components of EZF.Part ? The mechanism of action of EZF and its active compounds on diabetic nephropathyObjective:Elucidate the protective effect of EZF extract on renal injury in diabetic nephropathy rats.To elucidate the anti-diabetic nephropathy effect of ursolic acid and its protective mechanism against podocyte injury in diabetic nephropathy rats.To evaluate the protective effect of UMB on diabetic nephropathy and explore its possible mechanism.Methods:Methods One:Diabetic nephropathy rat model was established by high glucose and high fat diet combined with 1%streptozotocin(STZ).The effects of EZF extract on 24 h urinary protein content,kidney index,fasting blood glucose,pathological morphological changes of kidney tissue,the protein and mRNA expression of podocin,CD2AP in renal tissue were investigated.Methods Two:Diabetic nephropathy rats were induced by high-glucose and high-fat diet combined with 1%STZ.The effects of UA on fasting blood glucose,24-hour urinary protein,renal index,antioxidant and inflammatory factors in serum and kidney,pathomorphological changes of renal tissue,podocin and CD2AP eggs of podocyte membrane protein in renal tissue were studied.The expression level of white and m RNA finally elucidated the mechanism of UA against DN.Methods Three:STZ-induced diabetic SD rats were used to study the effects of UMB on blood glucose,insulin,uric acid,creatinine,triglyceride(TG),total cholesterol(TC),histological level and TLR/NF-kappa B signaling pathway related protein expression in STZ-induced diabetic rats.Results:Results One:After 8 weeks of treatment with EZF extract,the blood glucose levels of each dose group of EZF extract were significantly lower than those of model group at 16 weeks.At week 16,compared with the model group,the 24-hour urinary protein levels in the high-dose group(20.72±0.45 mg),the middle-dose group(24.88±0.42 mg)and the low-dose group(28.65±0.46 mg)were significantly decreased(p<0.05).The concentrations of IL-beta,TNF-a and IL-6 in kidneys of rats were significantly decreased in both high and middle doses of EZF(p<0.05),while MDA concentration in kidneys of rats was significantly decreased in both high and middle doses of EZF(p<0.05),and SOD activity was significantly increased(p<0.05).HE and PAS staining showed that EZF group could reduce the pathological changes of diabetic nephropathy rats to a certain extent,and the glomerular morphology was basically normal.The protein and gene expression of podocin and CD2AP in kidney tissue of EZF group were increased(p<0.01).Results Two:After 8 weeks of UA treatment,the UA dose groups showed hypoglycemic effect;by 16 weeks,compared with the model group,the blood glucose values of the UA high-dose group and low-dose group were significantly decreased(p<0.01);the UA dose groups improved the urinary protein content of diabetic rats,compared with the model group,the UA high-dose group and low-dose group(28.65±0.46 mg)24 days.The levels of urinary protein were significantly decreased(p<0.05),the body weight was significantly increased(p<0.05)and the renal index was significantly inhibited(p<0.05)in the high dose UA group,and the levels of IL-1 beta in serum and kidney were significantly decreased(p<0.05)in the diabetic rats.The level of IL-6 in serum and kidney of diabetic rats was decreased(p<0.05).The levels of TNF-a in serum and kidney of diabetic rats were significantly decreased by UA(p<0.01);The activities of SOD in kidney were significantly increased by UA high dose(21.07±0.76 U/mL)and low dose group(18.16±0.97 U/mL)(p<0.01);the activities of SOD in kidney were significantly increased by UA high dose(57.81±4.83 nmol/mL)and UA low dose group(63.75±4.56 nmol/mL).The results of HE and PAS staining showed that UA could alleviate the pathological changes of diabetic nephropathy rats to a certain extent,the glomerular morphology was basically normal,and the basement membrane was partly thickened,the mesangium was slightly hyperplasia,the degree of pathological changes was significantly lighter than that of the model group.The expression level of white pigment increased(p<0.01).Results Three:After a period of treatment with UMB(20,40 mg/kg),the levels of uric acid,creatinine,TC and TG in diabetic rats were significantly inhibited(p<0.05);after the intervention with UMB(20,40 mg/kg),the renal damage in diabetic rats was significantly inhibited.The asymptomatic symptoms were significantly alleviated.The results showed that UMB had protective effect on STZ-induced renal tissue loss in rats;UMB could significantly down-regulate the expression of TLR/NF-kappa B pathway-related proteins(TLR2,TLR4,MyD88,p-NF kBp65,NF-kBp65,p-IkBalpha)in a dose-dependent manner.Conclusion:? EZF has the effect of anti-diabetic nephropathy.It can reduce the amount of urinary protein by up-regulating the expression of podocin and CD2AP in podocyte hiatus membrane,repair the pathological damage of kidney caused by diabetes mellitus and protect renal function.? Ursolic acid,the main component of EZF,can inhibit inflammatory renal injury by inhibiting the production of inflammatory factors such as TNF-?,IL-6 and IL-lbeta.Ursolic acid can effectively reduce the urinary protein content of DN rats,significantly improve the blood glucose level of DN rats,and up-regulate the expression of CD2AP and podocin proteins in renal hiatal membrane of DN rats and protect the kidney of DN rats.? Umbrella lactone,the main component of EZF,can down-regulate the inflammatory factors related to TLR4/NF-kappa B pathway and protect the kidney from diabetes mellitus. |
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