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1.
Anal Chem ; 96(25): 10380-10390, 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38860916

ABSTRACT

To reduce the risk of atherosclerotic disease, it is necessary to not only diagnose the presence of atherosclerotic plaques but also assess the vulnerability risk of plaques. Accurate detection of the reactive oxygen species (ROS) level at plaque sites represents a reliable way to assess the plaque vulnerability. Herein, through a simple one-pot reaction, two near-infrared (NIR) fluorescent dyes, one is ROS responsive and the other is inert to ROS, are coassembled in an amphiphilic amino acid-assembled nanoparticle. In the prepared NIR fluorescent amino acid nanoparticle (named FANP), the fluorescent properties and ROS-responsive behaviors of the two fluorescent dyes are well maintained. Surface camouflage through red blood cell membrane (RBCM) encapsulation endows the finally obtained FANP@RBCM nanoprobe with not only further reduced cytotoxicity and improved biocompatibility but also increased immune escape capability, prolonged blood circulation time, and thus enhanced accumulation at atherosclerotic plaque sites. In vitro and in vivo experiments demonstrate that FANP@RBCM not only works well in probing the occurrence of atherosclerotic plaques but also enables plaque vulnerability assessment through the accurate detection of the ROS level at plaque sites in a reliable ratiometric mode, thereby holding great promise as a versatile tool for the diagnosis and risk assessment of atherosclerotic disease.


Subject(s)
Amino Acids , Fluorescent Dyes , Nanoparticles , Plaque, Atherosclerotic , Reactive Oxygen Species , Plaque, Atherosclerotic/diagnostic imaging , Animals , Reactive Oxygen Species/metabolism , Fluorescent Dyes/chemistry , Nanoparticles/chemistry , Mice , Amino Acids/chemistry , Humans , Risk Assessment , Optical Imaging , Infrared Rays , RAW 264.7 Cells
2.
Anal Chem ; 96(16): 6426-6435, 2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38604773

ABSTRACT

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.


Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , MicroRNAs , CRISPR-Cas Systems/genetics , Biosensing Techniques/methods , Humans , MicroRNAs/analysis , MicroRNAs/metabolism , Allosteric Regulation , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , HEK293 Cells
3.
Anal Chem ; 96(6): 2692-2701, 2024 02 13.
Article in English | MEDLINE | ID: mdl-38305871

ABSTRACT

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.


Subject(s)
Biosensing Techniques , Nucleic Acids , CRISPR-Cas Systems/genetics , Survivin/genetics , Biomarkers , Point-of-Care Testing
4.
Anal Chem ; 95(42): 15725-15735, 2023 10 24.
Article in English | MEDLINE | ID: mdl-37819747

ABSTRACT

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 µM, and 8.3 × 10-5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.


Subject(s)
Biosensing Techniques , Nucleic Acids , CRISPR-Cas Systems/genetics , Biological Assay , Image Processing, Computer-Assisted , Oligonucleotides
5.
Anal Chem ; 92(20): 13936-13944, 2020 10 20.
Article in English | MEDLINE | ID: mdl-32972135

ABSTRACT

The fat mass and obesity-associated enzyme (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) residues in mRNA, regulates the cellular level of m6A modification, and plays a critical role in human obesity and cancers. Herein, we develop a single-quantum-dot (QD)-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m6A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the QD surface, enabling the occurrence of FRET between the QD and Cy5. The presence of inhibitors can inhibit the FTO demethylation and consequently abolish FRET between the QD and Cy5. The inhibition effect of inhibitors upon FTO demethylation can be simply evaluated by monitoring the decrease of Cy5 counts. We use this nanosensor to screen several small-molecule inhibitors and identify diacerein as a highly selective inhibitor of FTO. Diacerein can inhibit the demethylation activity of endogenous FTO in HeLa cells. Interestingly, diacerein is neither a structural mimic of 2-oxoglutarate (2-OG) nor a chelator of metal ions, and it can selectively inhibit FTO demethylation by competitively binding the m6A-containing substrate.


Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO/antagonists & inhibitors , Biosensing Techniques/methods , Enzyme Inhibitors/chemistry , Fluorescence Resonance Energy Transfer , Quantum Dots/chemistry , Adenosine/analogs & derivatives , Adenosine/chemistry , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Anthraquinones/chemistry , Anthraquinones/metabolism , Binding Sites , Carbocyanines/chemistry , Catalytic Domain , DNA Demethylation , Enzyme Inhibitors/metabolism , HeLa Cells , Humans , Molecular Dynamics Simulation , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism
6.
Chem Commun (Camb) ; 56(69): 10083, 2020 08 27.
Article in English | MEDLINE | ID: mdl-32812579

ABSTRACT

Correction for 'Development of a bidirectional isothermal amplification strategy for the sensitive detection of transcription factors in cancer cells' by Yan Zhang et al., Chem. Commun., 2020, 56, 8952-8955, DOI: 10.1039/D0CC03134H.

7.
Chem Commun (Camb) ; 56(63): 8952-8955, 2020 Aug 14.
Article in English | MEDLINE | ID: mdl-32638710

ABSTRACT

We developed a new strategy to sensitively detect transcription factors (TFs) based on the integration of a bidirectional isothermal exponential amplification reaction (EXPAR) with endonuclease IV (endo IV)-assisted cycle digestion of signal probes. This assay exhibits ultrahigh sensitivity with a detection limit of 1.29 × 10-14 M, and it can measure endogenous NF-κB p50 in HeLa cell extracts. Moreover, this strategy can be applied to screen TF inhibitors and detect other TFs by simply changing the TF-binding sequence.


Subject(s)
NF-kappa B p50 Subunit/analysis , Nucleic Acid Amplification Techniques/methods , Deoxyribonuclease IV (Phage T4-Induced)/metabolism , HeLa Cells , Humans , Limit of Detection , Spectrometry, Fluorescence
8.
Chem Commun (Camb) ; 54(51): 6991-6994, 2018 Jun 21.
Article in English | MEDLINE | ID: mdl-29796498

ABSTRACT

We develop a label-free and high-throughput bioluminescence method for the sensitive detection of uracil DNA glycosylase (UDG) through enzyme-mediated tricyclic cascade signal amplification. This method exhibits high sensitivity with a detection limit as low as 0.00031 U mL-1, and it can be further applied for the measurement of enzyme kinetic parameters and the screening of UDG inhibitors as well as cancer cell analysis.


Subject(s)
Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Luminescent Measurements/methods , Uracil-DNA Glycosidase/analysis , Uterine Cervical Neoplasms/enzymology , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Female , HeLa Cells , Humans , Kinetics , Uracil-DNA Glycosidase/antagonists & inhibitors , Uracil-DNA Glycosidase/metabolism , Uterine Cervical Neoplasms/diagnosis
9.
Nan Fang Yi Ke Da Xue Xue Bao ; 37(11): 1489-1493, 2017 Nov 20.
Article in Chinese | MEDLINE | ID: mdl-29180329

ABSTRACT

OBJECTIVE: To study the effect of aldosterone on cell proliferation, alkaline phosphatase (AKP) activity and osteogenic gene expression in rat osteoblasts and explore the mechanisms. METHODS: Osteoblasts isolated from the skull of neonatal SD rats by enzyme digestion were cultured and treated with different concentrations of aldosterone. The cell proliferation and AKP activity were evaluated using CCK-8 assay kit and AKP assay kit, respectively. The effects of aldosterone on mRNA and protein expressions of the osteogenic genes and epithelial sodium channel (ENaC) gene were investigated using semi-quantitative PCR and Western blotting. RESULTS: Compared with the control cells, the cells treated with 0.01-1.0 µmol/L aldosterone showed obviously enhanced proliferation while lower (1×10-3 µmol/L) or higher (10 µmol/L) concentrations of aldosterone did not significantly affect the cell proliferation. Aldosterone within the concentration range of 1×10-3 to 10 µmol/L did not cause significant changes in AKP activity in the osteoblasts. Treatment with 0.01 to 1.0 µmol/L aldosterone significantly upregulated the expressions of the osteogenic genes and α-ENaC gene at both the mRNA and protein levels. CONCLUSION: Aldosterone within the concentration range of 0.01-1.0 µmol/L stimulates the proliferation and osteogenic gene expressions and enhances α-ENaC gene expression in rat osteoblasts in vitro, suggesting the possibility that ENaC participates in aldosterone-mediated regulation of osteoblast functions.


Subject(s)
Aldosterone/pharmacology , Alkaline Phosphatase/metabolism , Epithelial Sodium Channels/metabolism , Osteoblasts/drug effects , Alkaline Phosphatase/genetics , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Epithelial Sodium Channels/genetics , Gene Expression , Osteoblasts/cytology , Rats , Rats, Sprague-Dawley
10.
Zhongguo Zhong Yao Za Zhi ; 42(2): 280-284, 2017 Jan.
Article in Chinese | MEDLINE | ID: mdl-28948731

ABSTRACT

To determine the optimum aqueous extract protocol for Yugubao traditional Chinese medicines formula by using orthogonal experiment design. Through serum pharmacology research, L9(34) orthogonal design with single factor investigation was used to optimize the aqueous extract protocol for Yugubao formula. The effect of water extraction on activity of alkaline phosphatase (ALP) in osteoblast was referred as the evaluation index for investigating four factors: water consumption (A), heating time (B), soaking time (C), and number of decocting (D), analyzing the optimum extraction conditions, and verifying the effectiveness of this process. The optimum aqueous extract protocol for Yugubao was as follows: adding 8 times water into Chinese medical materials, heating for 60 min, soaking for 30 min, and decocting for 1 time. The drug serum of this aqueous extract of Yugubao could significantly up-regulate the osteogenic genes expression. The optimum aqueous extract protocol for Yugubao formula was established in this experiment, providing evidence for the development and utilization of Yugubao traditional Chinese medicines formula.


Subject(s)
Chemical Fractionation/methods , Drugs, Chinese Herbal/chemistry , Plant Extracts/chemistry , Medicine, Chinese Traditional , Research Design , Technology, Pharmaceutical , Water
11.
J Asian Nat Prod Res ; 19(2): 176-187, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28024413

ABSTRACT

Ferulic acid (FA) is an active component of the traditional Chinese herb Angelica sinensis. Numerous health benefits have been attributed to FA, but few studies have investigated the effects of FA on osteoblasts (Obs). Our work studied the effects of FA on proliferation, differentiation, and mineralization of rat calvarial Obs and examined the signaling pathways involved. Cell proliferation and differentiation were evaluated by Cell Counting Kit-8 (CCK-8) and alkaline phosphatase (ALP) assay kit, respectively. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKGII) expression was silenced by small interfering RNA (siRNA). The mRNA expression was investigated by semi-quantitative PCR. FA (40-2560 µM) promoted Ob proliferation and differentiation; at 40-640 µM, FA stimulated calcified nodule formation and increased the expression of osteogenic genes encoding osteopontin and collagen-l. FA (40-2560 µM) increased cGMP levels in Obs and upregulated the expression of PKGII, EnaCα, and ENaCγ mRNAs. Downregulated ENaCα mRNA expression in Obs transfected with the siRNA for PKGII was reversed when FA was introduced into Obs. These results demonstrated that FA promoted proliferation, differentiation, and mineralization of Obs in vitro, and enhanced osteogenic genes expression partly through the cGMP-PKGII-ENaC signaling pathway.


Subject(s)
Cell Proliferation/drug effects , Coumaric Acids/pharmacology , Osteoblasts/drug effects , Alkaline Phosphatase/metabolism , Animals , Cell Differentiation , Cyclic GMP/metabolism , Molecular Structure , Osteogenesis/drug effects , Osteopontin , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Rats , Signal Transduction/drug effects
12.
Nan Fang Yi Ke Da Xue Xue Bao ; 36(8): 1148-52, 2016 Aug 20.
Article in Chinese | MEDLINE | ID: mdl-27578589

ABSTRACT

OBJECTIVE: To explore the role of epithelial sodium channel (ENaC) in regulating the functional activity of osteoclasts. METHODS: Multinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor (M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENaC inhibitor amiloride, and the expression of ENaC on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts. RESULTS: s Exposure to different concentrations of amiloride significantly inhibited the expression of ENaC and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclast-specific gene cathepsin K. CONCLUSION: s ENaC may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENaC regulation for modulating bone metabolism.


Subject(s)
Bone Resorption , Epithelial Sodium Channels/metabolism , Osteoclasts/cytology , Animals , Bone Marrow Cells/cytology , Cathepsin K/metabolism , Cattle , Cell Differentiation , Macrophage Colony-Stimulating Factor/metabolism , RANK Ligand/metabolism , Rats , Signal Transduction
13.
Zhong Yao Cai ; 35(11): 1820-4, 2012 Nov.
Article in Chinese | MEDLINE | ID: mdl-23627098

ABSTRACT

OBJECTIVE: To study the influence of Plantaginis Semen on cell proliferation, differentiation and function of rat osteoblasts, and investigate the regulation effects of rat osteoblast epithelial sodium channel (ENaC) on bone formation. METHODS: The animal serum was prepared by serum pharmacology means. The cells were got by separating and inducing the SD neonatal rat's skull bone. Cell proliferation and differentiation were evaluated by CCK-8 assay kit and AKP assay kit respectively. Regulation effects on mRNA expression of ENaC and osteogenesis gene were investigated by semi-quantitative PCR. RESULTS: Plantaginis Semen stimulated the osteoblasts proliferation and differentiation,the difference between treatment group and control group had statistical significance (P < 0.01) in a dose-dependent manner. The effects of Plantaginis Semen serum on alpha-ENaC gene expression paralleled those on osteogenic gene (OC, ALP, OP) expression level. CONCLUSION: Plantaginis Semen stimulates proliferation, differentiation and the mRNA expression of ENaC and osteogenesis gene in rat osteoblasts. Our results suggest that ENaC participate in the effects of Plantaginis Semen serum on osteoblast bone formation. Regulation of ENaC channel expression and function may provide a new clue for research on treatment of osteoporosis with traditional Chinese medicine.


Subject(s)
Drugs, Chinese Herbal/pharmacology , Epithelial Sodium Channels/metabolism , Osteoblasts/drug effects , Osteogenesis , Plantago/chemistry , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/isolation & purification , Epithelial Sodium Channels/genetics , Gene Expression Regulation/drug effects , Osteoblasts/cytology , Osteoblasts/metabolism , Polymerase Chain Reaction/methods , RNA, Messenger/metabolism , Rats , Seeds/chemistry , Serum , Skull/cytology
14.
Nan Fang Yi Ke Da Xue Xue Bao ; 31(11): 1871-4, 2011 Nov.
Article in Chinese | MEDLINE | ID: mdl-22126768

ABSTRACT

OBJECTIVE: To study the effects of sodium on rat osteoblast function and explore the role of epithelial sodium channel (ENaC) in such effects. METHODS: The proliferation and differentiation of rat osteoblasts were evaluated following treatment with 1×10(-4) mol/L to 1 mol/L Na(+). The mRNA expressions of the osteogenic genes and ENaC-α gene in the treated cells were assessed using RT-PCR. RESULTS: Within the concentration of 1×10(-4) mol/L to 1 mol/L, Na(+) showed a two-way effect on the osteoblasts: low-concentration Na(+) (1×10(-4) mol/L) significantly promoted osteoblast differen- tiation, while at higher concentrations (0.5 and 1 mol/L), Na(+) produced an opposite effect. Sodium did not significantly affect osteoblast proliferation. Low-concentration Na(+) significantly increased the transcription of Cbfa1, OPN and OC, while high concentrations of Na(+) decreased their transcription. Low-concentration Na(+) also enhanced the mRNA expression of ENaC-α, but high-concentration Na(+) treatment lowered ENaC-α mRNA expression. CONCLUSION: Na(+) displays a direct dose-related effect on osteoblasts by affecting its differentiation, osteogenic gene expression profile, and ENaC-α gene expression, suggesting the involvement of ENaC in Na(+)-mediated functional modulation of rat osteoblasts.


Subject(s)
Cell Differentiation/drug effects , Epithelial Sodium Channels/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Sodium Chloride/pharmacology , Animals , Animals, Newborn , Cell Proliferation/drug effects , Cells, Cultured , Rats , Rats, Sprague-Dawley , Transcriptome
15.
Zhong Yao Cai ; 34(10): 1562-5, 2011 Oct.
Article in Chinese | MEDLINE | ID: mdl-22372146

ABSTRACT

OBJECTIVE: To study the effect of drug serum of prepared Radix Polygoni Multiflori on rat osteoblast (Ob) and its mechanism. METHODS: The animal serum was prepared by serum pharmacology means. The cells were getting by separating and inducing the SD neonatal rat skull bone. The proliferation and differentiation of Ob were detected by CCK-8, alkaline phosphatase (ALP) activity analysis. And RT-PCR method was used to determine the osteogenesis-related genes expression. RESULTS: Compared with control group, the groups with drug serum of prepared Radix Polygoni Mutiflori, 10%, 20% and 30% had an effect on promotion the proliferation significantly on Ob (P < 0.01). There was no concentration-related manner among groups. The 5% and 10% drug serum decreased ALP activity at the post-translation phase compared with control group, but higher doses (20% and 30%) did not have the same effect. However, drug serum of PR/MIN increased significantly osteogenesis-related genes (OC, ALP, Cbfalpha1) mRNA expression (P < 0.05). CONCLUSION: The drug serum of prepared Radix Polygoni Multiflori can stimulate osteoblast proliferation in vitro, and its mechanism may be associated with increasing osteogenesis-related genes expression.


Subject(s)
Alkaline Phosphatase/metabolism , Cell Proliferation/drug effects , Drugs, Chinese Herbal/pharmacology , Osteoblasts/drug effects , Polygonaceae/chemistry , Alkaline Phosphatase/genetics , Animals , Cell Differentiation/drug effects , Cells, Cultured , Core Binding Factors/genetics , Core Binding Factors/metabolism , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/isolation & purification , Female , Gene Expression Regulation/drug effects , Male , Osteoblasts/cytology , Osteoblasts/metabolism , Osteocalcin/genetics , Osteocalcin/metabolism , Plant Roots/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Serum , Skull/cytology
16.
Zhong Yao Cai ; 34(10): 1604-7, 2011 Oct.
Article in Chinese | MEDLINE | ID: mdl-22372152

ABSTRACT

OBJECTIVE: To study the best ultrasonic technology of the extraction of icariin of Hugu capsule. METHODS: Used the content of icariin as index, orthogonal experiment was carried out to investigate 4 influential factors as follows: the ultrasonic power (A), the ultrasonic frequency (B), the material fluid ratio (C), the time (D). RESULTS: The best extraction conditions were as follows: the ultrasonic power was 120 W, the ultrasonic frequency was 28 KHz, solid-liquid ratio of 1 : 35, the extraction time was 10 min. CONCLUSION: Optimization of extracting process is simple, quick and low energy consumption. Under these conditions, the extraction of icariin is 1.8 times higher than that of the traditional extraction method.


Subject(s)
Drugs, Chinese Herbal/chemistry , Flavonoids/isolation & purification , Technology, Pharmaceutical/methods , Ultrasonics , Capsules , Chromatography, High Pressure Liquid , Drug Combinations , Drugs, Chinese Herbal/isolation & purification , Flavonoids/analysis , Plants, Medicinal/chemistry , Solvents/chemistry , Time Factors
17.
Ying Yong Sheng Tai Xue Bao ; 22(10): 2732-40, 2011 Oct.
Article in Chinese | MEDLINE | ID: mdl-22263482

ABSTRACT

Taking the Hunhe River water system in Liaoning Province of Northeast China as a case, this paper investigated the community structure of periphyton and the physical and chemical characteristics of water environment at 62 sampling sites, and, by using the biological integrity index of periphyton (P-IBI) and the habitat environment quality index (QHEI), a health assessment on the aquatic ecosystem of the water system was conducted. There was a great spatial heterogeneity in the periphyton community structure, and the sampling sites could be divided into 4 groups, with significant differences in the community structure, species richness, density, and Shannon diversity index among the groups. Canonical correspondence analysis (CCA) showed that ammonia nitrogen (NH4(+)-N) and dissolved phosphorus (PO4(3-)-P) were the main water environment factors driving the formation of the periphyton community structure in the water system. The health assessment of the water system based on P-IBI and QHEI was basically in consistency, though the assessment of several reaches had large discrepancy. Overall, the Suzi River at the upper reaches of the water system had a better health status, while the upper reaches of Hunhe River and the main stream of Hunhe River at its middle and lower reaches were worse in health.


Subject(s)
Ecosystem , Environmental Monitoring/methods , Fresh Water/analysis , Phytoplankton/growth & development , Water Pollutants, Chemical/analysis , China , Nitrogen/analysis , Phosphorus/analysis , Phytoplankton/classification , Population Dynamics , Principal Component Analysis , Rivers
18.
Am J Physiol Renal Physiol ; 298(2): F323-34, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20007351

ABSTRACT

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.


Subject(s)
Cyclic GMP/analogs & derivatives , Epithelial Sodium Channels/metabolism , Oocytes/metabolism , Thionucleotides/administration & dosage , Animals , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Cyclic GMP/administration & dosage , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/drug effects , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , Cytosol/metabolism , Dose-Response Relationship, Drug , Electric Conductivity , Enzyme Activators/pharmacology , Female , Humans , Isoenzymes/drug effects , Isoenzymes/genetics , Isoenzymes/metabolism , Lithium/pharmacology , Mice , Oocytes/drug effects , Oocytes/physiology , Phosphorylation , Potassium/pharmacology , Protein Isoforms , Protein Kinases/metabolism , Protein Structure, Tertiary , RNA, Small Interfering/pharmacology , Rats , Thionucleotides/pharmacology , Up-Regulation , Xenopus laevis
19.
Zhonghua Yan Ke Za Zhi ; 44(5): 413-7, 2008 May.
Article in Chinese | MEDLINE | ID: mdl-18953895

ABSTRACT

OBJECTIVE: To investigate the effect of Rosiglitazone on the proliferation and the production of transforming growth factor-beta 1 (TGF-beta 1) in cultured humans Tenon's capsule fibroblasts (HTFs). METHODS: It was a experimental study. HTFs were cultured and subcultured in vitro. After HTFs were treated with different concentrations of Rosiglitazone, the proliferation was detected by MTT assay. The migration ability was detected by a scratch method. The production of TGF-beta 1 was determined by ELISA. TGF-beta 1 mRNA expression was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared with control group, Rosiglitazone significantly (F = 178.293, P < 0.01) inhibited the proliferation (0.33 +/- 0.01 vs 0.29 +/- 0.02) and the migration of HTFs in a dose-dependent manner with the concentrations ranged from 20 to 500 mg/L. Rosiglitazone also significantly (F = 86.404, P < 0.01) reduced the production of TGF-beta 1 in HTFs (323.48 +/- 13.69 vs 267.48 +/- 20.31). TGF-beta 1 mRNA was significantly (F = 176.102, P < 0.01) down-regulated (0.69 +/- 0.02 vs 0.55 +/- 0.02). CONCLUSIONS: This study suggests that the inhibition of the proliferation of Tenon's capsule and the migration of HTFs by Rosiglitazone is due, at least in part, to decrease the production of TGF-beta 1.


Subject(s)
Eye/drug effects , Eye/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Thiazolidinediones/pharmacology , Transforming Growth Factor beta1/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Eye/cytology , Humans , Rosiglitazone
20.
Di Yi Jun Yi Da Xue Xue Bao ; 23(11): 1117-20, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14625163

ABSTRACT

OBJECTIVE: To evaluate the effects of stanozolol on the bone mineral density (BMD) and bone biomechanical properties of rats with glucocorticoid (GC)-induced osteoporosis (OP). METHODS: Twenty-eight male Sprague-Dawley rats of 3-month old were randomly divided into Group A (the basal control group), Group B (the age-matched control group), Group C (GC-induced OP group) and Group D (stanozolol-administrated group), 7 in each group. The rats in Group A were killed when experiment commenced, and those in Group B were given normal saline ig., while those in Groups C and D received the prednisone acetate (4.5 mg/kg, twice a week) alone and in combination with stanozolol (0.5 mg/kg, 6 times a week), respectively. Ninety days later, the bilateral femur and the 5th lumbar vertebra of the rats were isolated for BMD test using dual-energy X-ray absorptiometry scanner, and the torsion test, three-point bending test and compression test using electronic testing device. RESULTS: Compared with Group B, the mean BMD of the femur and the 5th lumbar vertebra in Group C decreased by 14.64% (P<0.01), the BMD of the bilateral distal femoral segment and the 5th lumbar vertebra decreased by 21.42% (P<0.01), 19.62% (P<0.05) and 23.48% (P<0.01) respectively. The load that the femur withstood in three-point bending test decreased by 17.1% (P<0.05), and the other biomechanical parameters also declined. When compared with Group C, the BMD in Group D increased, the torsional angle of the femur increased by 72.5% (P<0.05) and the other biomechanical parameters also tended to increase. CONCLUSIONS: BMD and biomechanical properties of the rat femur and the 5th lumbar vertebra decrease in response to a long-term GC administration, which can be prevented by stanozolol.


Subject(s)
Bone Density/drug effects , Bone and Bones/drug effects , Osteoporosis/prevention & control , Stanozolol/pharmacology , Animals , Biomechanical Phenomena , Bone and Bones/physiology , Femur/drug effects , Femur/physiology , Glucocorticoids/adverse effects , Lumbar Vertebrae/drug effects , Lumbar Vertebrae/physiology , Male , Osteoporosis/metabolism , Rats , Rats, Sprague-Dawley , Stanozolol/therapeutic use
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