Protective Effect Of 8-methylene-tert-butylamine Hesperetin?MTBH? On CCl₄-induced Liver Fibrosis In Mice And Its Pharmacokinetic Studies In Rats

Liver fibrosis is a consequence of continuous wound-healing response, which caused by a variety of most chronic liver diseases such as hepatitis viral infection and autoimmune hepatitis, viruses, drugs, schistosomiasis and alcohol. It's mainly characterized by impaired liver function and the excessive deposition of extracellular matrix ?ECM? components and caused by the unbalanced fiber hyperplasia and decomposition. A variety of studies have indicated that the main source of excessive deposition of ECM is the activation of hepatic stellate cell, HSCs activation and proliferation are the central process of the development of liver fibrosis. Therefore, inhibiting HSC activation may alleviate the development of liver fibrosis. Carbon tetrachloride ?CCl₄?, a well-known hepatotoxin, is widely used in laboratory animals to induce toxic liver injuries including fibrosis.In the past decades, the prevention and treatment of liver fibrosis with traditional Chinese medicine has played key roles and gained increasing attention. Hesperidin, a flavanone found abundantly in fruit peels of the genus citrus, its aglycone form is called hesperetin, is one of the main active ingredients. Literatures showed that, hesperitin exhibit extensive biological and pharmacological properties, such as anti-inflammation, antioxidant, antiviral, anticarcinogenic activities and anti-hyperlipidemia. However, its poor water solubility, low bioavailability, and short half-life limit its wide application in medicine. In order to overcome these limitations, recently, a series of novel hesperitin derivatives containing Mannich base moieties were synthesized in our laboratory, the water solubility, anti-inflammatory and protective effect on liver fibrosis were evaluated. In our studies, we evaluated the role of MTBH in the activation and proliferation of TGF-? induced hepatic stellate cells. We found that MTBH might inhibit the activation and the proliferation of HSCs. Our results suggested that MTBH might serve as a promising natural supplement for liver protection drug. In order to confirming this idea, we conducted the following research. 1. The anti-fibrotic of MTBH in.liver fibrosis tissues of mice.To investigate the protective effects and its mechanism of MTBH on hepatic fibrosis in mice, the mice model of liver fibrosis was established by intraperitoneal injection of CCl₄. Sixty ICR mice were randomly divided into six groups:normal group, model group, colchicin control group ?0.1mg/kg?, MTBH prevention group ?25 mg/kg,50 mg/kg,100 mg/kg?. Except the normal group, all the other groups were injected intraperitoneally with CCl₄ to induce hepatic fibrosis, twice per week for 10 weeks. The prevention group was given supplemental MTBH, once per day during 6-10 weeks; the control group was given supplemental colchicin ?0.1 mg/kg?, twice per week during 6-10 weeks. HE, Masson and immunohistochemistry were used to test the injury of different groups of liver tissues. The Q-PCR and Western blot were used to measure the levels of a-SMA and Collagen I in the mouse liver fibrosis tissues. The results suggested that the treatment of different doses of MTBH ?50 mg/kg,100 mg/kg? attenuate the CCl₄-treated liver fibrosis and decreased the mRNA and protein expression of a-SMA and Collagen I in mouse liver.2. The effect of MTBH on the activation of HSC-T6 cellsHSC-T6 cells were treated with MTBH ?12.5,25,50,100,200,400 ?M?, then we used MTT assay to evaluate the role of MTBH on the viability of TGF-? activated HSC-T6 cells. HSC-T6 cells were pretreated with TGF-p ?10 ng/ml? for 2 h, then incubated with MTBH ?50, 100,200 ?M? for another 24 h. MTT were used to detect the role of MTBH on HSC-T6 cell proliferation. The expression of ?-SMA and Collagen I were measured by Q-PCR and Western blot. The results showed that MTBH might mainly function by negatively regulating TGF-? induced HSC-T6 cells activation by inhibiting the expression of ?-SMA and Collagen I.3. Pharmacokinetics and tissue distribution of MTBH in ratsIn order to investigate the pharmacokinetic characteristics and tissue distribution of MTBH and its conjugated metabolites in rats, male Sprague-Dawley ?SD? rats were orally administered ?25,50,100 mg/kg? or intravenously administered ?25 mg/kg? MTBH and blood samples were withdrawn at specific times. Plasma was obtained after centrifugation, and the concentration of free form, glucuronides, and glucuronides/sulfates of MTBH in plasma were assayed. In the tissue distribution study, after a single oral dose of MTBH ?200 mg/kg?, the rats were sacrificed at scheduled time points ?6 rats each?, tissues ?heart, liver, spleen, lung, kidney, stomach, intestine, brain and muscle? were collected before a systemically perfusing with cool normal saline at scheduled time points, then homogenized in normal saline, and the concentration of free form, glucuronides, and glucuronides/sulfates of MTBH in tissue homogenate were assayed. Identification of MTBH conjugated metabolites was performed on a PDA detector scanning from 250 to 700 nm, and an Agilent 6224 ESI/TOF mass spectrometer. Two main conjugates metabolites were MTBH-O-glucuronide and MTBH-O-sulfate. The concentrations of MTBH glucuronides or total conjugates ?glucuronides plus sulfates? in plasma and tissue homogenate were quantified from the amount of corresponding aglycone ?MTBH? liberated from the conjugated metabolites by hydrolysis with (3-glucuronidase or sulfatase ?containing ?-glucuronidase?. The results showed that the glucuronides/sulfates were extensively present in the plasma, moreover, the free form was detectable in the plasma, but in a small amount (AUC_o-t) equivalent to nearly 0.85-1.46% of the amount (AUC_o-t) of glucuronides/sulfates, the absolute bioavailability of MTBH was approximately 31.27%. The main pharmacokinetic parameters of MTBH after a single oral ?25,50,100 mg/kg? administration of MTBH to rats are as follows:free form MTBH T_max ?h?: 0.25 ± 0.06,0.50 ± 0.18,0.25 ± 0.06; t_1/2 ?h?:0.41 ± 0.16,0.46 ± 0.09,0.52± 0.06; C_max ??g/ml?:0.40 ± 0.07,0.83 ± 0.30,1.54 ± 0.37; AUC ??g/ml·h?:0.42 ± 0.08,0.82 ± 0.21,1.78 ± 0.25. Glucuronides of MTBH T_max ?h?:1.00 ± 0.19,1.00 ± 0.28,1.00 ± 0.19; t_1/2 ?h?:4.19 ± 0.75,4.32 ± 0.76,5.69 ± 0.80; C_max??g/ml?:4.86 ± 1.24,8.10 ± 1.82,19.23 ± 3.45; AUC ??g/ml·h?:24.05 ± 5.74,50.12 ± 13.19,118.79 ± 16.56. Glucuronides/sulfates of MTBH T_max ?h?:1.00 ± 0.22,1.00 ±0.18,1.00 ± 0.25; t_1/2 ?h?:4.43 ± 1.05,4.68 ± 0.64,6.20 ± 0.64; C_max ??g/ml?:5.80 ± 0.91,9.31 ± 2.30,24.28 ± 4.76; AUC ??g/ml·h?:28.69 ± 6.03,65.74 ± 12.27, 146.83 ± 22.51. The main pharmacokinetic parameters of MTBH after intravenous ?25 mg/kg? administration of MTBH to rats are as follows:free form MTBH T_max ?h?:.0.083 ± 0.02, t_1/2 ?h?:0.46 ± 0.13, C_max??g/ml?:11.07 ± 2.07, AUC0-24??gh/ml?:6.05 ± 0.93; Glucuronides of MTBH T_max ?h?:0.50 ± 0.02, t_1/2 ?h?:4.57 ± 1.03, C_max ??g/ml?:13.72 ± 2.45, AUC_0-24 ??gh/ml?:80.12 ± 9.80; Glucuronides/sulfates of MTBH T_max ?h?:0.50 ± 0.01, t_1/2 ?h?:4.63 ± 1.25, C_max ??g/ml?:15.92 ± 3.36, AUC0-24 ??gh/ml?:91.75 ± 15.42. In rat tissues, the free form appeared in all tissues examined, with trace amount in brain and muscle, and considerable concentration in stomach and lung. Glucuronides/sulfates were the major forms in intestine, kidney, and liver, whereas not detectable in heart, brain and muscle. The liver and intestine was found likely to accumulate MTBH at a high concentration among all tissues.4. In vitro and in vivo modulation of CYP enzymes by MTBHThe potential for herb-drug interactions has recently received greater attention worldwide, considering the fact that the use of MTBH for liver protection in the near future. The purpose of this work was to examine the potential for the metabolism-based drug interaction arising from MTBH and to investigate the inhibitory effect of MTBH on rat and human CYP450s. An in vitro cocktail approach was established to screen the activity of rat and human CYP1A2,2C9,2C19,2D6,2E1 and 3A4. Probes include phenacetin ?1A2?, diclofenac ?2C9?, omeprazole ?2C19?, dextromethorphan ?2D6?, chlorzoxazone ?2E1?, testosterone ?3A4?. All the probes were monitored with determining the corresponding metabolites of probes by a LC-MS/MS method. Probe substrates of cytochrome P450 enzymes were incubated in rat or human liver microsomes ?HLMs? with or without MTBH. In rat and human vitro trial, the results demonstrated that MTBH inhibited CYP3A4 and CYP2E1 in a concentration-dependent manner in rat liver microsomes. with IC50 values of 83.66 and 86.55 ?mol·L^-1, respectively. MTBH did not produce inhibition of CYPIA2, CYP2D6, CYP2C9 and CYP2C19 activities. In human liver microsomes, similarly, MTBH inhibited CYP3A4 and CYP2E1 in a concentration-dependent manner, with IC50 values of 93.73 and 88.40 ?mol·L^-1, respectively. MTBH did not produce inhibition of CYP1A2, CYP2D6, CYP2C9 and CYP2C19 activities. In rat vivo trial, the effect of 28 days of treatment with MTBH ?25,50,100 mg daily? on the expression of mRNA, protein, and the activities of six cytochrome P450 enzymes. Western blot and PCR analysis revealed that MTBH ?50 mg/kg,100 mg/kg? markedly decreased CYP3A4 and CYP2E1 mRNA and protein expression, MTBH ?100 mg/kg? markedly decreased CYP2C9 mRNA but not CYP2C9 protein expression. After 28 days of treatment with MTBH ?100 mg/kg?, the activity of hepaticmicrosomal CYP3A4 and CYP2E1 in rats was significantly reduced. However, MTBH had no effect on the activity of hepaticmicrosomal CYP1A2, CYP2D6, CYP2C9 and CYP2C19.5. Intestinal absorption mechanisms of MTBH in Caco-2 cellsThe human intestinal Caco-2 cell line, which is derived from human colonic adenocarcinoma, but exhibits many morphological and functional similarities to the normal human small intestinal epithelial cells when they are grown as polarized cells, has been extensively used as an in vitro model for intestinal drug absorption. The purpose of our work was to investigate the effect of time, concentration, pH, TEER values, temperature and different transporter inhibitors on the transport of MTBH in Caco-2 cell to elucidate the transport mechanisms of MTBH. Caco-2 cells were seeded on permeable filters to confluence and grown as fully differentiated mono layers. The integrity of the cell mono layer was checked before and after each experiment by measuring transepithelial electrical resistance ?TEER?. Transport medium containing MTBH ?7.5-480 ?M? was added either to the apical or basolateral compartment ?0.6 or 1.2 ml respectively?.100 ?L samples were collected at 30,60,90 and 120 min from the receiver compartment. The concentration of MTBH in transport medium was assayed by HPLC. The results demonstrated that MTBH was effectively absorbed by Caco-2 cells in a concentration and time-dependent manner in both directions at 7.5-480 ?M. Moreover, the absorption of MTBH is characterized by H+-driven polarized transport in A to B direction, which was pH dependent and TEER values independent transport in both directions. Transport of MTBH was obviously decreased in the presence of sodium azide ?an ATP inhibitor? or CCCP ?carbonyl cyanide m-chlorophenyl hydrazone, a proton-ionophore? in the presence of a pH-gradient ?apical pH,6.0; basolateral pH,7.4?. The decrease in pH with a increase in permeation has been shown to occur for the MCT and the peptide transporter 1 ?PepTl?, however, the model dipeptide substrate and inhibitor glycylsarcosine had no effect on MTBH transport; MTBH transport was markedly reduced by MCT inhibitors quercetin or phloretin, and the substrate analogs L-lactate or benzoic acid. We identified MCT1, MCT3, MCT4, MCT5, and MCT6 in Caco-2 cells by Western blot. Silence MCT1 with siRNA resulted in significant inhibition of MTBH uptake. The verapamil, a P-gp inhibitor, and Kol43, a BCRP inhibitor, had no effect on the transport of MTBH. However, MK-571 or probenecid, MRP2 inhibitors, led to an apparently decrease in the efflux of MTBH. In summary, MTBH was mainly absorbed by transcellular passive diffusion and a pH dependent mechanism mediated by MCT1. It was not a P-gp or BCRP substrate, but was substrate of the MRP2 efflux transporter.