Study On The Dose-effect-toxicity Relationship Of Tripterygium Glycoside Tablets In Rats With Diabetic Nephropathy

Diabetic nephropathy(DN)is the leading cause of end-stage renal failure,which is one of the most important current public health issues,representing a significant burden on the health system.The current therapy of diabetic nephropathy is mainly through blocking of the renin-angiotensin system,such as oral angiotensin-converting enzyme inhibitors(ACEI)/angio-tensin ? receptor blockers(ARB)drugs.Combination therapy with ACEI and ARB compared with ACEI or ARB monotherapy was shown to reduce proteinuria in patients.However,the antiprotein-uric effects of combination therapy do not seem to be sufficient for the prevention of renal disease progression.Therefore,in addition to ACEI/ARB drugs,it is necessary to delay the progress of renal disease with the underlying mechanism.Tripterygium glycosides(TG)tablets are made from the total glycosides extracted from the roots of Tripterygium wilfordii Hook.F.It is commonly used in the treatment of nephrotic syndrome and rheumatoid arthritis in clinical,and has been included in the treatment standard of DN.However,TG tablets have not only renal protective and anti-inflammatory effects,but also hepatotoxicity and nephrotoxicity,so there is controversy in the clinical medication plan.Therefore,to provide a reference for its clinical rational drug use program,especially to study the dosage-effect-toxicity relationship of TG tablets in DN rats.First,the method of measuring the blood concentration of TG tablets was established.Then,different dosage of TG tablets were given to drug groups,and the therapeutic indexes of different administration time were measured,such as unilateral renal index(URI),blood urea nitrogen(BUN),blood creatinine(SCR),and toxicity indexes,mainly liver toxicity indexes,such as alanine aminotransferase(ALT)and aspartate a:minotransferase(AST),the relative protein expression of autophagy related protein microtubule-associated protein light chain 3 beta(LC3B),phosphoinositide 3-kinases(PI3K),protein kinase B(Akt)and mammalian target of rapamycin(mTOR),the fluorescence expression of lysosome-associated membrane glycoprotein 1(LAMP1)and LC3 protein were determined,renal and liver pathological sections were observed and compared in each group.The dosage-effect relationship and its mechanism,the dosage-toxicity relationship and its mechanism were analyzed.The blood concentration of TG tablets in each group was measured,and the dosage-effect-toxicity relationship and its substance basis were analyzed.The main research items in this dissertation are as follows:(1)The determination method of the blood concentration of TG tabletsA method for the determination of triptolide,celastrol,triptonide,wilforlide A in plasma samples was established by UPLC-Q Extraction.The chromatographic column of ACQUITY UPLC BEH C18 was used.The mobile phase was ethanol-0.3%formic acid aqueous solution gradient elution.The flow rate was 0.2 mL�min-1.The column temperature was 40?,and the sample volume was 2 ?L.Full scan monitoring using electrospray(ESI)positive ion mode.Ethyl acetate was used to precipitate protein and acetonitrile was used for redissolution.The specificity was good,the endogenous substances did not interfere with the four components.The linear relationship of triptolide between 0.0125 and 3.2 ng�mL-1 was good(r=0.9993),the precision of triptolide within and between batches was 4.580%?7.706%,8.155%?9.191%respectively,the accuracy of triptolide within and between batches was 85.367%?1 11.538%,103.958%?107.609%respectively.The linear relationship of celastrol between 0.1719 and 44 ng�mL-1 was good(r=0.9981),the precision of celastrol within and between batches was 3.154%?8.996%,8.572%?12.025%respectively,the accuracy of celastrol within and between batches was 94.252%?1 14.098%,99.956%?108.576%respectively.The linear relationship of triptonide between 0.00625 and 1.6 ng�mL-1 was good(r=0.9990),the precision of triptonide within and between batches was 0.800%?9.591%,6.270%?14.162%respectively,the accuracy of triptonide within and between batches was 89.076%?107.817%,99.681%?107.311%respectively-The lin ear relationship of wilforlide A between 0.7656 and 196 ng�mL-1 was good(r=0.9995),the precision of wilforlide A within and between batches was 3.094%?4.152%,7.166%?10.708%respectively,the accuracy of wilforlide A within and between batches was 98.488%?103.163%,101.429%?104.476%respectively.The extraction recovery of each component was more than 50%,the matrix effect was about 70%,and the stability RSD was within 15%.(2)The dosage-effect relationship and mechanism of TG tablets in DN ratsThe renal function indexes of URI,BUN,Scr and creatinine clearance rate(Ccr)were measured at 1,2,4 and 8 weeks after administration in each group.The pathological sections of kidney were observed,the relative protein expression of autophagy related protein LC3B,PI3K,Akt and mTOR,the fluorescence expression of LAMP1 and LC3 protein were determined and compared in each group.It was found that 25 and 50 mg�kg-1 TG groups were in the early period(1?2 weeks)and 6.25 mg�kg-1 TG group was in the period of long-term administration(4?8 weeks),renal function indexes and glomerular filtration rate were improved,and renal pathological conditions were improved,which may be related to inhibition of PI3K/Akt/mTOR signaling pathway and promotion of autophagy.It is suggested that the common dosage should be used in the long-term treatment of DN,or dosage adjusted in a short period of time according to the patients' condition.(3)The dosage-toxicity relationship and mechanism of TG tablets in DN ratsCompared with the normal group,there was no significant difference in ALT,AST and liver pathology in the model group.Compared with the model group,there was no significant difference in ALT,AST and liver pathology in the 6.25 mg�kg-1 TG group;in the 12.5 mg�kg-1 TG group,ALT and AST increased significantly(P<0.05)at the 2nd week,hepatocyte cords were disordered;in the 25 mg�kg-1 TG group,ALT and AST increased significantly(P<0.05)at the 2nd and 4th weeks,hepatocyte cords were disordered,hepatocytes were swollen;in the 50 mg�kg-1 TG group ALT and AST increased significantly(P<0.05)at the 2nd,4th and 8th weeks,hepatocyte cords were disordered,hepatocytes were swollen,and inflammatory cell infiltration appeared in hepatic sinuses.After 8 weeks of administration,compared with the normal group,in the model group,the expression of LC3B decreased significantly(P<0.05),PI3K,Akt and mTOR increased significantly(P<0.05),while the expression of LAMP1 and LC3 decreased significantly(P<0.05).Compared with the model group,the expression of LC3B decreased gradually,PI3K,Akt and mTOR increased gradually,and the expression of LAMP1 and LC3 decreased gradually with the increase of dosage;and in the 50 mg�kg-1 TG group,the expression of LC3B decreased significantly(P<0.05),PI3K,Akt and mTOR increased significantly(P<0.05).This showed that the dosage is more than 12.5 mg�kg-1�d-1,and the administration time is more than 2 weeks,which can produce hepatotoxicity,and which may be related to activation of PI3K/Akt/mTOR signaling pathway.and reduced activity of autophagy.Or the combination of mTOR inhibitors should be considered to promote autophagy and reduce toxicity,which needs further study.(4)Study on the dosage-effect-toxicity relationship and blood concentration of TG tablets in DN ratsBy measuring the blood concentration of TG tablets in each group of rats,summarizing the dosage-effect and dosage-toxicity relationship of TG tablets in DN rats.The concentration of celastrol was measured,but the concentration of triptolide,triptonide and wilforlide A were lower than the lower limit of quantification.It was found that the Ccr of the model group and each administration group was lower than that of the normal group,the glomerular filtration rate was decreased.It was speculated that the chemical components in TG tablets might be partially accumulated in urine,and the high dose group had nephrotoxicity and hepatotoxicity.Therefore,the further study can be carried out for urine,kidney and liver.The 6.25 mg�kg-1 TG group improved renal function,played the role of renal protection,delayed the progression of diabetic nephropathy at the 4th and 8th weeks.In the 6.25 mg�kg-1 TG group,the concentration of celastrol in blood was about 2?4 ng�mL-1,and the effective substance maybe celastrol.The 12.5 mg�kg-1 TG group had hepatotoxicity in two weeks after administration.And in the 12.5 mg�kg-1 TG group,the concentration of celastrol in blood increased from 3.2 ng�mL-1 to 7.1 ng�mL-1.The 25 mg�kg-1 TG group had hepatotoxicity at the 2nd and 4th weeks after administration,nephrotoxicity at the 8th week.In the 25 mg�kg-1 TG group,the celastrol concentration in blood was more than 8 ng�mL-1 and decreased at the 8th week,which may be related to the decrease of Ccr,and celastrol may exist in urine.In the 50 mg�kg-1 TG group,the protective effect on the kidney was produced at the 1th and 2nd weeks,and the hepatotoxicity was produced at the 2nd,4th and 8th weeks.The concentration of celastrol in the blood was more than 7 ng�mL-1,and the toxic substances might be related to celastrol.It was found that there was a dosage-effect-toxicity relationship of TG tablets in DN rats and the blood concentration of celastrol can reflect the effect and toxicity of tripterygium glycoside tablets in diabetic nephropathy rats.The safe range of celastrol in vivo was about 2?7 ng�mL-1,when the drug concentration in the blood was more than 7 ng�mL-1 for more than 2 weeks,hepatotoxicity was produced.In conclusion,tripterygium glycosides tablets have a dosage-effect-toxicity relationship in diabetic nephropathy rats,6.25 mg�kg-1(clinical dosage is 1 mg�kg-1�d-1)TG can produce renal protective effect in 4?8 weeks and 50 mg�kg-1 TG in 1?2 weeks;when the dose is more than 12.5 mg�kg-1,the administration time is more than 2 weeks,the hepatotoxicity can be produced;celastrol in vivo safety range is about 2?7 ng�ml-1,when the drug concentration in blood is more than 7 ng�ml-1,and it had been stored for more than 2 weeks,the hepatotoxicity can be produced.And its effect and toxicity may be related to PI3K/Akt/mTOR signaling pathway,which could regulate the activity of autophagy.It is suggested that the commonly used dose(1 mg�kg-1�d-1)should be used in the treatment of diabetic nephropathy.If it is necessary to give multiple doses of tripterygium glycosides tablets in a short time according to the body condition of the clinical patients,it is suggested that the time should not exceed 2 weeks,and closely monitor the liver function,renal function,to prevent the occurrence of adverse reactions.In addition,when the administration time is more than 8 weeks,the dosage-effect relationship needs to be further studied;whether the combination of mTOR inhibitors can promote autophagy and reduce the toxicity of tripterygium glycoside tablets is worthy of further study.