Detection of Osteoarthritis Inflammation by Single-Photon Emission Computed Tomography Based on an Inflammation-Targeting Peptide cFLFLF.

PURPOSE: Although inflammation has been recognized as a key process in the pathogenesis of osteoarthritis (OA), there remains no clinical noninvasive imaging modality that can specifically diagnose inflammatory activity of OA. In this study, a formyl peptide receptor 1 (Fpr1) targeting probe cFLFLF-PEG-HYNIC-99mTc and single-photon emission computed tomography (SPECT) imaging was used to detect inflammatory activity by targeting macrophages involved in the pathogenesis of OA. PROCEDURES: In vitro experiments were performed to evaluate Fpr1 expression during macrophage inflammatory response. In the in vivo studies, anterior cruciate ligament transection (ACLT) surgery was performed, and magnetic resonance imaging (MRI) and histological data were assessed to analyze the OA model in both mice and rats. The radioactive probe cFLFLF-PEG-HYNIC-99mTc and SPECT imaging were used to corroborate OA-related inflammation and compare ACLT vs sham knees. RESULTS: In vitro macrophage activation resulted in a remarkable increase in Fpr1 expression. In vivo experiments in mice and rats produced similar results. MRI and histological analysis demonstrated significant joint degeneration in the ACLT knee. The ACLT knee produced a much stronger signal from the probe when compared to the sham knee. It is important to note that the ratio of ACLT/sham knee signal intensity decreased with OA progression, indicating greater differences earlier in the progression of OA. CONCLUSION: The radioactive probe cFLFLF-PEG-HYNIC-99mTc and SPECT imaging are effective for detecting and monitoring inflammation during OA progression by targeting Fpr1 expression in the knee joint.

Induction of caspase-3-dependent apoptosis in human leukemia HL-60 cells by δ-elemene.

  Î´-Elemene, an antitumor component, is a chemical compound isolated from Curcuma wenyujin, a Chinese traditional herb. We examined whether δ-elemene could inhibit cell growth and cell cycle progression and induce apoptosis in human leukemia HL-60 cells. The results demonstrated that δ-elemene induces significant apoptosis of HL-60 cells, as shown by MTT assay, annexin V (AnV) binding of externalized phosphatidylserine (PS), and the mitochondrial probe JC-1 using flow cytometry. HL-60 cells treated with δ-elemene showed high percentages in the early apoptotic and late apoptoctic/necrotic stages, as well as caspase-3 activation of HL-60 cells. By monitoring the changes in cell cycle profiles, we confirmed that δ-elemene could interfere with the cell cycle in the G2/M phase and induce apoptosis in HL-60 cells in a time-dependent manner. Caspase-3 plays a direct role in proteolytic cleavage of the cellular proteins responsible for progression to apoptosis. Therefore we examined apoptosis in HL-60 cells after exposure to δ-elemene and measured caspase-3 activities with or without Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, a broad-spectrum caspase inhibitor) pretreatment using flow cytometric analysis. The results showed that δ-elemene could induce caspase-3 activation as detected by the decrease in δ-elemene-induced caspase-3 activities after treatment with z-VAD-fmk. In the present study, δ-elemene activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an inhibitory effect of z-VAD-fmk on this cell death. During δ-elemene-induced apoptosis, cytochrome c and apoptosis-inducing factor were released into the cytosol and BAX was translocated from the cytosol to mitochondria. However, these were not prevented by z-VAD-fmk. In conclusion, our study demonstrated that δ-elemene could induce G2/M cell cycle transition and trigger apoptosis through a caspase-3-dependent pathway.

Cell apoptosis induced by delta-elemene in colorectal adenocarcinoma cells via a mitochondrial-mediated pathway.

The chemical compound delta-elemene, isolated from the Chinese herbal medicine plant Curcuma Wenyujin, has been known to exert antitumor activity. In this study we demonstrated that apoptotic cell death induced by delta-elemene in DLD-1 cells was concentration-and time-dependent, and had little inhibition of the normal human liver cell line WRL-68. Apoptosis was further confirmed and quantified by DNA fragmentation ELISA, Annexin V (AnV) binding of externalized phosphatidylserine and the mitochondrial probe JC-1 using flow cytometry. The rapid increase in intracellular reactive oxygen species (ROS) levels was involved in the mechanism of cell death. Western blot analysis demonstrated that delta-elemene activated the caspase-signaling pathway, leading to the proteolysis conversion of pro-caspase-3 to activate caspase-3, and the subsequent cleavage of the caspase substrate PARP. In the process of the induction of apoptotic cell death, Bax translocated into mitochondria, a reduction in Deltapsim was observed and a release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria into the cytosol occurred, indicating that cell death induced by delta-elemene was through a mitochondrial-mediated pathway.

Validated LC-MS/MS assay for the determination of felbinac: Application to a preclinical pharmacokinetics study of felbinac trometamol injection in rat.

A rapid and sensitive analytical method based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of felbinac in rat plasma, bile, urine, feces and tissue. Sample preparation involved liquid-liquid extraction with ethyl ether-dichloromethane (60:40, v/v). Chromatography of felbinac and the internal standard probenecid was performed within 2min on a Venusil MP C(18) column (100mmx4.6mm i.d., 5microm) with a mobile phase consisting of acetonitrile-5mM ammonium acetate containing 0.1% formic acid (pH 3.0) (80:20, v/v) at a flow rate of 1.2ml/min. Detection by electrospray negative ionization mass spectrometry and multiple-reaction monitoring of the transitions of felbinac at m/z 211.1-->167.0 and of probenecid at m/z 283.9-->239.9 was linear over the concentration range 5-5000ng/ml with a lower limit of quantitation of 5ng/ml using a sample volume of only 50microl. Intra- and inter-day precisions (as relative standard deviation, R.S.D.) were < or =7.3% and < or =6.4%, respectively, and accuracy (as relative error, R.E.) was in the range -2.1 to 7.4%. Recoveries and matrix effects were satisfactory in all the biological matrices examined. The method was applied to a preclinical pharmacokinetic study in rat involving a single intravenous injection of felbinac trometamol.

[Research on active part of Sambucus chinensis against hepatitis mice induced by CCl4].

OBJECTIVE: To investigate the best extraction process of Sambucus chinensis against hepatitis and research on its active part. METHODS: We studied the protective effects of different extracts of Sambucus chinensis distilled by different extraction process on mice acute hepatic injury induced by CCl4, and searched for the active part of Sambucus chinensis against hepatitis from the best extract by extraction with different solvent and purification with macroporous adsorption resin, then studied their protective effects on mice acute hepatic injury induced by CCl4. RESULTS: The extraction of Sambucus chinensis by 75% alcohol showed very significantly protective effects on mice acute hepatic injury induced by CCl4 and the effects were better than that of other extraction process. The extraction eluted by 30% alcohol after purification with macroporous adsorption resin and extracted by EtOAc in the extraction of Sambucus chinensis by 75% alcohol all showed significantly protective effects on mice acute hepatic injury induced by CCl4, and the effects of the extraction eluted by 30% alcohol after purification with macroporous adsoption resin were better than extraction with EtOAc. CONCLUSIONS: The best extraction solvent is 75% alcohol. The active part of Sambucus chinensis against hepatitis is the extraction eluted by 30% alcohol after purification with macroporous adsorption resin and extraction with EtOAc.

Effect of delta-elemene on Hela cell lines by apoptosis induction.

This study was designed to investigate the apoptosis-inducing activity of delta-elemene on Hela cells in vitro. MTT assay and Hoechst 33258/PI fluorescence microscopy were used for this investigation. Apoptosis was further confirmed and quantified by DNA fragmentation ELISA, Annexin V (AnV) binding of externalized phosphatidylserine and the mitochondrial probe JC-1 using flow cytometry. Generation of reactive oxygen species (ROS) was detected using CM-H2DCFDA. Western blots analysis was performed using antibodies against the pro-caspase-3, or PRAP (Poly (ADP-ribose) polymerase). The results showed that delta-elemene exhibited a marked antiproliferative effect on Hela cells in dose- and time-dependent manners, and had little inhibition to normal human liver cell line WRL-68. It was demonstrated that delta-elemene was capable of inducing DNA fragmentation in a dose- and time-dependent manner. AnV positivity and the disturbance of the polarized mitochondrial transmembrane potential (Deltapsim) suggested that delta-elemene induced apoptotic death of Hela cells. Western blot analysis demonstrated that delta-elemene activated the caspase-signaling pathway, leading to the proteolysis conversion of pro-caspase-3 to activate caspase-3, and the subsequent cleavage of the caspase substrate PARP. Further, it was noted that the apoptotic effect of delta-elemene could be attenuated by L-Glutathione (GSH) or z-DEVD-fmk. It suggested that the increase in ROS generation might be involved in the mechanism of delta-elemene induced cell apoptosis.

A new function of a previously isolated compound that stimulates activation and differentiation of myogenic precursor cells leading to efficient myofiber regeneration and muscle repair.

Muscle repair following severe injury is slow and incomplete due to the limited regenerative capacity of muscles comprising the function. In this study, one pure compound structurally corresponding to triterpenoid, which can directly induce the activation, proliferation and maturation of quiescent satellite cells into myocytes in vitro, was isolated from Geum japonicum. The potential effect of this compound on myogenesis was further tested in repair of severe muscle injury. It was found that this compound could significantly stimulate the regenerative potential of the damaged muscle resulting in regeneration of myotubes and myotube bundles time-dependently replacing the damaged muscle tissues. This compound-mediated active regeneration of new myofibers repairing damaged muscles was probably due to its direct action on activation and proliferation of quiescent myogenic precursor cells and enhancement of their maturation into regenerating myotubes, as was demonstrated in our primary myogenic precursor cells culture experiments. The up-regulated expression of endogenous phospho-Akt1 in compound-treated myogenic precursor cells may also contribute to the process of myofiber regeneration and muscle repair probably via promoting myogenic cell survival capacity.

[Protective effects of the granule of Sambucus chinensis lindl on acute hepatic injury].

OBJECTIVE: To investigate the protective effects of the granule of Sambucus chinensis Lindl on acute hepatic injury. METHODS: The acute liver-injury model induced by CCl4 or D-GalN or ConA was established in mice or rats. RESULTS: The granule of Sambucus chinensis Lindl showed significantly protective effects on mice acute hepatic injury induced by CCl4 ,and the effects were concern of antagonizing lipoperoxidation and improving energy of Ca2+ -ATPase of theca mitochondria and microsome. It showed significantly protective effects on mice or rats acute hepatic injury induced by D-GalN or Con A, too. CONCLUSION: The granule of Sambucus chinensis Lindl showed significantly protective effects against many kinds of chemical and immunological liver injuries.