The Research Of Bushen Tongluo Formula’s Efficacy Material Base And Action Mechanism Based On TGF-β1/CTGF Cytokines

Objective:1. To establish an pretreatment and HPLC method for simultaneous determination of loganin, ferulic acid and stibene glucoside in BSTLF extract.2.To establish an HPLC method for extracting and determining the loganin, ferulic acid and stilbene glucoside in rat plasma after oral administered with BSTLF, and to study the pharmacokinetics characteristic of loganin, stilbene glucoside and ferulic acid, for studying the efficacy material base of BSTLF.3. To study the action mechanism of BSTLF contra renal interstitial fibrosis, through the researh of plasma pharmacology in vitro.Methods:1.The content of ferulic acid, stibene glucoside and loganin was used as index, orthogonal design was applied to study the water extract-alcohol extraction method of BSTLF. L9(34) orthogonal table was used with three factors:the amount of ethanol, ethanol concentration and ethanol precipitation time. HPLC isocratic elution was adopted, with the Hypersil C18(4.6mm×250mm,5μm) as chromatographic column; the acetic acid aqueous solution (0.01%):methanol(65:35) as mobile phase; The detection wavelength was set at240nm (loganin),320nm (stibene glucoside and ferulic acid), the column temperature was30℃, the flow rate was1.0mL/min, and the injection volume was10μL.2. Make holder device to blood by snipping tail on male SD rats for pharmacokinetics. To collect0.5mL blood each time point before and the5min,10min,20min,30min,45min,60min,90min,2h,3h,6h,10h,14h,18h,23h after oral administrated BSTLF, then centrifugate to get plasma.The plasma was treated by using liquid-liquid extraction technique, the plasma concentrations of loganin, ferulic acid and stilbene glucoside in rat plasma at different time were determined by HPLC-UV. Johnson. spherigel C18column (250mm×4.6mm,5μm) was adopted, the composed of methanol-water containing0.01%glacial acetic acid as mobile phase,the flow rate was1.0mL/min, The detection wavelength was set at235nm (loganin),320nm (stibene glucoside and ferulic acid), the column temperature was30℃, and the injection volume was10μL. The pharmacokinetic parameters calculated by DAS2.0software.3. To oral administrate the high school low doses BSTLF for14days on male SD rats.To collect blood by abdominal aorta about1h after oral administrated the last dose, then centrifugate to get plasma.Glomerular mesangial cells as test object, rat interleukin as inflammatory intervention factor, fosinopril as positive drug. Blank control group, interleukin group, fosinopril group, BSTLF high dose group, BSTLF middle dose group, BSTLF low dose group been designed. The Glomerular mesangial cells proliferation induced by interleukin were detected by MTT method. The mRNA expression of TGF-β1and CTGF in glomerular mesangial cells were detected by RT-PCR. The protein expression of TIEG1,TGF-β1and CTGF in glomerular mesangial cells were detected by ELISA.Results:1. The optimum extraction condition was follow:6times70%alcohol extraction24h; At concentrations ranging between5～250,0.5～25,6～300μg/mL, peak area of loganin, ferulic acid and stibene glucoside showed good linear relationship0=0.9999,0.9999,0.9998); The limit of quantification was0.5,0.08and0.2μg/mL; The limit of quantitation was5,0.5and6μg/mL; The RSD of precisions were less than2%;The RSD of stability and repeatability all were less than3%; The average recoveries were98.3%(RSD=1.9%, n=6),96.3%(RSD=1.9%, n=6),100.8%(RSD=1.9%, n=6) respectively.2. The device was easy to be made and handled, while each of20rats was treated by this device6times one day,no mouse was dead and no person was hurted during the experimentl course. The method showed good linearity and no endogenous material interfered with the marked compounds. The linear range of loganin, ferulic acid and stilbene glucoside was0.03～3.00、0.03～3.00and0.02～2.00μg/mL respectively (r=0.9962,0.9956,0.9963), the limit of quantification was0.020,0.015and0.010μg/mL,the limit of quantitation was0.030,0.030and0.020μg/mL, the extraction recovery was over82%, over76%and over60%, within-day and between-day precisions were less than10%,7.8%and9.4%. The pharmacokinetic parameters of loganin, ferulic acid and stilbene glucoside were as follows:Cmax was0.369±0.042, 0.387±0.071and0.233±0.044μg/mL; tmax was0.226±0.022,0.282±0.031and0.233±0.044h; t1/2(β) was6.89±0.20,10.7±0.11and6.93±0.09h;AUC0-∞was1.91±0.36,3.22±0.52and1.52±0.33μg·h/mL; AUC0-t was1.62±0.33,2.58±0.43and1.30±0.30μg·h/mL;CL was20.2±4.0,1.39±0.23and31.7±6.9L/h/kg.3. Statistical differences between blank control group and rIL-1β group (P<0.01), rIL-1β can induce GMCs proliferation, promote TGF-β1and CTGF gene mRNA expression, promote TIEG1, TGF-β1and CTGF protein production. Statistical differences between rIL-1β group and BSTLF/fosinopril group (P<0.05or P<0.01), BSTLF and fosinopril can reduce GMCs’proliferation, reduce the mRNA expression of CTGF and TGF-β1gene, reduce the protein production of TIEG1, CTGF and TGF-β1,which mediated by interleukin. The part of BSTLF group and fosinopril group had statistical difference between control group when been cultured for24h(P<0.05). BSTLF group and fosinopril group had no signigicant difference between control group when been cultured for48h(P>0.05).There is no signigicant difference among the BSTLF high middle low dose groups with fosinopril group at the same time.Conclusion:1. The optimal extraction method is stable and feasible, The method is accurate,sensitive and can be used for the quality control of BSTLF of three ingredients above.2. The device is a safe and ideal equipment for blood collection by tail snipping in rat. The established method is accurate, sensitive and convenient for detecting blood concentration of loganin, ferulic acid and stilbene glucoside. The results showed all the three components’concentration-time curves were conformed to two compartment models. All the three components could be absorbed in blood quickly, loganin and stilbene glucoside could be moderate excreted, ferulic acid could be excreted slowly(but the highest bioavailability). BSTLF can be made into common oral preparation, which been taken for3times a day can maintain plasma concentration.3. BSTLF can reverse the rat mesangial cells’proliferation, reduce the mRNA expression of TGF-β1and CTGF gene, reduce the protein production of TIEG1,TGF-β1and CTGF,the efficacy appears time and dose dependence. The research result suggest that there is closely related between the rat mesangial cells’ proliferation and the cytokines,such as IL-1,TIEG1,TGF-β1and CTGF; Restrain the TGF-β1/CTGF signaling pathway and adjust the function of rat mesangial cells maybe is the possible action mechanism of BSTLF contra nephrotic syndrome and its derivatives disease, then anti TGF-β1and CTGF is the effective target for chinese medicine in the treatment of nephropathy.