Effect of LGR4 on synovial cells and inflammatory factors in rats with traumatic osteoarthritis.

OBJECTIVE: Traumatic arthritis is one of the most common diseases in orthopedics. LGR4 is involved in bone formation and bone development. However, the role of LGR4 in synovial cells of rats with traumatic osteoarthritis has not been reported. MATERIALS AND METHODS: Sprague Dawley (SD) rats were randomly divided into the control group and model group. The Real Time-Polymerase Chain Reaction (RT-PCR), Western blot, and Enzyme-Linked Immunosorbent Assay (ELISA) were used to analyze the expression of LGR4 in synovial tissue and synovial fluid. Synovial cells were isolated and cultured, followed by transfection of LGR4-pcDNA3.1 plasmid into cells. Cell proliferation was analyzed by MTT and EdU assay, and the Caspase-3 activity was assessed using the Caspase-3 activity kit. The secretion of the inflammatory factors interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was detected by ELISA. NF-κB signaling pathway changes were evaluated by the Western blot. RESULTS: In the model group, LGR4 mRNA expression in synovial tissue was significantly decreased, and the secretion of LGR4 in the synovial fluid was significantly decreased compared with the control group (p<0.05). LGR4 protein expression in the synovial membrane in the model group tissue was reduced. The transfection of LGR4-pcDNA3.1 plasmid into synovial cells promoted the LGR4 expression, inhibited the proliferation of synoviocytes, increased the Caspase-3 activity, the secretion of IL-1, TNF-α, and IL-6, as well as the decreased expression of NF-κB with a statistical significance, compared with the control group (p<0.05). CONCLUSIONS: LGR4 expression is reduced in the rat model of traumatic osteoarthritis. The upregulation of LGR4 expression can inhibit the secretion of the inflammatory factors and inhibit the proliferation of the synovial cells by regulating NF-κB signaling pathway, which may alleviate the development of the joint inflammation.

Matrix metallopeptidase 2 (MMP2) mediates MHC class I polypeptide-related sequence A (MICA) shedding in renal cell carcinoma.

INTRODUCTION: The MHC class i chain-related molecule A (MICA) is a ligand for the natural killer group 2, member D (NKG2D) immunoreceptor activation. The engagement of tumor cell surface MICA to NKG2D stimulates the NK and T cell antitumor immunity. Shedding of MICA by tumor cells facilitates tumor immune evasion, which might partially contribute to tumor progression. MATERIAL AND METHODS: Inmunohistochemistry was performed on both normal and neoplastic renal tissue. Human renal carcinoma cell lines 786-0 and ACHIN were transfected and target sequences to silence human MMP2 by shRNA expression were established. The degree of MICA shedding was measured and quantitative real-time PCR and Western-blot analysis were performed. RESULTS: The membrane type matrix metalloproteinase 2 (MMP2) mediated the MICA shedding, which was blocked by suppression of MMP2 expression. Concomitantly, MMP2 over-expression enhanced the MICA shedding, indicating that MMP2 was involved in the renal cell carcinoma-associated proteolytic release of soluble MICA (sMICA), which facilitated the tumor immune escape. CONCLUSIONS: These findings suggested that MMP2 might be a new potential target for tumor immune therapy. Elucidation of the mechanisms by which tumors shed MICA could be of a great importance for cancer treatment in order to reinforce the NK and T cell antitumor immunity.

GATA-3 is down-regulated in patients with clear cell renal carcinoma.

BACKGROUND: GATA-3 is a transcription factor involved in human growth and development. Recent studies found its association with breast cancer, however, its expression profile in renal cell carcinoma (RCC) has not been investigated. MATERIAL AND METHOD: The study included 35 patients submitted to radical nephrectomy with confirmed pathological diagnosis of RCC. Normal control kidney tissues were obtained from 25 living kidney donors and tissues were biopsied before implantation. The majority of RCC samples were diagnosed as clear cell renal cell carcinoma (94.3%) except for 1 case of papillary RCC and 1 case of collecting duct carcinoma. GATA-3 expression was evaluated by quantitative PCR and Western blotting (WB) in RCC samples and normal kidneys respectively, immunohistochemical staining was performed as well. Meanwhile, the GATA-3 expression in two cancer cell lines (786-O, ACHN) and normal kidney epithelial cells (HK-2) was detected by PCR and WB. In addition, renal cancer cells and HK-2 cells were cultivated and detected by confocal microscopy for the exact intra-cellular localization of GATA-3. RESULTS: Data showed a significant down-regulation of GATA-3 expression present in neoplastic tissues compared with normal tissues; similarly, GATA-3 was significantly attenuated in all renal cancer cell lines compared with normal HK-2 cells. Confocal displayed a strong cytoplasmic immno-fluorescence activity of GATA-3 with peri-nuclear zone in HK-2, whereas the intensity in cancer cells was markedly weaker than that of HK-2. CONCLUSIONS: In summary, our present study clarifies that the aberrant expression profile of GATA-3 in human RCC is possibly involved with tumorigenesis, and the complicated mechanism is worthy of further investigation.

Separation of cis- and trans-Asarone from Acorus tatarinowii by Preparative Gas Chromatography.

A preparative gas chromatography (pGC) method was developed for the separation of isomers (cis- and trans-asarone) from essential oil of Acorus tatarinowii. The oil was primarily fractionated by silica gel chromatography using different ratios of petroleum ether and ethyl acetate as gradient elution solvents. And then the fraction that contains mixture of the isomers was further separated by pGC. The compounds were separated on a stainless steel column packed with 10% OV-101 (3 m × 6 mm, i.d.), and then the effluent was split into two gas flows. One percent of the effluent passed to the flame ionization detector (FID) for detection and the remaining 99% was directed to the fraction collector. Two isomers were collected after 90 single injections (5 uL) with the yield of 178 mg and 82 mg, respectively. Furthermore, the structures of the obtained compounds were identified as cis- and trans-asarone by (1)H- and (13)C-NMR spectra, respectively.

Fractionation of volatile constituents from curcuma rhizome by preparative gas chromatography.

A preparative gas chromatography (pGC) method was developed for the separation of volatile components from the methanol extract of Curcuma rhizome. The compounds were separated on a stainless steel column packed with 10% OV-101 (3 m × 6 mm, i.d.), and then, the effluent was split into two gas flows. One percent of the effluent passed to the flame ionization detector (FID) for detection and the remaining 99% were directed to the fraction collector. Five volatile compounds were collected from the methanol extract of Curcuma rhizome (5 g/mL) after 83 single injections (20 uL) with the yield of 5.1-46.2 mg. Furthermore, the structures of the obtained compounds were identified as ß-elemene, curzerene, curzerenone, curcumenol, and curcumenone by MS and NMR spectra, respectively.

Determination of nucleotides, nucleosides and their transformation products in Cordyceps by ion-pairing reversed-phase liquid chromatography-mass spectrometry.

An ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) was developed for the determination of nucleotides, nucleosides and their transformation products in Cordyceps. Perfluorinated carboxylic acid, namely pentadecafluorooctanoic acid (PDFOA, 0.25mM), was used as volatile ion-paring agent and a reversed-phase column (Agilent ZORBAX SB-Aq column) was used for the separation of three nucleotides namely uridine-5'-monophosphate (UMP, 0.638-10.200microg/mL), adenosine-5'-monophosphate (AMP, 0.24-7.80microg/mL) and guanosine-5'-monophosphate (GMP, 0.42-13.50microg/mL), seven nucleosides including adenosine (0.55-8.85microg/mL), guanosine (0.42-6.75microg/mL), uridine (0.33-10.50micro/mL), inosine (0.21-6.60microg/mL), cytidine (0.48-15.30microg/mL), thymidine (0.20-6.30microg/mL) and cordycepin (0.09-1.50microg/mL), as well as six nucleobases, adenine (0.22-6.90microg/mL), guanine (0.26-4.20microg/mL), uracil (0.38-12.15microg/mL), hypoxanthine (0.13-4.20microg/mL), cytosine (0.39-12.45microg/mL) and thymine (0.26-8.25microg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01-0.16microg/mL and 0.04-0.41microg/mL for the 16 analytes, respectively. The contents of 16 investigated compounds in natural and cultured Cordyceps were also determined and compared after validation of the developed IP-RP-LC-MS method. The transformations of nucleotides and nucleosides in Cordyceps were evaluated based on the quantification of the investigated compounds in three extracts, including boiling water extraction (BWE), 24h ambient temperature water immersion (ATWE) and 56h ATWE extracts. Two transformation pathways including UMP-->uridine-->uracil and GMP-->guanosine-->guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. The pathway of AMP-->adenosine-->inosine-->hypoxanthine was proposed in natural C. sinensis, while AMP-->adenosine-->adenine in cultured C. militaris. However, the transformation of nucleotides and nucleosides was not found in commercial cultured C. sinensis.

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis.

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.

Analysis of sterols and fatty acids in natural and cultured Cordyceps by one-step derivatization followed with gas chromatography-mass spectrometry.

Ten free fatty acids namely lauric acid, myristic acid, pentadecanoic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, docosanoic acid and lignoceric acid and four free sterols including ergosterol, cholesterol, campesterol and beta-sitosterol in natural (wild) Cordyceps sinensis, Cordyceps liangshanensis and Cordyceps gunnii, as well as cultured C. sinensis and Cordyceps militaris were first determined using pressurized liquid extraction (PLE), trimethylsilyl (TMS) derivatization and GC-MS analysis. The conditions such as the amount of reagent, temperature and time for TMS derivatization of analytes were optimized. Under the optimum conditions, all calibration curves showed good linearity within the tested ranges. The intra- and inter-day variations for 14 investigated compounds were less than 3.4% and 5.2%, respectively. The results showed that palmitic acid, linoleic acid, oleic acid, stearic acid and ergosterol are main components in natural and cultured Cordyceps which could be discriminated by hierarchical clustering analysis based on the contents of 14 investigated compounds or the 4 fatty acids, where the contents of palmitic acid and oleic acid in natural Cordyceps are significantly higher than those in the cultured ones.

Effects of sample preparation methods on the quantification of nucleosides in natural and cultured Cordyceps.

Sample preparation is the first and very important step, which can greatly influence the repeatability and accuracy of the analysis. To date, several sample preparation methods with different solvents have been used for quantitative determination of nucleosides in Cordyceps, but their data are greatly various. In this study, five nucleosides, including adenosine, guanosine, inosine, uridine and cordycepin, in Cordyceps were determined using three extraction methods i.e. organic solvent pressurized liquid extraction, boiling water extraction and ambient temperature water extraction and high performance liquid chromatography (HPLC)-diode array detection (DAD). The similar results were obtained when organic solvent pressurized liquid extraction and boiling water extraction were applied. However, the amounts of nucleosides in natural C. sinensis and cultured C. militaris extracted with ambient temperature water were greatly increased except those of adenosine in natural C. sinensis and cordycepin in cultured C. militaris. In addition, the amount of investigated nucleosides in cultured C. sinensis had no obvious variation among the three extraction methods. The results suggest that sample preparation has significant effect on the quantification of nucleosides in Cordyceps.

Quantitative determination of eight components in rhizome (Jianghuang) and tuberous root (Yujin) of Curcuma longa using pressurized liquid extraction and gas chromatography-mass spectrometry.

Curcuma longa (Zingiberaceae) is a native plant of southern Asia and is cultivated extensively throughout the warmer parts of the world. Jianghuang and Yujin are rhizome and tuberous root of C. longa, respectively, which were traditionally used as two Chinese medicines. In this paper, pressurized liquid extraction (PLE) and gas chromatography-mass spectrometry (GC-MS) were developed for quantitative determination/estimation of eight characteristic compounds including beta-caryophyllene, ar-curcumene, zingiberene, beta-bisabolene, beta-sesquiphellandrenendrene, ar-turmerone, alpha-turmerone and beta-turmerone in Jianghuang and Yujin. A HP-5MS capillary column (30 m x 0.25 mm i.d.) coated with 0.25 microm film 5% phenyl methyl siloxane was used for separation and selected ion monitoring (SIM) method was used for quantitation. Hierarchical cluster analysis based on characteristics of eight identified peaks in GC-MS profiles showed that 10 samples were divided into two main clusters, Jianghuang and Yujin, respectively. Four components such as ar-curcumene, ar-turmerone, alpha-turmerone and beta-turmerone were optimized as markers for quality control of rhizome (Jianghuang) and tuberous root (Yujin), which are two traditional Chinese medicines, from Curcuma longa.

Optimization and comparison of three methods for extraction of volatile compounds from Cyperus rotundus evaluated by gas chromatography-mass spectrometry.

The essential oil of Cyperus rotundus has multiple pharmacological activities. Therefore, the extraction with high yield and quality is very important for preparation of essential oil of C. rotundus. In this paper, three methods, namely hydrodistillation (HD), pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE), for extraction of volatile compounds from C. rotundus were optimized and compared by gas chromatography-mass spectrometry. Among eight identified compounds in C. rotundus, five components including alpha-copaene, cyperene, beta-selinene, beta-cyperone and alpha-cyperone were quantitatively determined or estimated using alpha-cyperone as standard, which showed that PLE had the highest extraction efficiency, while SFE had the best selectivity for extraction of beta-cyperone and alpha-cyperone. The contents of ingredients from C. rotundus extracted with HD, PLE and SFE are significantly different, which suggest that comparison of chemical components and pharmacological activities of different extracts is helpful to elucidate the active components in C. rotundus and control its quality.

Fast simultaneous determination of 14 nucleosides and nucleobases in cultured Cordyceps using ultra-performance liquid chromatography.

Determination of nucleosides and their metabolic compounds is important for physiological and pharmacological studies. Herein, a rapid ultra-performance liquid chromatography (UPLC) method was developed for the simultaneous determination of 14 nucleosides and nucleobases, namely adenine, adenosine, cytosine, cytidine, uracil, uridine, guanine, guanosine, hypoxanthin, inosine, thymine, thymidine, 2'-deoxyuridine and cordycepin. The separation was performed on Waters Acquity UPLC system with Acquity UPLC BEH C(18) column and gradient elution of 0.5mM acetic acid and acetonitrile in 5min. The correlation coefficients of 14 analytes were high (R(2)>0.9995) within the test ranges. The LOD and LOQ were lower to 11.9 and 47.0ng/ml with 1mul of injection volume, respectively. The overall R.S.D. for intra- and inter-day of 14 analytes were less than 1.8%. The developed method was applied for the analysis of nucleosides and nucleobases in cultured Cordyceps, which also could be used for the fast determination of the analytes in pharmaceutical products and biological fluids.

Quality control of Cordyceps sinensis, a valued traditional Chinese medicine.

Cordyceps sinensis, a well-known and valued traditional Chinese medicine, is also called DongChongXiaCao (winter worm summer grass) in Chinese. It is commonly used to replenish the kidney and soothe the lung for the treatment of fatigue, night sweating, hyposexualities, hyperglycemia, hyperlipidemia, asthemia after severe illness, respiratory disease, renal dysfunction and renal failure, arrhythmias and other heart disease, and liver disease. As the rarity and upstanding curative effects of natural Cordyceps, several mycelial strains have been isolated from natural Cordyceps and manufactured in large quantities by fermentation technology, and they are commonly sold as health food products in Asia. In addition, some substitutes such as Cordyceps militaris also have been used and adulterants also confused the market. Therefore, quality control of C. sinensis and its products is very important to ensure their safety and efficacy. Herein, markers and analytical methods for quality control of Cordyceps were reviewed and discussed.

Identification and quantitation of eleven sesquiterpenes in three species of Curcuma rhizomes by pressurized liquid extraction and gas chromatography-mass spectrometry.

In this paper, GC-MS and pressurized liquid extraction (PLE) was developed for identification and quantitative determination/estimation 11 sesquiterpenes including germacrene D, curzerene, gamma-elemene, furanodienone, curcumol, isocurcumenol, furanodiene, germacrone, curdione, curcumenol and neocurdione in Ezhu which are derived from three species of Curcuma, i.e., Curcuma phaeocaulis, Curcuma wenyujin and Curcuma kwangsiensis by using an analogue as standard. The results showed the methodology could quantitatively compare the quality of three species of Curcuma. The contents of investigated sesquiterpenes in three species of Curcuma were high variant. Hierarchical clustering analysis based on characteristics of 11 identified peaks in GC profiles showed that 18 samples were divided into two main clusters, C. phaeocaulis and C. wenyujin, respectively. C. kwangsiensis showed the characters closed to C. phaeocaulis or C. wenyujin based on its location. Five components such as furanodienone, germacrone, curdione, curcumenol and neocurdione were optimized as markers for quality control of Ezhu.

[Synthesis and cardiovascular activities of peptidyl dopamine derivatives].

Amino acids and small peptides with different steric configurations and lipophilicities were appended to dopamine. Eighteen compounds have been synthesized. The attachment of D-amino acids or N-methyl amino acids onto the dopamine molecule caused a marked decrease in cardiovascular activity by intravenous injection, while the introduction of lipophilic amino acids caused a marked increase in myocardial contractility and blood pressure in anesthetized dogs. The durations of action were also prolonged.