The Material Foundation And Action Mechanism Of Er Zhi Fang(EZF)in Reversing Diabetic Nephropathy

Diabetes is one of the three chronic diseases in the world.As of 2017,over 98 million400 thousand of the diabetic patients in China which have become the world's largest diabetes mellitus,with an incidence rate of 11.6%.Diabetes itself is not terrible,but its complications can greatly reduce the living standards of patients,and even end their lives.Diabetic nephropathy(DN)is a glomerulopathy caused by diabetes mellitus,which is mainly caused by microvascular damage.It is one of the main fatal and disabling microvascular complications of diabetes mellitus.About 30%-45%of patients eventually lead to end-stage renal disease.DN patients are facing great physical and psychological pain and expensive medical costs.Diabetes belongs to the endocrine system disease.The treatment of traditional Chinese medicine can achieve satisfactory results.In“Huangdi Neijing”,the main manifestations,etiology and pathogenesis of diabetes are systematically discussed.It is considered that they belong to the category of"Xiao Ke"in traditional Chinese medicine.DN is the change syndrome of diabetes,which is developed on the basis of yin and fluid loss and excessive heat.The key of DN pathogenesis is deficiency of both spleen and kidney.Zhong Jing Zhang,a famous doctor in the Eastern Han Dynasty,put forward the theory of"spleen can damage kidney"in his synopsis of golden chamber.He thought that the deficiency of water and grain essence and the loss of kidney essence caused deficiency of kidney yin and hyperactivity of fire in liver and kidney.The hyperactivity of heart fire and fire in liver and kidney is one of the direct sources of"Yin Fire".Er Zhi formula(EZF)is recorded in“Yibian”written by Sancai Wang,a famous doctor in Ming Dynasty:"the first prescription for clearing the upper part of the body and tonifying the lower part of the body is cheap and effective",which is an important medicine for treating yin deficiency of liver and kidney.Fructus Ligustri Lucidi and Herba Ecliptae can nourish the liver and kidney.In recent years,a large number of basic studies have shown that EZF has a significant role in regulating renal function.EZF can not only regulate blood glucose and lipid,but also improve renal function and prevent DN.Compared with western medicine,it also has the advantages of less adverse reactions,good compliance,good effect and low treatment cost.In this study,we will take EZF as the research object,using a variety of chromatographic analysis methods to analyze the main chemical components and content of EZF,combined with in vivo pharmacokinetic methods to explore the material basis of its anti-diabetic nephropathy effect,and investigate the anti DN mechanism of EZF extract and its main active monomer.The research work of this paper provides important data for the application of EZF in the treatment of diabetic nephropathy and the development of effective fractions/monomers.Part I:Pharmacodynamic material basis of Er Zhi Fang(EZF)in anti-diabetic nephropathy1.Determination of Umbelliferide,Oleanolic acid and Ursolic acid in Erzhi prescription and their pharmacokinetics in ratsObjective:Objective to determine the contents of umbelliferide,oleanolic acid and ursolic acid in Erzhi formula(EZF)and elucidate their pharmacokinetics in rats..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/m L,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 and40.10 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;T_max of oleanolic acid and ursolic acid were 0.5 h,C_max 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.t_1/2z were 2.93±0.03 and 3.19±0.02h respectively.The C_max of umbelliferol was 100.30±3.81mg/L,AUC_(0-)?was 316.89±15.01 mg/L*h,t_1/2zwas 2.87±0.37.The results showed that the content of each component in EZF was oleanolic acid>ursolic acid>umbelliferide;The C_max and AUC_(0-)?of EZF after oral administration were ursolic acid>oleanolic acid>umbelliferide;In kidney,ursolic acid>umbelliferide>oleanolic acid.Conclusion:Umbelliferolide,oleanolic acid and ursolic acid can be absorbed into the blood,and can be distributed to the kidney tissue and reach a certain concentration.The distribution of ursolic acid and umbelliferolide in the target tissue(kidney)is higher,which is worth further exploring the mechanism of action.2.The pharmacokinetic characteristics of EZF according to the contents of Specnuezhenide and Wedelolactone in rats.Objective: To elucidate the pharmacodynamic basis of EZF according to the contents of Specnuezhenide and Wedelolactone 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 C_max of Wedelolactone was 30.24 ± 1.65 ug/L,the C_max of Specnuezhenide was 6.39 ± 0.05 ug/L,the AUC(0-?)of Wedelolactone was 123.30 ± 2.68 ug/L*h,the AUC(0-?)of Specnuezhenide was 16.56 ± 0.98 ug/L*h.The concentration of Specnuezhenide in lung,liver and kidney was 8.56,1.52 and 7.19 ?g/L respectively,but not in spleen and heart.The concentrations of wedelion in spleen,lung,kidney and liver were 5.78,0.089,0.20 and 0.057 ?g/L,respectively,but not detected in heart.The results showed that the content of EZF was Specnuezhenide > wedelolactone;C_max and AUC(0-?)after oral administration were wedelolactone > Specnuezhenide;And in the kidney was Specnuezhenide > wedelolactone.Conclusions: The method was suitable for the pharmacokinetic study of Specnuezhenide and Wedelolactone in rats.Meanwhile,Specnuezhenide and Wedelolactone are important pharmacodynamic components of EZF.Part II The mechanism of action of EZF extract 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 m RNA expression of podocin,CD2 AP 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 CD2 AP 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-? 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 CD2 AP 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-? 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/m L)and low dose group(18.16 ± 0.97 U/m L)(p < 0.01);the activities of SOD in kidney were significantly increased by UA high dose(57.81 ± 4.83 nmol/m L)and UA low dose group(63.75 ± 4.56 nmol/m L).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,My D88,p-NF k Bp65,NF-k Bp65,p-Ik Balpha)in a dose-dependent manner.Conclusion:(1)EZF has the effect of anti-diabetic nephropathy.It can reduce the amount of urinary protein by up-regulating the expression of podocin and CD2 AP in podocyte hiatus membrane,repair the pathological damage of kidney caused by diabetes mellitus and protect renal function.(2)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-1beta.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 CD2 AP and podocin proteins in renal hiatal membrane of DN rats and protect the kidney of DN rats.(3)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.Conclusion1.Umbelliferide,oleanolic acid,ursolic acid,specnuezhenide and wedelolactone are the main components of EZF,and umbelliferide,oleanolic acid,ursolic acid,specnuezhenide and wedelolactone can directly reach the target tissue and reach a certain concentration;2.EZF can reduce the pathological damage of kidney caused by hyperglycemia,reduce urinary protein,protect renal function,protect podocytes by regulating the expression of CD2 AP and podocin,and then repair renal function and resist diabetic nephropathy;3.Ursolic acid can effectively reduce the amount of urinary protein and significantly improve the level of blood glucose in DN rats;Ursolic acid can up regulate the expression of CD2 AP and podocin in renal hiatal membrane of DN rats,which indicates that ursolic acid can protect the kidney of DN rats by up regulating the expression of podocin and CD2 AP.To sum up,ursolic acid's effects on reducing urinary protein,reducing pathological damage of kidney caused by hyperglycemia,and protecting renal function are closely related to its protective effect on podocytes and regulation of CD2 AP and podocin;4.Umbelliferide has the effect of anti STZ induced diabetes in rats,and can play the role of renal protection by down regulating the protein expression of TLR4 / NF-? B pathway.In conclusion,umbelliferide,oleanolic acid,ursolic acid,specnuezhenide and wedelolactone in EZFcan be absorbed into the blood and transported to the kidney,which is an important material basis for EZF to play an anti-diabetic nephropathy role.In addition,EZF can up regulate the expression of pore membrane proteins(CD2AP,podocin).It can protect podocyte injury and down regulate the expression of TLR4/NF-?B pathway protein,inhibit inflammatory reaction,and finally show the effect of anti-diabetic nephropathy.