Evaluation on the Metabolic Activity of Two Carboxylesterase Isozymes in Mouse Liver Microsomes by a LC-MS/MS Method.

An applicable method for the precise measurement of major carboxylesterase (CESs) activity in liver still limited. Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively. Clopidogrel is metabolized to the inactive metabolite clopidogrel carboxylate (CCAM) by CES1. Irinotecan is metabolized to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by CES2. In the present study, the LC-MS/MS method for the determination of CCAM and SN-38 were separately developed to characterize the metabolic activities of CES1 and CES2 in mouse liver microsomal. CCAM was separated on a Ecosil ODS column with an isocratic mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile (15:85, V:V) at a flow rate of 0.4mL/min. SN-38 was separated on a Waters symmetry C18 column with an gradient mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min. Calibration curves were linear within the concentration range of 100-20,000 ng/mL for CCAM and 1-200 ng/mL for SN-38. The results of method showed excellent accuracy and precision. The recovery rate, matrix effect and stability inspection results were within the acceptance criteria. The optimized incubation conditions were as follows: protein concentration of microsomes were all 0.1 mg/mL, incubation time was 60 min for clopidogrel and 30 min for irinotecan, respectively. This method was sensitive and applicable for the determination of the activity of CESs in the mouse liver microsomes.

Ginsenosides Restore Lipid and Redox Homeostasis in Mice with Intrahepatic Cholestasis through SIRT1/AMPK Pathways.

Intrahepatic cholestasis (IC) occurs when the liver and systemic circulation accumulate bile components, which can then lead to lipid metabolism disorders and oxidative damage. Ginsenosides (GS) are pharmacologically active plant products derived from ginseng that possesses lipid-regulation and antioxidation activities. The purpose of this study was to evaluate the possible protective effects of ginsenosides (GS) on lipid homeostasis disorder and oxidative stress in mice with alpha-naphthylisothiocyanate (ANIT)-induced IC and to investigate the underlying mechanisms. A comprehensive strategy via incorporating pharmacodynamics and molecular biology technology was adopted to investigate the therapeutic mechanisms of GS in ANIT-induced mice liver injury. The effects of GS on cholestasis were studied in mice that had been exposed to ANIT-induced cholestasis. The human HepG2 cell line was then used in vitro to investigate the molecular mechanisms by which GS might improve IC. The gene silencing experiment and liver-specific sirtuin-1 (SIRT1) knockout (SIRT1LKO) mice were used to further elucidate the mechanisms. The general physical indicators were assessed, and biological samples were collected for serum biochemical indexes, lipid metabolism, and oxidative stress-related indicators. Quantitative PCR and H&E staining were used for molecular and pathological analysis. The altered expression levels of key pathway proteins (Sirt1, p-AMPK, Nrf2) were validated by Western blotting. By modulating the AMPK protein expression, GS decreased hepatic lipogenesis, and increased fatty acid ß-oxidation and lipoprotein lipolysis, thereby improving lipid homeostasis in IC mice. Furthermore, GS reduced ANIT-triggered oxidative damage by enhancing Nrf2 and its downstream target levels. Notably, the protective results of GS were eliminated by SIRT1 shRNA in vitro and SIRT1LKO mice in vivo. GS can restore the balance of the lipid metabolism and redox in the livers of ANIT-induced IC models via the SIRT1/AMPK signaling pathway, thus exerting a protective effect against ANIT-induced cholestatic liver injury.

Estrogen cholestasis induces gut and liver injury in rats involving in activating PI3K/Akt and MAPK signaling pathways.

BACKGROUNDS: Estrogen and its metabolites often lead to intrahepatic cholestasis in susceptible women with pregnancy, administration of oral contraceptives and postmenopausal hormone replacement therapy. Recently, dysfunction of the gut-liver axis has been suggested to play a pivotal role in the progression of cholestasis, but details about estrogen cholestasis (EC)-induced gut and liver injury are still largely unknown. This study aims to gain insight into EC-induced gut and liver injury and cell signaling implicated. METHODS: Male rats were exposed to 5 and 10 mg/kg of 17α-ethinylestradiol via subcutaneous injection for 5 successive days to simulate human EC. RESULTS: By detection of these estrogen cholestatic rats, we found that EC induced inflammation in the liver but not in the intestine through activating NF-κB signaling pathway. EC strongly induced oxidative stress in both the liver and intestine, and activated the hepatic Nrf2/Gclm/Gclc pathway and the intestinal Nrf2/Ho-1 pathway, respectively, for adaptively regulating oxidative stress. EC increased cell apoptosis in both the liver and intestine. Additionally, EC elevated phosphorylation of Akt, ERK1/2, and p38 in the liver and increased phosphorylation of p38 in the intestine. CONCLUSIONS: EC induces liver inflammation, both gut and liver oxidative stress and apoptosis, involving in activating PI3K/Akt and MAPK signaling pathways. Investigation of EC-induced gut and liver injury contributes to the development of new potential therapeutic strategies.

Protective effect of Andrographolide on 5-Fu induced intestinal mucositis by regulating p38 MAPK signaling pathway.

AIMS: Intestinal mucositis is the most common side effect of 5-fluorouracil (5-Fu) treatment in cancer patients. Previous research suggested that andrographolide (Andro) attenuated the intestinal injury in colitis or diarrhea in mice. The present study was aimed at investigating the protective effect of Andro against 5-Fu induced intestinal mucositis and the underlying mechanism. MAIN METHODS: BALB/C mice were injected 5-Fu at a dose of 100 mg/kg for 5 days to induce intestinal mucositis. Andro at different doses (25, 50, 100 mg/kg/day) was administered. Weight loss, diarrhea score, cellular apoptosis and proliferation were evaluated. Apoptosis related proteins were detected by Western blotting. Then, NCM460 cells were used to explore the possible mechanism in vitro. The effect of Andro on the anti-tumor efficacy of 5-Fu was investigated in H22 tumor-bearing mice. KEY FINDINGS: Andro significantly ameliorated the 5-Fu induced weight loss and diarrhea. The apoptosis of intestinal cells was also attenuated by Andro treatment both in vivo and in vitro. Besides, Andro markedly down-regulated the 5-Fu-induced protein expression of caspase8/3, Bax and the phosphorylation of p38. Moreover, 5-Fu significantly reduced the viability of NCM460 cells, which was restored by the Andro pretreatment. Furthermore, asiatic acid, an agonist of p38 MAPK, reversed the anti-apoptotic effect of Andro in NCM460 cells. Andro did not weaken the anti-H22 tumor effect of 5-Fu in vivo. SIGNIFICANCE: We have demonstrated that p38 MAPK inhibition mediates anti-apoptotic effects of Andro against 5-Fu induced intestinal mucositis, suggesting that Andro may benefit the patients undergoing 5-Fu based chemotherapy.

Bioinformatics­based identification of key pathways and candidate genes for estrogen­induced intrahepatic cholestasis using DNA microarray analysis.

Estrogen­induced intrahepatic cholestasis (EIC) has increased incidence during pregnancy, and within women taking oral contraception and postmenopausal hormone replacement therapy. However, the pathology underlying EIC is not well understood. The aim of the present study was to identify key pathways and candidate genes in estrogen­induced intrahepatic cholestasis (EIC) that may be potential targets for diagnosis and treatment. A whole­genome microarray (4x44K) analysis of a 17α­ethinylestradiol (EE)­induced EIC rat liver model was performed. Bioinformatics­based methods were used to identify key pathways and candidate genes associated with EIC. The candidate genes were validated using a reverse transcription quantitative polymerase chain reaction assay. A total of 455 genes were differentially expressed (P<0.05 and fold change >2.0) following EE treatment, including 225 downregulated genes and 230 upregulated genes. Sulfotransferase family 1E member 1, cytochrome P450 family 3 subfamily A member 2, carbonic anhydrase 3, leukotriene C4 synthase and ADAM metallopeptidase domain 8 were the 5 candidate genes identified to be differentially expressed and involved in the metabolism of estrogens and bile acids and the regulation of inflammation and oxidative stress. The Analyses of Gene Ontology enrichment, Kyoto Encyclopedia of Genes and Genomes pathways and protein­protein interaction network associated­modules identified several key pathways involved in the homeostasis of lipids and bile acids and in AMPK, p53 and Wnt signaling. These key pathways and candidate genes may have critical roles in the pathogenesis of EIC. In addition, reversing the abnormal expression of candidate genes or restoring the dysfunction of key pathways may provide therapeutic opportunities for patients with EIC.

Reasons for Laser in Situ Keratomileusis in China: A Qualitative Study.

SIGNIFICANCE: Myopia is a major health issue in East Asian countries, especially in China. By identifying Chinese patients' motivations for laser in situ keratomileusis (LASIK) surgery, our results are expected to help clinicians counsel patients before LASIK surgery and to maximize patients' post-operative LASIK surgery satisfaction, improving the quality of LASIK surgery services. PURPOSE: Laser in situ keratomileusis has become a popular type of refractive surgery for the correction of myopia worldwide. This study uses qualitative inquiry approaches to understand the motives and processes of patients' LASIK surgery decision making. METHODS: A purposive sample of 45 patients who had decided to undergo LASIK was recruited. Our qualitative study used in-depth interviews and used content analysis to interpret the data. RESULTS: Among 45 participants, 48.9% reported that career requirements were the most important reason for seeking LASIK surgery. The inconvenience of wearing glasses or lenses during activities of daily life was also a primary motive. Improving facial appearance was a main reason for female but not male respondents. Potential complications of spectacles and contact lenses in addition to maturation of LASIK technology were also reported motives to seek surgery. Participants gave multiple, overlapping reasons for LASIK surgery. CONCLUSIONS: These findings suggest that motives to seek LASIK surgery are not only a desire to correct refractive error but also social factors and confidence in improved surgical technology. The implications for clinicians are to be aware of these multiple motives for LASIK to improve the quality and effectiveness of health services for myopia patients.

Hepatoprotective effect of calculus bovis sativus on nonalcoholic fatty liver disease in mice by inhibiting oxidative stress and apoptosis of hepatocytes.

Calculus bovis (CB, niu-huang) is a high-class therapeutic drug that is often used in traditional Chinese medicine. CB helps to eliminate heat and toxic components, and prevents the accumulation of phlegm and blood stasis in the liver. In Asian countries, CB Sativus (CBS), an ideal substitute for natural CB, is presently extensively used for long-term treatment of chronic liver diseases. The present study aimed to evaluate the effects and potential mechanism(s) of action of CBS on mice with fructose-induced nonalcoholic fatty liver disease (NAFLD). The NAFLD model was established in C57BL/6 mice by exclusively feeding fluids containing 30% fructose for 8 consecutive weeks. After these 8 weeks, mice were given CBS (50 mg/kg/day or 100 mg/kg/day) for 2 consecutive weeks. Treatment with CBS reversed the fructose-induced impaired glucose tolerance. Compared with the model group, in which mice received 8 weeks of high-fructose diet and 2 weeks of 0.5% sodium carboxymethyl cellulose, CBS treatment significantly decreased the levels of fasting serum glucose, fasting insulin, triglyceride, and total cholesterol, and increased levels of high-density lipoprotein-cholesterol. CBS treatment also significantly decreased the levels of triglyceride, total cholesterol, and free fatty acid in the liver. The activity of superoxide dismutase in the liver was increased after treatment with CBS, however, levels of malondialdehyde and reactive oxygen species decreased. Histopathological examination showed that liver steatosis and injury were significantly reduced in CBS-treated mice. The expression of fatty acid synthase, nuclear factor kappa-light-chain-enhancer of activated B cells, Cysteinyl aspartate-specific proteinase-3, and synonyms B-cell leukemia/lymphoma-2 gene-associated X protein were downregulated after treatment with CBS, whereas the expression of nuclear factor erythroid-2-related factor 2 was upregulated. In conclusion, CBS treatment exerted therapeutic effects in the liver of mice with NAFLD, which may be associated with amelioration of metabolic disorders, enhanced antioxidant effects, and alleviation of apoptosis.

Upregulation of PDZK1 by Calculus Bovis Sativus May Play an Important Role in Restoring Biliary Transport Function in Intrahepatic Cholestasis.

Intrahepatic cholestasis is a main cause of hepatic accumulation of bile acids leading to liver injury, fibrosis, and liver failure. Our previous studies proved that Calculus Bovis Sativus (CBS) can restore biliary transport function through upregulating the multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) in 17α-ethynylestradiol- (EE-) induced intrahepatic cholestasis rats. The regulation mechanism of CBS on these transporters, however, remains unclear. This study was designed to evaluate the possible relationship between the effect of CBS on transport activities and the regulation of CBS on the expression of PDZK1, a mainly scaffold protein which can regulate MRP2 and BCRP. Intrahepatic cholestasis model was induced in rats with injection of EE for five consecutive days and then the biliary excretion rates and cumulative biliary excretions were measured. The mRNA and protein expression levels of PDZK1 were detected by reverse transcription-quantitative real-time polymerase chain reaction, western blot, and immunohistochemical analysis. When treated with CBS, cumulative biliary excretions and mRNA and protein expressions of PDZK1 were significantly increased in intrahepatic cholestasis rats. This study demonstrated that CBS exerted a beneficial effect on EE-induced intrahepatic cholestasis rats by restoring biliary transport function, which may result from the upregulation of PDZK1 expression.

Protective Effect of Calculus Bovis Sativus on Dextran Sulphate Sodium-Induced Ulcerative Colitis in Mice.

Calculus Bovis Sativus (CBS) is a commonly used traditional Chinese medicine, which has been reported to exhibit antispasmodic, fever-reducing, anti-inflammatory, and gallbladder-repairing effects. The present study aims to investigate the protective effect of CBS on dextran sulphate sodium- (DSS-) induced ulcerative colitis (UC) in mice. C57BL/6 male mice were exposed to 5% DSS in drinking water. CBS was given orally at 50 and 150 mg/kg once per day for 7 days. Body weight, disease activity index (DAI), colon length, colonic myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and nitric oxide (NO) levels were measured. Administration of CBS significantly reserved these changes, decreased the MPO activity and MDA and NO level, and increased the SOD activity in the colon tissue. Histological observation suggested that CBS alleviated edema, mucosal damage, and inflammatory cells infiltration induced by DSS in the colon. Moreover, CBS significantly downregulated the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1ß and IL-6 in the colon tissue. Our data suggested that CBS exerted protective effect on DSS-induced UC partially through the antioxidant and anti-inflammatory activities.

Beneficial effect of Calculus Bovis Sativus on 17α-ethynylestradiol-induced cholestasis in the rat.

AIMS: Calculus Bovis Sativus (CBS) shares similar pharmacological effects with Calculus Bovis like relieving hepatobiliary diseases. This study aims to investigate the effect and mechanism of CBS on 17α-ethynylestradiol (EE)-induced cholestasis in the rat. MAIN METHODS: CBS (50 and 150 mg/kg per day) was intragastrically (i. g.) given to experimental rats for 5 consecutive days in coadministration with EE. The levels of serum biomarkers, hepatic malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by biochemical methods. The bile flow in 2h was measured. The histopathology of the liver tissue was evaluated. The expression of transporter was studied by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. KEY FINDINGS: CBS treatment significantly prevented EE-induced increases in serum levels of biomarkers. Decreased bile flow by EE was restored with CBS treatment. The tissue lesions were also relieved with CBS treatment. Western blot studies indicated that EE significantly decreased the protein expression of multidrug resistance-associated protein 2 (Mrp2) and breast cancer resistance protein (Bcrp), but notably increased P-glycoprotein (P-gp) protein, compared with the control group. CBS treatment significantly increased the protein expression of P-gp, Mrp2 and Bcrp compared with the EE group. RT-qPCR studies indicated that EE down-regulated Bcrp at transcriptional level. CBS up-regulated the mRNA expression of P-gp, Mrp2 and Bcrp compared with the EE group. SIGNIFICANCE: The present study indicated that CBS exerted a beneficial effect on EE-induced cholestasis in the rat, which may result from its induction of P-gp, Mrp2 and Bcrp expression.

Utility values among myopic patients in mainland China.

PURPOSE: To elicit utility values of adult myopic patients in mainland China. METHODS: A valid sample of 442 myopia patients (spherical equivalent at least -0.5 diopters) aged 17 to 44 years who were scheduled to undergo refractive surgery were recruited. Information on time trade-off ([TTO] years of life willing to sacrifice for treatment of myopia) and standard gamble (SG) for blindness (risk of blindness from therapy, willing to sacrifice for treatment of myopia) utility values and sociodemographic and clinical data were obtained. RESULTS: The mean utility values based on TTO and SG were 0.96 ± 0.05 (95% confidence interval [CI], 0.95 to 0.96; median, 0.98) and 0.93 ± 0.09 (95% CI, 0.92 to 0.94; median, 0.97), respectively. Myopic patients using contact lens had significantly higher TTO utility values than those wearing glasses (p < 0.001). There was no significant difference in the TTO and SG utility values by age, sex, occupation, educational levels, residence, reasons for refractive surgery, and severity and duration of myopia (p > 0.05). CONCLUSIONS: The TTO and SG produce similar mean utility values, but there is poor agreement between results for individuals from the two methods. Utility values associated with myopic patients obtained in this study or reported in the literature seem to be higher than those obtained for other ophthalmic conditions.

Reduced bioavailability of cyclosporine A in rats by mung bean seed coat extract

Mung bean seed coat (MBSC) is a healthcare product in Asian countries. The aim of this study was to investigate the effect of an MBSC ethanol extract on the bioavailability of cyclosporine A (CsA) in rats. Rats were orally dosed with CsA alone or in combination with MBSC ethanol extracts (500 mg/kg, p.o.). The blood levels of CsA were assayed by liquid chromatography with an electrospray ionization source and tandem mass spectrometry (LC-MS/MS). The everted rat intestinal sac technique was used to determine the influence of MBSC on the absorption of CsA. The results reveal that combined CsA intake with MBSC decreased the Cmax, AUC0-t, t1/2z and MRT0-t values of CsA by 24.96%, 47.28%, 34.73% and 23.58%, respectively (P<0.05), and significantly raised the CL/F by 51.97% (P<0.01). The in vitro results demonstrated that significantly less CsA was absorbed (P<0.05). The overall results indicate that after being concomitantly ingested, MBSC reduced the bioavailability of CsA, at least partially, in the absorption phase.

O tegumento da semente de feijão-mungo (MBSC) é um produto para tratamento de saúde em países asiáticos. O objetivo deste estudo foi investigar o efeito de extrato etanólico de MBSC na biodisponibilidade da ciclosporina A (CVsA) em ratos. Administrou-se aos ratos CsA sozinha ou em associação com extrato etanólico de MBSC (500 mg/kg, p.o.), por via oral. Os níveis sanguíneos de CSA foram determinados por cromatografia a líquido com ionização por electrospray, associada à espectrometria de massas (LC-MS/MS). Utilizou-se a técnica de inversão do saco intestinal de rato para determinar a influência do MBSC na absorção de CsA. Os resultados revelaram que a ingestão combinada de CsA e MBSC diminuiu os valores de Cmax, AUC0-t, t1/2z e MRT0-t de CsA em 24%, 47,28%, 34,73% e 23,58%, respectivamente (P<0.05), e aumentou, significativamente, CL/F em 51,79% (P<0.05). Os resultados in vitro demostraram que, significativamente, menos CsA foi absorvida (P<0.05). Os resultados totais indicaram que após ser concomitantemente ingerida, a MBSC reduziu, ao menos parcialmente, a biodisponibilidade de CsA, na fase de absorção.

Evaluation of intestinal absorption of amtolmetin guacyl in rats: breast cancer resistant protein as a primary barrier of oral bioavailability.

AIMS: The purpose of the present study was to investigate the role of efflux transporters on the intestinal absorption of amtolmetin guacyl (MED-15). MAIN METHODS: The effects of P-glycoprotein (P-gp), multiple resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on intestinal absorption amount of MED-5 (tolmetin-glycine amide derivative), the metabolite formed from MED-15 in the intestinal epithelial cells were studied in the in vitro everted gut sac experiments. Moreover, the in situ single-pass intestine perfusion was adopted to clarify the role of efflux transporters in excreting MED-5 in knockout mice. The plasma concentration of MED-5 and tolmetin, the metabolite formed from MED-5 was determined in Bcrp1 knockout mice and wild-type mice. KEY FINDINGS: BCRP inhibitor Ko143 (50 µM and 100 µM) significantly increased the intestinal absorption amount in jejunum, ileum and colon (p<0.05). However, no effect was observed in the presence of P-gp inhibitor verapamil and MRP2 inhibitor MK571 in each intestinal segment. Furthermore, the plasma concentration MED-5 and tolmetin, metabolites of MED-15, increased 2-fold and 4-fold, respectively, in Bcrp1 knockout mice compared with wild-type mice after the single-pass perfusion of small intestine with MED-15. SIGNIFICANCE: It may be concluded that BCRP plays an important role in the intestinal efflux of MED-5 and limits the bioavailability after oral administration of MED-15.

Preparation of anti-HER2 monoclonal antibody-paclitaxel immunoconjugate and its biological evaluation.

Anti-HER2 monoclonal antibody (Sc7301)-paclitaxel (TAX) immunoconjugate was prepared and its specific binding to tumor cells was investigated in this study. Sc7301 was conjugated to TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained. After purification and labeling by Cyano-fluorescein isothiocyanate (FITC), the specific binding of TAX-Sc7301 to HER2-positive tumor cells (SKOV3) and HER2-negative tumor cells (HepG2) was evaluated respectively. TAX-Sc7301 (20 nmol/L) showed distinct specific binding to SKOV3 cells rather than HepG2 cells. And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-Sc7301 concentration (3-48 nmol/L) and the incubation time (P<0.05). It was concluded that the TAX-Sc7301 immunoconjugate is potentially applicable as a targeted agent against HER2-positive tumor cells.