2014 Vol. 45 No. 4
2014, 45(4): 383-391.
DOI: 10.11665/j.issn.1000-5048.20140401
Abstract：
The novel incretin-based diabetic drug, glucagon like peptide-1(GLP-1), has a range of physiological effects. The widespread distribution of GLP-1 receptors also determines the pleiotropy of GLP-1 receptor agonists. Recent studies suggest that, in addition to anti-diabetic use, GLP-1 receptor agonists exert neuroprotective and anti-inflammatory effects in both central and peripheral nervous systems. Therefore, GLP-1 receptor agonists show great potential as novel preventive or therapeutic agents for neurodegenerative conditions. This article mainly reviews the recent pre-clinical and clinical studies about GLP-1 receptor agonists applied in Alzheimer′s disease, Parkinson′s disease, stroke and other neurological diseases.
The novel incretin-based diabetic drug, glucagon like peptide-1(GLP-1), has a range of physiological effects. The widespread distribution of GLP-1 receptors also determines the pleiotropy of GLP-1 receptor agonists. Recent studies suggest that, in addition to anti-diabetic use, GLP-1 receptor agonists exert neuroprotective and anti-inflammatory effects in both central and peripheral nervous systems. Therefore, GLP-1 receptor agonists show great potential as novel preventive or therapeutic agents for neurodegenerative conditions. This article mainly reviews the recent pre-clinical and clinical studies about GLP-1 receptor agonists applied in Alzheimer′s disease, Parkinson′s disease, stroke and other neurological diseases.
Abstract：
Novel hybrid compounds 7a-7p were designed and synthesized via conjugating farnesylthiosalicylic acid(FTS)and hydroxylcinnamic acids, and their in vitro biological activities were evaluated. It was found that most compounds displayed good to moderate in vitro antitumor activities against six human cancer cells. Especially, the antitumor activities of compound 7b , with the IC50 values of 5. 51-9. 25 μmol/L range against all tested cancer cells, were superior to those of FTS and sorafenib, and compound 7b could selectively inhibit the proliferation of tumor cells but not that of human normal cell. Furthermore, compound 7b significantly induced SMMC-7721 cell apoptosis in a dose-dependent manner.
Novel hybrid compounds 7a-7p were designed and synthesized via conjugating farnesylthiosalicylic acid(FTS)and hydroxylcinnamic acids, and their in vitro biological activities were evaluated. It was found that most compounds displayed good to moderate in vitro antitumor activities against six human cancer cells. Especially, the antitumor activities of compound 7b , with the IC50 values of 5. 51-9. 25 μmol/L range against all tested cancer cells, were superior to those of FTS and sorafenib, and compound 7b could selectively inhibit the proliferation of tumor cells but not that of human normal cell. Furthermore, compound 7b significantly induced SMMC-7721 cell apoptosis in a dose-dependent manner.
2014, 45(4): 400-404.
DOI: 10.11665/j.issn.1000-5048.20140403
Abstract：
As an important intermediate of quinolone antibacterial agents such as sitafloxacin and olamufloxacin, 7-(S)-tert-butoxycarbonylamino-5-azaspiro［2. 4］heptane has received much attention. Previous syntheses suffered from low yielding and poor stereoselectivity. In this study, an asymmetric synthetic approach was developed to optically pure amine-azaspiro compound with convenient manipulation, excellent yields and high stereoselectivity.
As an important intermediate of quinolone antibacterial agents such as sitafloxacin and olamufloxacin, 7-(S)-tert-butoxycarbonylamino-5-azaspiro［2. 4］heptane has received much attention. Previous syntheses suffered from low yielding and poor stereoselectivity. In this study, an asymmetric synthetic approach was developed to optically pure amine-azaspiro compound with convenient manipulation, excellent yields and high stereoselectivity.
2014, 45(4): 405-409.
DOI: 10.11665/j.issn.1000-5048.20140404
Abstract：
In this paper quantitative structure-activity relationships(QSAR)were developed on HBV DNA polymerase inhibitors to uncover the relationship between biological activity and the key structural features. The 2D QSAR model of fingerprint fragments was built using genetic function approximation(GFA)method with good internal(adjusted R-squared: 0. 911 9)and external prediction(r2pred = 0. 848 9). The features of 8 fingerprint fragments obtained are consistent with 3D pharmacophore. These fingerprints are notably more effective than those based on a fragment dictionary or hashing library. The combination of these fingerprint fragments to novel molecule library provides an effective and efficient approach to virtual screening and de novo drug design.
In this paper quantitative structure-activity relationships(QSAR)were developed on HBV DNA polymerase inhibitors to uncover the relationship between biological activity and the key structural features. The 2D QSAR model of fingerprint fragments was built using genetic function approximation(GFA)method with good internal(adjusted R-squared: 0. 911 9)and external prediction(r2pred = 0. 848 9). The features of 8 fingerprint fragments obtained are consistent with 3D pharmacophore. These fingerprints are notably more effective than those based on a fragment dictionary or hashing library. The combination of these fingerprint fragments to novel molecule library provides an effective and efficient approach to virtual screening and de novo drug design.
2014, 45(4): 410-412.
DOI: 10.11665/j.issn.1000-5048.20140405
Abstract：
A new norlignan, named sauchinolide A( 1 ), together with two known tetrahydrofuran lignans( 2 and 3 ), were isolated from the 95% EtOH extract of the whole plants of Saururus chinensis. All of them were isolated from the family Saururaceae for the first time. The effect of the obtained compounds on hypoxia/reoxygenation(H/R)injury in human endothelial EA. hy926 cells were preliminarily evaluated. The results showed that compound 2 exhibited moderate protective effect against H/R injury in EA. hy926.
A new norlignan, named sauchinolide A( 1 ), together with two known tetrahydrofuran lignans( 2 and 3 ), were isolated from the 95% EtOH extract of the whole plants of Saururus chinensis. All of them were isolated from the family Saururaceae for the first time. The effect of the obtained compounds on hypoxia/reoxygenation(H/R)injury in human endothelial EA. hy926 cells were preliminarily evaluated. The results showed that compound 2 exhibited moderate protective effect against H/R injury in EA. hy926.
2014, 45(4): 413-419.
DOI: 10.11665/j.issn.1000-5048.20140406
Abstract：
The aim of this research was to prepare and evaluate thermo-sensitive complex nano-hydrogel(HP/CN)comprising of hyaluronic acid derivative(HA-PN)and thiolated chitosan(CMCS-NAC)for the ocular delivery of cyclosporine A(CsA). The CsA complex nano-hydrogel(CsA HP/CN)was prepared by incubation, then the size, Zeta potential, low critical solution temperature(LCST)and drug-loading capacity were characterized. The rabbit eye irritation effect, tissue adhesiveness and pharmacokinetic studies were investigated to evaluate the ocular compatibility and bioavailability of CsA HP/CN formulation. CsA HP/CN was found to be spherical in shape with a mean diameter of(122. 7±17. 8)nm, the Zeta potential of -(27. 87±2. 16)mV and LCST of 28. 4 °C with a drug-loading capacity up to 15. 08%. The rabbit eye irritation experiment showed no irritation on rabbit eyes. The CsA HP/CN group displayed significantly higher adhesive effect(1. 97- fold)on eye tissue as compared to the control group(drug carrier alone)after 15 min of ocular administration. Rabbit eyes pharmacokinetic data confirmed significantly higher AUC(1. 67- fold)and MRT(5. 17- fold)compared to the control group. Therefore, CsA HP/CN with small particle size and high drug loading capacity could obviously extend the drug retention time and increase its bioavailability on eyes, thus with potential application prospect in ocular drug delivery system.
The aim of this research was to prepare and evaluate thermo-sensitive complex nano-hydrogel(HP/CN)comprising of hyaluronic acid derivative(HA-PN)and thiolated chitosan(CMCS-NAC)for the ocular delivery of cyclosporine A(CsA). The CsA complex nano-hydrogel(CsA HP/CN)was prepared by incubation, then the size, Zeta potential, low critical solution temperature(LCST)and drug-loading capacity were characterized. The rabbit eye irritation effect, tissue adhesiveness and pharmacokinetic studies were investigated to evaluate the ocular compatibility and bioavailability of CsA HP/CN formulation. CsA HP/CN was found to be spherical in shape with a mean diameter of(122. 7±17. 8)nm, the Zeta potential of -(27. 87±2. 16)mV and LCST of 28. 4 °C with a drug-loading capacity up to 15. 08%. The rabbit eye irritation experiment showed no irritation on rabbit eyes. The CsA HP/CN group displayed significantly higher adhesive effect(1. 97- fold)on eye tissue as compared to the control group(drug carrier alone)after 15 min of ocular administration. Rabbit eyes pharmacokinetic data confirmed significantly higher AUC(1. 67- fold)and MRT(5. 17- fold)compared to the control group. Therefore, CsA HP/CN with small particle size and high drug loading capacity could obviously extend the drug retention time and increase its bioavailability on eyes, thus with potential application prospect in ocular drug delivery system.
Abstract：
Hydrocaffeoylchitosan(HCAC)was synthesized and characterized with 15. 6% substitution degree of hydrocaffeic acid. Ternary coordination supermolecule HCAC-Cu-Dox was subsequently fabricated through the solvent-induced precipitation method in the presence of HCAC, Cu(NO3)2 ·3H2O and Dox. The contents of Dox and Cu were(7. 8±0. 3)% and(5. 2±0. 2)% determined by high-performance liquid chromatography(HPLC)and inductively coupled plasma-atomic emission spectrometry(ICP-AES), respectively. The supermolecule was in spherical shape with an average particle size of(68. 7±3. 9)nm observed by TEM. The binding constant of supermolecule was measured, and thermodynamic property was evaluated, indicating that Dox installed in supermolecule existed in amorphous or molecular state. Besides, in vitro release profile indicated that supermolecule possessed satisfactory stable in physiological conditions, and only 13. 5% of Dox were detected in 24 h. Furthermore, the near-infrared fluorescence(NIRF)imaging showed that the supermolecule could be specifically delivered to kidney after intravenous injection, while hardly accumulated in other organs. Cytotoxicity assay by MTT method showed that A498 cells were significantly inhibited after incubation with HCAC-Cu-Dox. In conclusion, HCAC-Cu-Dox ternary coordination supermolecule has potential for renal targeting and treatment.
Hydrocaffeoylchitosan(HCAC)was synthesized and characterized with 15. 6% substitution degree of hydrocaffeic acid. Ternary coordination supermolecule HCAC-Cu-Dox was subsequently fabricated through the solvent-induced precipitation method in the presence of HCAC, Cu(NO3)2 ·3H2O and Dox. The contents of Dox and Cu were(7. 8±0. 3)% and(5. 2±0. 2)% determined by high-performance liquid chromatography(HPLC)and inductively coupled plasma-atomic emission spectrometry(ICP-AES), respectively. The supermolecule was in spherical shape with an average particle size of(68. 7±3. 9)nm observed by TEM. The binding constant of supermolecule was measured, and thermodynamic property was evaluated, indicating that Dox installed in supermolecule existed in amorphous or molecular state. Besides, in vitro release profile indicated that supermolecule possessed satisfactory stable in physiological conditions, and only 13. 5% of Dox were detected in 24 h. Furthermore, the near-infrared fluorescence(NIRF)imaging showed that the supermolecule could be specifically delivered to kidney after intravenous injection, while hardly accumulated in other organs. Cytotoxicity assay by MTT method showed that A498 cells were significantly inhibited after incubation with HCAC-Cu-Dox. In conclusion, HCAC-Cu-Dox ternary coordination supermolecule has potential for renal targeting and treatment.
Abstract：
A coated capillary with 3-aminopropyltriethoxysilane modified magnetic nanoparticles(APTES-MNPs)as the stationary phase was constructed and applied in enantio-separation in capillary electrophoresis. The magnetic nanoparticles coating inside the capillary columns could be easily generated by applying and removing the external magnetic field. The APTES-MNPs-coated capillary showed long lifetime, superior chemical stability and good reproducibility. It endured during more than 80 replicated analyses and was tolerant to HCl(0. 01 mol/L), NaOH(0. 001 mol/L), CH3OH and CH3CN. The APTES-MNPs-coated capillary can be applied to enantioseparation of basic drugs. Compared with the uncoated capillary system, improved separation of the tested enantiomers(ofloxacin and propranolol)were obtained in the system based on APTES-MNPs-coated capillary. Several primary parameters such as chiral selector concentration, running buffer pH, organic solvent and applied voltage, were systematically optimized in order to obtain successful chiral separation. The results indicate that the application of magnetic nanoparticles is a promising way in chiral separation.
A coated capillary with 3-aminopropyltriethoxysilane modified magnetic nanoparticles(APTES-MNPs)as the stationary phase was constructed and applied in enantio-separation in capillary electrophoresis. The magnetic nanoparticles coating inside the capillary columns could be easily generated by applying and removing the external magnetic field. The APTES-MNPs-coated capillary showed long lifetime, superior chemical stability and good reproducibility. It endured during more than 80 replicated analyses and was tolerant to HCl(0. 01 mol/L), NaOH(0. 001 mol/L), CH3OH and CH3CN. The APTES-MNPs-coated capillary can be applied to enantioseparation of basic drugs. Compared with the uncoated capillary system, improved separation of the tested enantiomers(ofloxacin and propranolol)were obtained in the system based on APTES-MNPs-coated capillary. Several primary parameters such as chiral selector concentration, running buffer pH, organic solvent and applied voltage, were systematically optimized in order to obtain successful chiral separation. The results indicate that the application of magnetic nanoparticles is a promising way in chiral separation.
2014, 45(4): 434-437.
DOI: 10.11665/j.issn.1000-5048.20140409
Abstract：
A UPLC method was established for the simultaneous determination of protocatechuic acid, chlorogenic acid, pumiloside, 3-epi-pumiloside, strictosamide and vincosamide in Nauclea officinalis. The experiment was performed with an Acquity BEH C18(2. 1 mm ×100 mm, 1. 7 μm)column and the mobile phase consisting of acetonitrile and 0. 1% formic acid with gradient elution. The flow rate of 0. 4 mL/min and the injection volume of 2 μL as well as the column temperature of 40 ℃ were employed. Under the above-mentioned condition, 6 components in Nauclea officinalis were separated completely and the method was fully validated. Good linearity(R2> 0. 999 6), precision of inter- and intra-day(RSD≤4. 05%)together with average recoveries of 96. 59%-101. 8% were obtained. The method was proved to be rapid and accurate and can be used for the quality control of Nauclea officinalis.
A UPLC method was established for the simultaneous determination of protocatechuic acid, chlorogenic acid, pumiloside, 3-epi-pumiloside, strictosamide and vincosamide in Nauclea officinalis. The experiment was performed with an Acquity BEH C18(2. 1 mm ×100 mm, 1. 7 μm)column and the mobile phase consisting of acetonitrile and 0. 1% formic acid with gradient elution. The flow rate of 0. 4 mL/min and the injection volume of 2 μL as well as the column temperature of 40 ℃ were employed. Under the above-mentioned condition, 6 components in Nauclea officinalis were separated completely and the method was fully validated. Good linearity(R2> 0. 999 6), precision of inter- and intra-day(RSD≤4. 05%)together with average recoveries of 96. 59%-101. 8% were obtained. The method was proved to be rapid and accurate and can be used for the quality control of Nauclea officinalis.
Abstract：
A specific ion-pairing HPLC method was developed for the determination of related substances in atropine sulfate. Their structures were identified by LC-MS/MS. HPLC separation was carried out on a Thermo BDS Hypersil C18 column with linear gradient elution by a mobile phase consisting of acetonitrile and phosphate buffer solution(containing sodium heptane-sulphonate, adjusting pH to 5. 0 with phosphoric acid). The flow rate was 0. 8 mL/min and UV detection wavelength was 225 nm. Atropine sulfate and its related substances can be completely separated under the established HPLC condition. And they were characterized by hyphenated chromatographic techniques with accurate mass determination using high resolution LC-TOF/MS methods as well as product MS spectra scanning through a volatile mobile phase. Twelve related substances were detected and identified as by-products from synthesis and degradations of atropine sulfate. Six of them were related substances from EP and the other six were unknown related substances. This newly established HPLC method is effective in the separation and identification of the related substances in atropine sulfate and the results are useful for its quality control and process optimization.
A specific ion-pairing HPLC method was developed for the determination of related substances in atropine sulfate. Their structures were identified by LC-MS/MS. HPLC separation was carried out on a Thermo BDS Hypersil C18 column with linear gradient elution by a mobile phase consisting of acetonitrile and phosphate buffer solution(containing sodium heptane-sulphonate, adjusting pH to 5. 0 with phosphoric acid). The flow rate was 0. 8 mL/min and UV detection wavelength was 225 nm. Atropine sulfate and its related substances can be completely separated under the established HPLC condition. And they were characterized by hyphenated chromatographic techniques with accurate mass determination using high resolution LC-TOF/MS methods as well as product MS spectra scanning through a volatile mobile phase. Twelve related substances were detected and identified as by-products from synthesis and degradations of atropine sulfate. Six of them were related substances from EP and the other six were unknown related substances. This newly established HPLC method is effective in the separation and identification of the related substances in atropine sulfate and the results are useful for its quality control and process optimization.
2014, 45(4): 444-449.
DOI: 10.11665/j.issn.1000-5048.20140411
Abstract：
An affinity capillary electrophoresis(ACE)method with bovine serum albumin(BSA)as chiral selector was developed to test the enantiomeric impurity in esomeprazole. The mechanism of enantioseparation of esomeprazole and its enantiomer via BSA was investigated by fluorescence and circular dichroism spectrum for thermodynamic parameters, enthalpy entropy-driven process and the conformational changes of BSA. The optimal separation condition obtained was a running buffer consisting of 20 mmol/L sodium dihydrogen phosphate and disodium hydrogen phosphate with 250 μmol/L BSA and 7% n-propanol at pH 8. 0, a positive voltage of 30 kV, separation temperature at 25 ℃ and detection wavelength at 302 nm. The resolution of esomeprazole and its enantiomer was more than 1. 8. The LOD and LOQ were 0. 8 μg/mL and 2. 0 μg/mL respectively, for the enantiomer of esomeprazole, and the linear range was 2-20 μg/mL. The average recovery of R-omeprazole was 100. 4%(RSD< 2. 0%). This method could be applied to test the enantiomeric impurity of esomeprazole drug substances.
An affinity capillary electrophoresis(ACE)method with bovine serum albumin(BSA)as chiral selector was developed to test the enantiomeric impurity in esomeprazole. The mechanism of enantioseparation of esomeprazole and its enantiomer via BSA was investigated by fluorescence and circular dichroism spectrum for thermodynamic parameters, enthalpy entropy-driven process and the conformational changes of BSA. The optimal separation condition obtained was a running buffer consisting of 20 mmol/L sodium dihydrogen phosphate and disodium hydrogen phosphate with 250 μmol/L BSA and 7% n-propanol at pH 8. 0, a positive voltage of 30 kV, separation temperature at 25 ℃ and detection wavelength at 302 nm. The resolution of esomeprazole and its enantiomer was more than 1. 8. The LOD and LOQ were 0. 8 μg/mL and 2. 0 μg/mL respectively, for the enantiomer of esomeprazole, and the linear range was 2-20 μg/mL. The average recovery of R-omeprazole was 100. 4%(RSD< 2. 0%). This method could be applied to test the enantiomeric impurity of esomeprazole drug substances.
2014, 45(4): 450-455.
DOI: 10.11665/j.issn.1000-5048.20140412
Abstract：
Felodipine has been widely coadministered with simvastatin for the treatment of cardiovascular disease(CVD). However, in vitro and in vivo interactions between them have not been systemically understood. Herein, the effects of simvastatin on the in vitro metabolism and oral pharmacokinetic profile of felodipine were investigated. Felodipine was incubated in rat liver microsomes(RLMs)with or without simvastatin. Simvastatin was found to be a non-competitive inhibitor(inhibition constant(Ki): (9. 86±0. 27)μmol/L). Following oral combination of felodipine and simvastatin, the peak plasma concentration(cmax)and area under the plasma concentration-time curve(AUC0-∞)of felodipine increased significantly, and the clearance(CLz/F)decreased obviously, while the mean residence time(MRT0-∞)and biological half-life(t1/2)were not statistically different. The altered pharmacokinetic profile of felodipine was probably due to the promoting effect of simvastatin on felodipine absorption. These findings provide some useful information for possible drug-drug interactions(DDIs)in rats, which should be paid particular attention to when felodipine and simvastatin are coadministered in clinical.
Felodipine has been widely coadministered with simvastatin for the treatment of cardiovascular disease(CVD). However, in vitro and in vivo interactions between them have not been systemically understood. Herein, the effects of simvastatin on the in vitro metabolism and oral pharmacokinetic profile of felodipine were investigated. Felodipine was incubated in rat liver microsomes(RLMs)with or without simvastatin. Simvastatin was found to be a non-competitive inhibitor(inhibition constant(Ki): (9. 86±0. 27)μmol/L). Following oral combination of felodipine and simvastatin, the peak plasma concentration(cmax)and area under the plasma concentration-time curve(AUC0-∞)of felodipine increased significantly, and the clearance(CLz/F)decreased obviously, while the mean residence time(MRT0-∞)and biological half-life(t1/2)were not statistically different. The altered pharmacokinetic profile of felodipine was probably due to the promoting effect of simvastatin on felodipine absorption. These findings provide some useful information for possible drug-drug interactions(DDIs)in rats, which should be paid particular attention to when felodipine and simvastatin are coadministered in clinical.
2014, 45(4): 456-461.
DOI: 10.11665/j.issn.1000-5048.20140413
Abstract：
To investigate the anti-metastasis effect of wogonin on human alveolar adenocarcinoma cell A549 induced by IL-6 and its potential mechanism, the growth inhibition effect of wogonin by MTT assay and the anti-metastasis effect of wogonin by transwell invasion assay were investigated. The immunofluorescence and Real-time PCR assay were employed to observe wogonin regulating metastasis marker protein E-cadherin, N-cadherin, Vimentin protein and mRNA level expression. Snail and Twist mRNA level expression and transcription factors of metastasis were also detected. In order to explore the potential mechanism of wogonin, PcDNA3. 1-Flag-STAT3 was transfected into A549 to detect metastasis marker protein mRNA level expression. Data showed that wogonin inhibited A549 cell invasion and metastasis, and regulated metastasis-related transcription factors and metastasis marker protein expression in a dose-dependent manner. In conclusion, all these results showed that wogonin can suppress A549 cells invasion andmetastasis induced by IL-6. It is possible that wogonin inhibits A549 cell invasion and metastasis by regulating the activity of transcription factor Snail and Twist.
To investigate the anti-metastasis effect of wogonin on human alveolar adenocarcinoma cell A549 induced by IL-6 and its potential mechanism, the growth inhibition effect of wogonin by MTT assay and the anti-metastasis effect of wogonin by transwell invasion assay were investigated. The immunofluorescence and Real-time PCR assay were employed to observe wogonin regulating metastasis marker protein E-cadherin, N-cadherin, Vimentin protein and mRNA level expression. Snail and Twist mRNA level expression and transcription factors of metastasis were also detected. In order to explore the potential mechanism of wogonin, PcDNA3. 1-Flag-STAT3 was transfected into A549 to detect metastasis marker protein mRNA level expression. Data showed that wogonin inhibited A549 cell invasion and metastasis, and regulated metastasis-related transcription factors and metastasis marker protein expression in a dose-dependent manner. In conclusion, all these results showed that wogonin can suppress A549 cells invasion andmetastasis induced by IL-6. It is possible that wogonin inhibits A549 cell invasion and metastasis by regulating the activity of transcription factor Snail and Twist.
2014, 45(4): 462-468.
DOI: 10.11665/j.issn.1000-5048.20140414
Abstract：
The aim of the study was to evaluate and explore the inhibition effect of emodin on the adriamycin-resistance in K562/ADM cell line. In this study, verapamil and adriamycin were used as positive drug and tool drug, respectively. Cell viability was measured by MTT. The effect of emodin combined with adriamycin on cell cycle and apoptosis was determined by flowcytometry. The protein expression of P-gp, Bcr-Abl and STAT5 was analyzed by Western blot. Emodin inducing conformational change of P-gp with UIC2 antibody was detected by FCM. The results showed that emodin dramatically enhanced the cytotoxic effect of adriamycin in a concentration-dependent manner. Emodin promoted cell cycle arrest in G2/M phase and adriamycin-induced apoptosis. It down-regulated the protein expression levels of P-gp, Bcr-Abl and STAT5. The results of the UIC2 binding experiment showed that emodin may not be the substrate of P-gp. It could effectively inhibit adriamycin-resistance in vivo. The mechanism of emodin inhibiting drug-resistance of K562/ADM may be related to the decrease of P-gp, Bcr-Abl and STAT5 protein expression, instead of competitive inhibition of P-gp.
The aim of the study was to evaluate and explore the inhibition effect of emodin on the adriamycin-resistance in K562/ADM cell line. In this study, verapamil and adriamycin were used as positive drug and tool drug, respectively. Cell viability was measured by MTT. The effect of emodin combined with adriamycin on cell cycle and apoptosis was determined by flowcytometry. The protein expression of P-gp, Bcr-Abl and STAT5 was analyzed by Western blot. Emodin inducing conformational change of P-gp with UIC2 antibody was detected by FCM. The results showed that emodin dramatically enhanced the cytotoxic effect of adriamycin in a concentration-dependent manner. Emodin promoted cell cycle arrest in G2/M phase and adriamycin-induced apoptosis. It down-regulated the protein expression levels of P-gp, Bcr-Abl and STAT5. The results of the UIC2 binding experiment showed that emodin may not be the substrate of P-gp. It could effectively inhibit adriamycin-resistance in vivo. The mechanism of emodin inhibiting drug-resistance of K562/ADM may be related to the decrease of P-gp, Bcr-Abl and STAT5 protein expression, instead of competitive inhibition of P-gp.
2014, 45(4): 469-474.
DOI: 10.11665/j.issn.1000-5048.20140415
Abstract：
To evaluate the hepatotoxicity and its mechanism of the peptide-rich fraction(Fr-2)of the root of Aster tataricus in human liver L-02 cells. Cell viability was assessed by MTT assay and the contents of LDH, ROS, GSH, cytochrome C and Caspase-9/3 were assessed by commercially available kits. Cell apoptosis and mitochondrial membrane potential were measured by flow cytometry. The protein expressions of p-JNK, Bax, Bcl-2 were analysed by Western blot assay. The results demonstrated that the Fr-2 fraction induced apoptosis in a concentration- and time-dependent manner and provoked oxidative stress-associated inflammation in L-02 cells. It was characterized by loss of mitochondrial membrane potential and release of cytochrome c from mitochondria. The enhanced Bax/Bcl-2 ratio and the activated Caspase-9/3 indicated that the Fr-2 fraction induces apoptosis via mitochondria-dependent pathway in liver L-02 cells.
To evaluate the hepatotoxicity and its mechanism of the peptide-rich fraction(Fr-2)of the root of Aster tataricus in human liver L-02 cells. Cell viability was assessed by MTT assay and the contents of LDH, ROS, GSH, cytochrome C and Caspase-9/3 were assessed by commercially available kits. Cell apoptosis and mitochondrial membrane potential were measured by flow cytometry. The protein expressions of p-JNK, Bax, Bcl-2 were analysed by Western blot assay. The results demonstrated that the Fr-2 fraction induced apoptosis in a concentration- and time-dependent manner and provoked oxidative stress-associated inflammation in L-02 cells. It was characterized by loss of mitochondrial membrane potential and release of cytochrome c from mitochondria. The enhanced Bax/Bcl-2 ratio and the activated Caspase-9/3 indicated that the Fr-2 fraction induces apoptosis via mitochondria-dependent pathway in liver L-02 cells.
2014, 45(4): 475-479.
DOI: 10.11665/j.issn.1000-5048.20140416
Abstract：
The in-situ recirculating rat perfused liver model in our lab was taken as an example. A set of methods were introduced for evaluating the viability of isolated liver perfusion. Firstly, a series of measurements including gross appearance of the liver, pressure of portal vein, pH of perfusate, concentration of potassium, levels of cytosolic enzymes and morphology were suggested to provide an adequate general assessment of viability. The liver should be a homogeneous, pinkish-brown color during perfusion; white spots caused by air emboli and red spots due to nonhomogeneous perfusion should be absent. The pressure of portal vein should not go beyond 8-14 mmHg or raise more than 0. 5 mmHg/30 min. The pH of perfusate should not fluctuate more than 0. 1/30 min and be 7. 4-7. 2 all the time. Sudden increase in perfusate potassium was forbidden. Significant changes of ALT(alanine aminotransferase)and AST(aspartate aminotransaminase)levels along with the perfusion time were not allowed. The perfused liver should show no other pathological changes except vacuolization after the whole perfusion. Secondly, further observation and examination of functional applicability were extremely necessary for different study purposes; obvious differences of potential markers and indexes between the group perfused with K-H perfusate only and that with medicine-contained perfusate were needed. In this paper, the isolated perfused liver model was used for studying drug-induced liver injury. Significant differences of perfusate ALT, AST and LDH(lactate dehydrogenase)levels, the damage markers, between the control and isoniazid groups were observed. Morphological analysis of the perfused liver in isoniazid group showed more serious vacuolization than the control group with slight necrosis, which means qualified functional viability for liver injury research.
The in-situ recirculating rat perfused liver model in our lab was taken as an example. A set of methods were introduced for evaluating the viability of isolated liver perfusion. Firstly, a series of measurements including gross appearance of the liver, pressure of portal vein, pH of perfusate, concentration of potassium, levels of cytosolic enzymes and morphology were suggested to provide an adequate general assessment of viability. The liver should be a homogeneous, pinkish-brown color during perfusion; white spots caused by air emboli and red spots due to nonhomogeneous perfusion should be absent. The pressure of portal vein should not go beyond 8-14 mmHg or raise more than 0. 5 mmHg/30 min. The pH of perfusate should not fluctuate more than 0. 1/30 min and be 7. 4-7. 2 all the time. Sudden increase in perfusate potassium was forbidden. Significant changes of ALT(alanine aminotransferase)and AST(aspartate aminotransaminase)levels along with the perfusion time were not allowed. The perfused liver should show no other pathological changes except vacuolization after the whole perfusion. Secondly, further observation and examination of functional applicability were extremely necessary for different study purposes; obvious differences of potential markers and indexes between the group perfused with K-H perfusate only and that with medicine-contained perfusate were needed. In this paper, the isolated perfused liver model was used for studying drug-induced liver injury. Significant differences of perfusate ALT, AST and LDH(lactate dehydrogenase)levels, the damage markers, between the control and isoniazid groups were observed. Morphological analysis of the perfused liver in isoniazid group showed more serious vacuolization than the control group with slight necrosis, which means qualified functional viability for liver injury research.
2014, 45(4): 480-485.
DOI: 10.11665/j.issn.1000-5048.20140417
Abstract：
In traditional Chinese medicine, toad skin-origin materials including toad skin and its secretions have long been used to treat inflammation, sores and even many kinds of cancers. To clarify the polypeptides included in these toad-origin materials, screening of cDNAs encoding polypeptides was carried out from the plasmid skin cDNA library of Japanese toad(Bufo japonicus formosus)by colony polymerase chain reaction(PCR). As the result, a transcript of 958 bp(GenBank accession number: KF769458)was obtained consisting of 13 bp 5′ untranslated region(UTR), 498 bp 3′ UTR and an open reading frame(ORF)of 447 bp encoding a polypeptide of 148 amino acid residues with high homology with endothelial differentiation-related factor-1(EDF-1)of other animals. Based on the EDF-1 cDNA sequence of Japanese toad, a pair of primers were designed for cloning Chinese toad(B. gargarizans) EDF-1 and a DNA segment was obtained with an ORF of 447 bp(GenBank accession number: KF769459)encoding a polypeptide of 148 amino acids, among which three amino acid residues were different from that of Japanese toad. For two Bufo species, the similarity is 95% with human EDF-1 and 93%-97% with other animals indicating that EDF-1 is evolutionarily highly conserved. Besides, phosphorylation site prediction indicated 9 potential sites suggesting its activity being upstream regulated which is consistent with previous reports. Due to the involement in the process of endothelial cell differentiation, EDF-1 expressed in toad skin might be one of the important ingredients with antitumor effects.
In traditional Chinese medicine, toad skin-origin materials including toad skin and its secretions have long been used to treat inflammation, sores and even many kinds of cancers. To clarify the polypeptides included in these toad-origin materials, screening of cDNAs encoding polypeptides was carried out from the plasmid skin cDNA library of Japanese toad(Bufo japonicus formosus)by colony polymerase chain reaction(PCR). As the result, a transcript of 958 bp(GenBank accession number: KF769458)was obtained consisting of 13 bp 5′ untranslated region(UTR), 498 bp 3′ UTR and an open reading frame(ORF)of 447 bp encoding a polypeptide of 148 amino acid residues with high homology with endothelial differentiation-related factor-1(EDF-1)of other animals. Based on the EDF-1 cDNA sequence of Japanese toad, a pair of primers were designed for cloning Chinese toad(B. gargarizans) EDF-1 and a DNA segment was obtained with an ORF of 447 bp(GenBank accession number: KF769459)encoding a polypeptide of 148 amino acids, among which three amino acid residues were different from that of Japanese toad. For two Bufo species, the similarity is 95% with human EDF-1 and 93%-97% with other animals indicating that EDF-1 is evolutionarily highly conserved. Besides, phosphorylation site prediction indicated 9 potential sites suggesting its activity being upstream regulated which is consistent with previous reports. Due to the involement in the process of endothelial cell differentiation, EDF-1 expressed in toad skin might be one of the important ingredients with antitumor effects.
2014, 45(4): 486-490.
DOI: 10.11665/j.issn.1000-5048.20140418
Abstract：
The objective of the present study was to develop a duplex real-time reverse transcription polymerase chain reaction(rRT-PCR)assay for the simultaneous detection of both hemagglutinin(HA)and neuraminidase(NA)genes of H9N2 avian influenza viruses. In order to design primers and probes for the detection of HA and NA genes of avian influenza virus subtype H9N2, current available HA and NA gene sequence of avian influenza virus subtype H9N2 from GenBank were aligned, and two different fluorescein(FAM and JOE)were used to label HA and NA probe, respectively. A one-step duplex Taq Man rRT-PCR assay, which could detect both HA and NA genes of the avian influenza virus subtype H9N2 simultaneously was then established. The amplification curve showed that the duplex rRT-PCR assay had a very good amplification efficiency. The specificity detection showed that the duplex rRT-PCR assay could identify HA and NA gene of the avian influenza virus subtype H9N2 specifically, with no cross reactivity being observed with other influenza virus subtypes or respiratory tract viruses. The sensitivity of the duplex rRT-PCR assay was determined to be 10 RNA copies per reaction for both HA and NA genes. Sixty H9N2-infected mice and sixty environmental specimens were tested and compared with conventional PCR. the sensitivity of the duplex rRT-PCR was 100-fold higher than conventional PCR. The results of the duplex rRT-PCR were completely consistent with those of virus isolation. It was concluded that the duplex rRT-PCR assay established in this study was a highly specific and sensitive method that could be used for the diagnosis of avian influenza virus subtype H9N2.
The objective of the present study was to develop a duplex real-time reverse transcription polymerase chain reaction(rRT-PCR)assay for the simultaneous detection of both hemagglutinin(HA)and neuraminidase(NA)genes of H9N2 avian influenza viruses. In order to design primers and probes for the detection of HA and NA genes of avian influenza virus subtype H9N2, current available HA and NA gene sequence of avian influenza virus subtype H9N2 from GenBank were aligned, and two different fluorescein(FAM and JOE)were used to label HA and NA probe, respectively. A one-step duplex Taq Man rRT-PCR assay, which could detect both HA and NA genes of the avian influenza virus subtype H9N2 simultaneously was then established. The amplification curve showed that the duplex rRT-PCR assay had a very good amplification efficiency. The specificity detection showed that the duplex rRT-PCR assay could identify HA and NA gene of the avian influenza virus subtype H9N2 specifically, with no cross reactivity being observed with other influenza virus subtypes or respiratory tract viruses. The sensitivity of the duplex rRT-PCR assay was determined to be 10 RNA copies per reaction for both HA and NA genes. Sixty H9N2-infected mice and sixty environmental specimens were tested and compared with conventional PCR. the sensitivity of the duplex rRT-PCR was 100-fold higher than conventional PCR. The results of the duplex rRT-PCR were completely consistent with those of virus isolation. It was concluded that the duplex rRT-PCR assay established in this study was a highly specific and sensitive method that could be used for the diagnosis of avian influenza virus subtype H9N2.
2014, 45(4): 491-495.
DOI: 10.11665/j.issn.1000-5048.20140419
Abstract：
Maculosin is a kind of cyclic dipeptides resulting from double condensations between L-proline and L-tyrosine. It was purified from the fermentation broth of strain CGD12, an Aspergillus fungi isolated from Rhizophora stylosa rhizosphere soil in the previous study. In the present study, lactate dehydrogenase(LDH)assay was applied to detect the cytotoxicity of Maculosin in lung fibroblasts. Real-time quantitative RT-PCR was used to assess its effects on the fibrotic-related gene expression. The results showed that Maculosin inhibits the activation of lung fibroblasts and attenuates the accumulation of extracellular matrix(ECM)induced by exogenous TGF-β2. Further study suggested that the anti-fibrotic activity of Maculosin may be related to repressing the expression of inflammatory cytokines and promoting the process of ECM degradation.
Maculosin is a kind of cyclic dipeptides resulting from double condensations between L-proline and L-tyrosine. It was purified from the fermentation broth of strain CGD12, an Aspergillus fungi isolated from Rhizophora stylosa rhizosphere soil in the previous study. In the present study, lactate dehydrogenase(LDH)assay was applied to detect the cytotoxicity of Maculosin in lung fibroblasts. Real-time quantitative RT-PCR was used to assess its effects on the fibrotic-related gene expression. The results showed that Maculosin inhibits the activation of lung fibroblasts and attenuates the accumulation of extracellular matrix(ECM)induced by exogenous TGF-β2. Further study suggested that the anti-fibrotic activity of Maculosin may be related to repressing the expression of inflammatory cytokines and promoting the process of ECM degradation.
2014, 45(4): 496-503.
DOI: 10.11665/j.issn.1000-5048.20140420
Abstract：
Aspirin is a widely used anti-platelet drug. However, patients continue to experience atherothromboembolic events despite aspirin therapy, which is known as “aspirin resistance(AR)”. Besides the traditional explanation from COX-1—dependent and COX-1—independent mechanism of aspirin resistance, the mechanism under physiological and pathological conditions related to aspirin resistance has been made further progress recently. This review provides a general overview regarding current knowledge about aspirin resistance from the perspective of the pharmacokinetic and pharmacodynamic mechanism under physiological and pathological conditions, respectively. These advances could guide future research toward aspirin resistance and provides potential strategies to improve aspirin efficacy.
Aspirin is a widely used anti-platelet drug. However, patients continue to experience atherothromboembolic events despite aspirin therapy, which is known as “aspirin resistance(AR)”. Besides the traditional explanation from COX-1—dependent and COX-1—independent mechanism of aspirin resistance, the mechanism under physiological and pathological conditions related to aspirin resistance has been made further progress recently. This review provides a general overview regarding current knowledge about aspirin resistance from the perspective of the pharmacokinetic and pharmacodynamic mechanism under physiological and pathological conditions, respectively. These advances could guide future research toward aspirin resistance and provides potential strategies to improve aspirin efficacy.
2014, 45(4): 504-510.
DOI: 10.11665/j.issn.1000-5048.20140421
Abstract：
In recent years, techeniqus as zinc-finger nucleases(ZFNs), transcription activator-like effector nucleases(TALENs)and clustered regulatory interspaced short palindromic repeats CRISPR/Cas-based RNA-guided DNA endonucleases(CRISPR/Cas)have been applied for genomics editing scale. According to their high efficiency and customizable possibility, such techeniques have a significant influence upon the following research aspects, gene therapy, cell model, protein glycoengineer technology, cell engineering technology, and ect. , which have also been promoting several novel strategies and study methods involved rapidly. This review summerizes the development of genome editing technology and its practical application inside the field of gene therapy and biophamarceutical industry.
In recent years, techeniqus as zinc-finger nucleases(ZFNs), transcription activator-like effector nucleases(TALENs)and clustered regulatory interspaced short palindromic repeats CRISPR/Cas-based RNA-guided DNA endonucleases(CRISPR/Cas)have been applied for genomics editing scale. According to their high efficiency and customizable possibility, such techeniques have a significant influence upon the following research aspects, gene therapy, cell model, protein glycoengineer technology, cell engineering technology, and ect. , which have also been promoting several novel strategies and study methods involved rapidly. This review summerizes the development of genome editing technology and its practical application inside the field of gene therapy and biophamarceutical industry.
