HOXA7 plays a critical role in metastasis of liver cancer associated with activation of Snail.

BACKGROUND: Liver cancer is one of the main causes of cancer-related death in human. HOXA7 has been proved to be related with several cancers. METHOD: The expression levels of HOXA7 were examined by Western blot, qRT-PCR or ICH. MTT was used to detect the proliferative rate of liver cancer cells. The invasive abilities were examined by matrigel and transwell assay. The metastatic abilities of liver cancer cells were revealed in BALB/c nude mice. RESULTS: In this report, we revealed that HOXA7 promoted metastasis of HCC patients. First, increased levels of HOXA7 were examined in liver cancer especially in metastatic liver cancer. Moreover, higher expression level of HOXA7 was associated with poorer prognosis of liver cancer patients. Overexpression of HOXA7 significantly enhanced proliferation, migration, invasion in vitro and tumor growth and metastasis in vivo meanwhile silencing HOXA7 significantly inhibited the aboves abilities of liver cancer cells. In this research, we identified that HOXA7 performed its oncogenic characteristics through activating Snail. CONCLUSION: Collectively, we identify the critical role and possible mechanism of HOXA7 in metastasis of liver cancer which suggest that HOXA7 may be a potential therapeutic target of liver cancer patients.

Deficiency of female sex hormones augments PGE2 and CGRP levels within midbrain periaqueductal gray.

The midbrain periaqueductal gray (PAG) is a substantial component of the descending modulatory network to control on nociceptive transmission and autonomic functions. Also, accumulated evidence has suggested that the PAG plays a crucial role in regulating migraine headache, a neurovascular disorder. The purpose of this study was to employ ELISA methods to examine the levels of prostaglandin E2 (PGE2) and calcitonin-gene related peptide (CGRP) in the PAG of rats who received ovariectomy and subsequent hormone replacement with 17ß-estradiol, progesterone, or the combination of 17ß-estradiol and progesterone. In addition, using Western blot analysis we examined expression of subtypes of PGE2 receptor in the PAG of rats with different conditions of female sex hormones. Results of our study demonstrated that lack of female sex hormones significantly increased the levels of PGE2 and CGRP in the dorsolateral PAG (P < 0.05) as well as expression of PGE2 EP3 receptors (P < 0.05). Furthermore, a liner relationship was observed between PGE2 and CGRP in the PAG (r = 092, P < 0.01). Also, inhibiting EP3 receptors by chronic administration of L-798106 (EP3 antagonist) into the lateral ventricles significantly attenuated expression of CGRP in the PAG of ovariectomized animals (P < 0.05 vs. vehicle control). Overall, our findings for the first time show that (1) circulating 17ß-estradiol and/or progesterone influences the levels of PGE2 and CGRP in the PAG; (2) a lower level of 17ß-estradiol and/or progesterone augments PGE2 and its EP3 receptor; and (3) PGE2 plays a role in regulating expression of CGRP in the PAG.

Simultaneous determination of three dipeptides (JBP485, Gly-Sar and JBP923) in the cell lysates by liquid chromatography-tandem mass spectrometry: application to identify the function of the PEPT1 transfected cell.

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates using methanol as a deproteinization solvent was developed and validated. Detection was performed by turbo ionspray ionization in multiple reaction monitoring mode using the transitions of m/z 147.1 → m/z 90.1 for Gly-Sar, m/z 201.1 → m/z 86.1 for JBP485, m/z 219.1 → m/z 86.1 for JBP923 and m/z 152.0 → m/z 110.0 for paracetamol (internal standard). The analytes were separated on a Hypersil ODS C18 HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water-methanol (97:3, v/v) at a flow rate of 0.2 mL/min. The calibration curves were demonstrated to be linear over the concentration range of 5.00-5000 nm with coefficient of 0.9968 for Gly-Sar, 0.9975 for JBP485 and 0.9952 for JBP923. The intra- and inter-day precisions were <10.2% for each quality contro; level, and the accuracy was within ±5.6% for each analyte. The matrix effect, the extraction recovery and stabilities of LC-MS/MS analysis were also investigated. This validated method was successfully applied to the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates for identification of stably transfected HeLa cells with human PEPT1.

Wogonoside ameliorates lipopolysaccharide-induced acute lung injury in mice.

Wogonoside has been reported to have anti-inflammatory properties. In this study, we evaluated the effect of wogonoside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice with ALI, induced by intranasal instillation of LPS, were treated with wogonoside 1 h prior to LPS exposure. Mice treated with LPS alone showed significantly increased TNF-α, IL-6, and IL-1ß levels in the bronchoalveolar lavage fluid (BALF). When pretreated with wogonoside, the TNF-α, IL-6, and IL-1ß levels were significantly decreased. Meanwhile, wogonoside significantly inhibited LPS-induced increases in the macrophage and neutrophil infiltration of lung tissues and markedly attenuated myeloperoxidase activity. Furthermore, wogonoside inhibited the TLR4 expression and the phosphorylation of NF-κB p65, and IκB induced by LPS. In conclusion, our results indicate that wogonoside exhibits a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways.

The oligopeptide transporter 2-mediated reabsorption of entecavir in rat kidney.

We investigated whether entecavir is a substrate of the oligopeptide transporter 2 (PEPT2) and whether reabsorption of entecavir is mediated by PEPT2 as well as what is the contribution of PEPT2 to entecavir reabsorption during urinary excretion. Entecavir uptake in transfected cells and rat kidney slices, changes in urine entecavir concentrations following isolated kidney perfusion and in vivo entecavir plasma and urine concentrations were determined with LC-MS/MS. In hPEPT2-HELA cells, entecavir uptake was significantly higher compared to vector-HELA cells and was sharply inhibited by Gly-sar and JBP485, and there were two distinct transport systems. The Km and Vmax of entecavir were 427 (µM) and 1.60 (nmol/mg protein/30s) (low-affinity, high-velocity system) and 24.0 (µM) and 0.296 (nmol/mg protein/30s) (high-affinity, low-velocity system). In rat kidney slices, uptake of entecavir was not markedly inhibited by Gly-sar. In isolated kidney perfusion experiments, entecavir cumulative urinary excretion was statistically significant at 45 and 60 min. CLR(4 °C), CLR(37 °C Control) and CLR(37°C Experiment) were 12.6, 27.6 and 36 (ml/min/kg), respectively. CLTS and TR rate (for PEPT2) were 25.3 and 9.4 (ml/min/kg). In vivo, the cumulative urinary excretion of entecavir had statistical significance at 3 and 4h with CLR(Control) and CLR(Experiment) values of 31 and 42 (ml/min/kg), respectively. The CLTS and TR rate (for PEPT2) were 32 and 11.6 (ml/min/kg), respectively. The present study demonstrated that entecavir is a substrate of PEPT2. Moreover, reabsorption of entecavir is mediated by PEPT2, and 25% of urinary entecavir is reabsorbed by PEPT2.

JBP485 improves gentamicin-induced acute renal failure by regulating the expression and function of Oat1 and Oat3 in rats.

We investigated the effects of JBP485 (an anti-inflammatory dipeptide and a substrate of OAT) on regulation of the expression and function of renal Oat1 and Oat3, which can accelerate the excretion of accumulated uremic toxins (e.g. indoxyl sulfate) in the kidney to improve gentamicin-induced ARF in rats. JBP485 caused a significant decrease in the accumulation of endogenous substances (creatinine, blood urea nitrogen and indoxyl sulfate) in vivo, an increase in the excretion of exogenous compounds (lisinopril and inulin) into urine, and up-regulation of the expressions of renal Oat1 and Oat3 in the kidney tissues and slices via substrate induction. To determine the effect of JBP485 on the accelerated excretion of uremic toxins mediated by Oat1 and Oat3, the mRNA and protein expression levels of renal basolateral Oats were assessed by quantitative real-time PCR, western blot, immunohistochemical analysis and an immunofluorescence method. Gentamicin down-regulated the expression of Oats mRNA and protein in rat kidney, and these effects were reversed after administration of JBP485. In addition, JBP485 caused a significant decrease in MPO and MDA levels in the kidney, and improved the pathological condition of rat kidney. These results indicated that JBP485 improved acute renal failure by increasing the expression and function of Oat1 and Oat3, and by decreasing overoxidation of the kidney in gentamicin-induced ARF rats.

Inhibitory effect of 1α,25-dihydroxyvitamin D3 on excretion of JBP485 via organic anion transporters in rats.

The aim of this study was to investigate the pharmacokinetic mechanism of interaction between JBP485 and 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Rats were injected intraperitoneally with 0.64 nmol/kg/day 1,25(OH)(2)D(3) in 1 ml/kg corn oil for 5 days. The plasma and urine concentrations of JBP485 after intravenous administration and the uptake of JBP485 in kidney slices in vitro were determined by liquid chromatography/tandem mass spectrometry. Quantitative polymerase chain reaction, western blotting, immunohistochemical analysis and immunofluorescence were used to determine the changes in the expression of organic anion transporter (Oat)1 and Oat3 in rat kidney in response to 1,25(OH)(2)D(3) treatment. The plasma concentrations and AUCs of JBP485 were significantly increased, while the renal clearance of JBP485 and uptake of JBP485 in kidney slices were significantly decreased after 1,25(OH)(2)D(3) treatment. These results confirmed that 1,25(OH)(2)D(3) inhibited renal excretion of JBP485. Moreover, 1,25(OH)(2)D(3) decreased expression of Oat1 and Oat3 in rat kidney. Our results are novel in demonstrating an interaction between JBP485 and 1,25(OH)(2)D(3) when they are co-administered. The mechanism of interaction between JBP485 and 1,25(OH)(2)D(3) could be explained at least in part by inhibitory effect of 1,25(OH)(2)D(3) on expression of Oats in rat kidney.

Changes in expression of renal Oat1, Oat3 and Mrp2 in cisplatin-induced acute renal failure after treatment of JBP485 in rats.

The purpose of this study is to investigate whether the effect of cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) on acute renal failure (ARF) induced by cisplatin is related to change in expression of renal Oat1, Oat3 and Mrp2 in rats. JBP485 reduced creatinine, blood urea nitrogen (BUN) and indoxyl sulfate (IS) in plasma and malondialdehyde (MDA) in kidney, and recovered the glomerular filtration rate (GFR) and the activity of superoxide dismutase (SOD) in cisplatin-treated rats. The plasma concentration of PAH (para-aminohippurate) determined by LC-MS/MS was increased markedly after intravenous administration of cisplatin, whereas cumulative urinary excretion of PAH and the uptake of PAH in kidney slices were significantly decreased. qRT-PCR and Western-blot showed a decrease in mRNA and protein of Oat1 and Oat3, an increase in mRNA and protein of Mrp2 in cisplatin-treated rats, and an increase in IS (a uremic toxin) after co-treatment with JBP485. It indicated that JBP485 promoted urinary excretion of toxins by upregulating renal Mrp2. This therefore gives in part the explanation about the mechanism by which JBP485 improves ARF induced by cisplatin in rats.

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats.

The objective was to determine whether protective effects of JBP485 on biliary obstruction induced by alpha-naphthylisothiocyanate (ANIT) are mediated by the organic anion transporters Oat1, Oat3 and the multidrug resistance-associated protein Mrp2. The ANIT-induced increases in bilirubin (BIL), alanine aminotransferase (ALT) and aspartate transaminase (AST) in rat serum were inhibited significantly by oral administration of JBP485. The plasma concentration of JBP485 which is the substrate of Oat1 and Oat3 determined by LC-MS/MS was markedly increased after intravenous administration in ANIT-treated rats, whereas cumulative urinary excretion of JBP485 in vivo and the uptake of JBP485 in kidney slices were decreased remarkably. RT-PCR and Western blot showed the decreased expression of Oat1 and Oat3, increased expression of Mrp2 in ANIT-induced rats, meanwhile, the expression levels of Mrp2 and Oat1 were up-regulated after administration of JBP485. The up-regulation of Mrp2 and Oat1 was associated with a concomitant increase in urinary BIL after treatment with JBP485 in ANIT-treated rats. The mechanism for JBP485 to restore liver function might be related to improvement of the expression and function for Oat1 and Mrp2 as well as facilitation of urinary excretion for hepatoxic substance.

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2.

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.

Peptide cotransporter 1 in intestine and organic anion transporters in kidney are targets of interaction between JBP485 and lisinopril in rats.

The purpose of this study was to clarify the pharmacokinetic mechanism of interaction between JBP485 (cyclo-trans-4-L-hydroxyprolyl-L-serine, a dipeptide with antihepatitis activity) and lisinopril (an angiotensin-converting enzyme inhibitor) in vitro and in vivo. When JBP485 and lisinopril were administered orally simultaneously, the plasma concentrations of the two drugs were decreased significantly, but few changes were observed after simultaneous intravenous administration of the two drugs. The uptake of JBP485 and lisinopril in everted intestinal sacs and in HeLa cells transfected with human peptide cotransporter 1 (PEPT1), as well as absorption of JBP485 and lisinopril after jejunal perfusion were reduced after simultaneous drug administration, which suggested that the first target of drug interaction was PEPT1 in the intestine during the absorption process. The cumulative urinary excretions and renal clearance of the two drugs were decreased after intravenous co-administration, while uptakes of the two drugs in kidney slices and hOAT1/hOAT3-transfected HEK293 cells were decreased. These results indicated that the second target of drug-drug interaction was located in the kidney. These findings confirmed that the pharmacokinetic mechanism of interaction between JBP485 and lisinopril could be explained by their inhibition of the same transporters in the intestinal mucosa (PEPT1) and kidneys (OATs).

Inhibitory effect of zinc on the absorption of JBP485 via the gastrointestinal oligopeptide transporter (PEPT1) in rats.

The aim of this study was to investigate the pharmacokinetic mechanism of interaction between JBP485 and zinc. The plasma concentration of JBP485 after oral administration in vivo, the plasma concentration of JBP485 from the portal vein after jejunal perfusions in situ, the serosal fluid concentration of JBP485 in everted small intestine preparations and the uptake of JBP485 by HeLa-hPEPT1 cells in vitro were determined by LC-MS/MS. RT-PCR and Western blotting were used to determine the mRNA and protein levels of Pept1 in the intestinal mucosa. The AUCs of JBP485 in in vivo, in vitro and in situ studies were significantly decreased after zinc pre-administration. Kinetic analysis showed that zinc inhibits the uptake of JBP485 by decreasing the affinity of JBP485 for PEPT1 in HeLa-hPEPT1 cells. RT-PCR and Western blotting indicated that zinc had no effect on basal intestinal Pept1 expression. Our results are novel in demonstrating for the first time that zinc ions, but not zinc gluconate, can inhibit the transport activity of PEPT1. In addition, the uptake of JBP485 was not affected by changes in pH values after zinc treatment. Zinc decreases the absorption of JBP485 by inhibiting the transport activity of PEPT1; however, basal intestinal Pept1 expression does not change.

Preparation of the traditional Chinese medicine compound recipe Shuxiong sustained-release capsules by multiparticulate time-controlled explosion technology.

In this study the traditional Chinese medicine compound recipe (TCMCR) Shuxiong sustained-release capsules (SXSRC) were prepared by multiparticulate time-controlled explosion technology. First, Shuxiong pellets were prepared with the refined medicinal materials containing in the recipe of Shuxiong tablets. Then, the pellets were coated sequentially with an inner swelling layer containing low-substituted hydroxypropylcellulose as the swelling agent and an outer rupturable layer of ethylcellulose. Finally, SXSRC were developed by encapsulating five kinds of pellets whose respective coating level of outer layer was 0%, 9%, 15%, 18% and 20% at equivalent ratio in hard gelatin capsules. Under the simulated gastrointestinal pH conditions, the in vitro release test of SXSRC was carried out. The value of similarity factor (f2) of hydroxysafflor yellow A and Panax notoginseng saponins, hydroxysafflor yellow A and ferulic acid, Panax notoginseng saponins and ferulic acid was 90.1, 77.3, 87.0, respectively. The release profiles of these three compositions from SXSRC showed a characteristic of obvious sustained-release and no significant difference between them. The results indicated that using multiparticulate time-controlled explosion technology various components in TCMCR with vastly different physicochemical properties could be released synchronously while sustained-releasing. That complies with the organic whole conception of compound compatibility of TCMCR.