Earthworm extract attenuates silica-induced pulmonary fibrosis through Nrf2-dependent mechanisms.

Silicosis is an occupational pulmonary fibrosis caused by inhalation of silica (SiO2) and there are no ideal drugs to treat this disease. Earthworm extract (EE), a natural nutrient, has been reported to have anti-inflammatory, antioxidant, and anti-apoptosis effects. The purpose of the current study was to test the protective effects of EE against SiO2-induced pulmonary fibrosis and to explore the underlying mechanisms using both in vivo and in vitro models. We found that treatment with EE significantly reduced lung inflammation and fibrosis and improved lung structure and function in SiO2-instilled mice. Further mechanistic investigations revealed that EE administration markedly inhibited SiO2-induced oxidative stress, mitochondrial apoptotic pathway, and epithelial-mesenchymal transition in HBE and A549 cells. Furthermore, we demonstrate that Nrf2 activation partly mediates the interventional effects of EE against SiO2-induced pulmonary fibrosis. Our study has identified EE to be a potential anti-oxidative, anti-inflammatory, and anti-fibrotic drug for silicosis.

Induction of apoptosis by opium in some tumor cell lines.

The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

Evaluation of the cytotoxic effects of CAM therapies: an in vitro study in normal kidney cell lines.

The purpose of this current study was to justify the incorporation of complementary and alternate medicine (CAM) in current cancer treatments. The major drawback of anticancer drugs is their nonselective killing, which ultimately leads to attrition of normal cells. Keeping this as the foundation of our study, we made an effort to compare the cytotoxicity associated with a known chemotherapeutic drug 5-Fluorouracil (5-FU), with certain CAM therapies previously reported to have anticancer activity. The parameters chosen for the study were based on antiproliferative and cytotoxic effects on normal, kidney epithelial cells (NRK-52E). The MTT assay, colony formation assay, DNA fragmentation, and differential staining using AO/EB, following treatment with either 5-FU or CAM therapies, were performed. The CAM therapies under study were various extracts of wheatgrass, roots of Achyranthes aspera (AA), mushroom extracts (Pleurotus ostreatus, Macrolepiota procera, and Auricularia polytricha), and a homeopathic drug, Ruta graveolens (Ruta). The results showed that treatment of normal cells with the CAM therapies led to minimum cell damage in comparison to 5-FU. This evidence-based study will lead to greater acceptance of alternative therapies against cancer.

Exploring the effects of homeopathic Apis mellifica preparations on human gene expression profiles.

BACKGROUND: Diluted preparations obtained from Apis mellifica are reported in the homeopathic literature to have anti-inflammatory activity. The present study was designed to explore the effects on global gene expression profiles of human cells by means of microarrays, using Apis mellifica mother tincture (TM) and its 3C, 5C, 7C dynamized dilutions; the technique employed allowed us to study the changes in gene expression at concentrations much lower than those associated with pharmacological responses. METHODS: An RWPE-1 cell line (human immortalized prostate epithelial cells) was used to study the effects on global gene expression by transcriptomic analysis. RESULTS: Apis mellifica TM and its 3C, 5C, 7C dynamized dilutions modulated hundreds of genes; using cluster analysis we observed groups of genes up- or down-regulated with similar expression profiles among treatments; other genes showed opposite regulation profiles at low and high dilutions of Apis mellifica, suggesting a hormetic response. In particular, genes involved in cytokine expression, inflammatory processes, anti-oxidative responses and proteasome degradation were differentially, and sometimes divergently expressed by the TM or by Apis mellifica 3C, 5C and 7C dilutions. We confirmed these data by RT-PCR analyses on 5 selected candidate genes (IL1ß, CD46, ATF1, UBE2Q2 and MT1X). CONCLUSIONS: Apis mellifica TM modifies gene expression in human cells and has inhibitory effects on regulatory processes of inflammation; in addition, extremely diluted dynamized dilutions (3C, 5C and 7C) still exert significant effects on genes involved in inflammation and oxidative stress.

Arsenic album 30C exhibits crystalline nano structure of arsenic trioxide and modulates innate immune markers in murine macrophage cell lines.

Macrophages are associated with innate immune response and M1-polarized macrophages exhibit pro-inflammatory functions. Nanoparticles of natural or synthetic compounds are potential triggers of innate immunity. As2O3 is the major component of the homeopathic drug, Arsenic album 30C.This has been claimed to have immune-boosting activities, however, has not been validated experimentally. Here we elucidated the underlying mechanism of Ars. alb 30C-mediated immune priming in murine macrophage cell line. Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) used for the structural analysis of the drug reveals the presence of crystalline As2O3 nanoparticles of cubic structure. Similarly, signatures of M1-macrophage polarization were observed by surface enhanced Raman scattering (SERS) in RAW 264.7 cells with concomitant over expression of M1 cell surface marker, CD80 and transcription factor, NF-κB, respectively. We also observed a significant increase in pro-inflammatory cytokines like iNOS, TNF-α, IL-6, and COX-2 expression with unaltered ROS and apoptosis in drug-treated cells. Enhanced expression of Toll-like receptors 3 and 7 were observed both in transcriptional and translational levels after the drug treatment. In sum, our findings for the first time indicated the presence of crystalline As2O3 cubic nanostructure in Ars. alb 30C which facilitates modulation of innate immunity by activating macrophage polarization.

Indication-Specific Effect of a Phytotherapeutic Remedy on Human Fetal Osteoblastic Cells: An in vitro Analysis.

BACKGROUND: Impaired fracture healing is a recurring interdisciplinary medical challenge. Alternative treatment concepts, apart from conventional medicine, are popular, but scientific evidence on their effects is still lacking. Plant-derived substances are widely assumed to support bone homeostasis. To clarify the effects on bone healing mechanisms, a commercially available, homeopathic-spagyric remedy, containing inter alia two herbal substances with assumed osteogenic potential, equisetum arvense and bellis perennis, was analyzed. METHODS: Human fetal osteoblastic (hFOB) 1.19 cells were incubated with the test substance in serial dilutions from 10 to 0.00001%. Cell viability has been evaluated through ATP level (CTG assay) and MTT tetrazolium reduction. Cell proliferation was analyzed by BrdU incorporation and cell migration by wound healing assay (WHA) via image analysis. Additionally, determination of the expression of key genes via real-time PCR and proteins via proteome array for inflammation, cell proliferation, and angiogenesis were performed. RESULTS: An incubation of hFOB 1.19 cells with the test substance for 24/72 h showed no reduction in cell number, viability, or proliferation. Cell migration was unimpaired. The test substance induced inflammatory genes and growth factors along with genes of osseous regeneration (ALP, Col1, IL-1α, IL-6, IL-8, IL-10, Osteocalcin, Osteonectin, RUMX2, TGF, VEGFA). Increased protein expression was found in multiple cytokines, chemokines, and acute phase proteins. CONCLUSION: The test substance did not impair cell vitality parameters (MTT, CTG, BrdU, and WHA). A tendency to activate growth factors, bone regeneration genes, and proteins was shown for osteoblasts, indicating a possible positive effect on osteogenic processes.Störungen des komplexen Prozesses der Knochenheilung stellen auch heutzutage noch eine interdisziplinäre Herausforderung dar. Es existieren zahlreiche alternative Therapiekonzepte, deren Evidenz jedoch häufig nicht belegt ist. Es wird davon ausgegangen, dass pflanzliche Substanzen die Knochenheilung unterstützen können. Wir analysierten die Wirkung eines kommerziellen, homeopathisch-spagyrischen Heilmittels, welches unter anderen zwei Pflanzenstoffe enthält, denen ein osteogenes Potential zugeschrieben wird (Equisetum arvense und Bellis perennis).Es erfolgte eine Inkubation humaner fetaler Osteoblastenzellen (hFOB 1.19) mit der Testsubstanz in absteigender Verdünnung von 10 bis 0.00001%. Die Zellvitalität wurde anhand der Zellzahlbestimmung durch ATP-abhängige metabolische Aktivität mittels CellTiter-Glo® (CTG) Test sowie durch Tetrazolium Reduktion (MTT) evaluiert. Die Zellproliferation wurde durch Inkorporation von Bromdesoxyuridin (BrdU) in die DNA aktiver Zellen analysiert. Der Wound Healing Assay (WHA) diente der Quantifizierung der Zellmigration. Zusätzlich wurde die Expression bestimmter Schlüsselgene mittels real-time PCR und die Proteinexpression via proteom array für Inflammation, Zellproliferation und Angiogenese erhoben.Die Inkubation von hFOB 1.19 mit der Testsubstanz für 24/72 Stunden führte zu keiner Reduktion von Zellzahl, -vitalität oder -proliferation. Auch die Zellmigration war unbeeinträchtigt. Es zeigte sich eine Induktion inflammatorischer Gene, Wachstumsfaktoren sowie Genen der knöchernen Regeneration (ALP, Col1, IL-1α, IL-6, IL-8, IL-10, Osteocalcin, Osteonectin, RUMX2, TGF, VEGFA). Verschiedene Zytokine, Chemokine und Akute Phase Proteine wurden vermehrt exprimiert.Die Testsubstanz hatte keine negativen Auswirkungen auf die gemessenen Zellvitalitätsparameter (MTT, CTG, BrdU and WHA). Es zeigte sich eine Aktivierungstendenz für Wachstumsfaktoren, Gene und Proteine der Knochenregeneration, die auf einen möglichen positiven Effekt der Substanz auf den Prozess des Knochenheilung hinweisen.

H3N2 homeopathic influenza virus solution modifies cellular and biochemical aspects of MDCK and J774G8 cell lines.

BACKGROUND: Influenza viruses cause highly contagious acute respiratory illnesses with significant mortality, especially among young children, elderly people, and individuals with serious medical conditions. This encourages the development of new treatments for human flu. Biotherapies are diluted solutions prepared from biological products compounded following homeopathic procedures. OBJECTIVES: To develop a biotherapy prepared from the infectious influenza A virus (A/Aichi/2/68 H3N2) and to verify its in vitro response. METHODS: The ultradiluted influenza virus solution was prepared in the homeopathic dilution 30dH, it was termed Influenzinum RC. The cellular alterations induced by this preparation were analyzed by optical and electron microscopy, MTT and neutral red assays. Glycolytic metabolism (PFK-1) was studied by spectrophotometric assay. Additionally, the production of tumor necrosis factor-α (TNF-α) by J774.G8 macrophage cells was quantified by ELISA before and after infection with H3N2 influenza virus and treatment. RESULTS: Influenzinum RC did not cause cytotoxic effects but induced morphological alterations in Madin-Darby canine kidney (MDCK) cells. After 30 days, a significant increase (p < 0.05) in mitosis rate was detected compared to control. MDCK mitochondrial activity was changed after treatment for 10 and 30 days. Treatment significantly diminished (p < 0.05) PFK-1 activity. TNF-α in biotherapy-stimulated J774.G8 macrophages indicated a significant (p < 0.05) increase in this cytokine when the cell supernatant was analyzed. CONCLUSION: Influenzinum RC altered cellular and biochemical features of MDCK and J774G8 cells.

Pearl extract enhances the migratory ability of fibroblasts in a wound healing model.

CONTEXT: For 2000 years, traditional Chinese medicine has been used as a remedy for general health improvement, including the fight against aging. Pearl powder has recently been used as a health food that has antioxidant, antiaging, antiradioactive, and tonic activities for cells; it is also applied to cure aphthous ulcer, gastric ulcer, and duodenal ulcer on clinical therapy. In addition, the mother of pearl, nacre, could enhance the cell adhesion and tissue regeneration of skin fibroblasts. OBJECTIVE: Fibroblast is regarded as indispensable in the processes of wound healing. Therefore, the effect of pearl extract (PL) on fibroblasts is investigated in this study. MATERIALS AND METHODS: PL is produced by a room temperature super extraction system (Taiwan patent no. I271 220). DMEM medium containing PL (300 µg/mL) was used to examine the effect of migration-promoting potential on human fibroblast cell line or human primary fibroblast cells in a wound healing model in vitro. RESULTS: Medium containing PL (300 µg/mL) demonstrated that the migratory cell numbers of fibroblasts were three times more than that without PL, and mRNA expression of collagen type III was higher than in collagen type I in fibroblasts. It revealed a migration-promoting potential of human fibroblasts in a wound healing model in vitro. DISCUSSION AND CONCLUSION: The present study found that the migration-promoting effect in PL, which could be a supplement in cell culture. These data suggest PL could be useful for enhancing the wound healing of fibroblasts.

[Anti-hepatic fibrosis effects research on serum containing extracts of Periplaneta americana in vitro].

OBJECTIVE: To study the anti-hepatic fibrosis effect of serum containing extracts of Periplaneta americana. METHODS: The serum contained extracts of Periplaneta americana was prepared with serologic pharmacological method. MTT method was used to observe the effect of serum containing extracts from periplaneta americana on hepatic stellate cells (HSC), and Elisa method was used to detect the contents of TGF-beta1 and collagen I in supernatant. RESULTS: Serum containing extracts I and II (15%) of Periplaneta americana had inhibitory effect on HCS (P < 0.05) after HSC were cultured with serum containing extracts of different concentration of Periolaneta americana for 24, 48 and 72 h. At 24 and 48 h, serum containing extracts I and II of Periplaneta americana decreased the content of collagen I in supernatant without significant difference (P < 0.05). Serum containing extracts I (15%, 9%, 5.4%) of Periplaneta americana could reduce generation of TGF-beta1 in supernatant for 24 h (P < 0.05). As for 48 h, only high concentration serum containing extracts I (15%) deceased the content of TGF-beta1 in supernatant. For 24 and 48 h,serum containing extracts II couldn't reduce the content of TGF-beta1 in supernatant (P < 0.05). CONCLUSION: It has definite effect on anti-hepatic fibrosis with serum containing extracts of Periplaneta americana in vitro. The mechanism may be related to inhibiting HSC propagation and reducing the production of TGF-beta1.

[Ganoderma lucidum extract protects dopaminergic neurons through inhibiting the production of inflammatory mediators by activated microglia].

Abundant evidence has suggested that neuroinflammation participates in the pathogenesis of Parkinson's disease (PD). The emerging evidence has supported that microglia may play key roles in the progressive neurodegeneration in PD and might be a promising therapeutic target. Ganoderma lucidum (GL), a traditional Chinese medicinal herb, has been shown potential neuroprotective effect in our clinical trials that lead us to speculate that it might possess potent anti-inflammatory and immunomodulating properties. To test this hypothesis, the present study investigated the potential neuroprotective effect of GL and underlying mechanism through inhibiting microglial activation using co-cultures of dopaminergic neurons and microglia. The cultures of microglia or MES23.5 cells alone or together were treated for 24 h with lipopolysaccharide (LPS, 0.25 µg/mL) as a positive control, GL extracts (50-400 µg/mL) or MES23.5 cell membrane fragments (150 µg/mL) were used in treatment groups. Microglia activation, microglia-derived harmful factors and [(3)H]dopamine ([(3)H]DA) uptake of MES23.5 cells were analyzed. The results showed that microglia were activated by LPS and MPP(+)-treated MES23.5 cell membrane fragments, respectively. Meanwhile, GL extracts significantly prevented the production of microglia-derived proinflammatory and cytotoxic factors, including nitric oxide, tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß), in a dose-dependent manner and down-regulated the TNF-α and IL-1ß expressions on mRNA level. In addition, GL extracts antagonized the reduction of [(3)H]DA uptake induced by MPP(+) and microglial activation. In conclusion, these results suggest that GL may be a promising agent for the treatment of PD through anti-inflammation.

[Effect of Cordyceps Cinensis extractant on apoptosis and expression of Toll-like receptor 4 mRNA in the ischemia-reperfusion injured NRK-52E cells].

OBJECTIVE: To observe the apoptosis and expression of Toll-like receptor (TLR)-4's mRNA in ischemia-reperfusion injured rat renal tubular epithelia cells (NRK-52E) in vitro, and effect of Cordyceps Sinensis (CS) extractant. METHODS: Cultured NRK-52E cells were divided into a control group, a model group, and a CS preincubated group. All first removed media and incubated with antimycin A for 1 h, and then recovered the media to simulate the ischemia-reperfusion injury in vitro. We detected the apoptosis ratio of cells by flow cytometer and the mRNA expression of TLR-4, Bax, Bcl-2 gene by reverse transcription-polymerase chain reaction at different time points. RESULTS: In ischemia-reperfusion injured NRK-52E, the apoptosis ratio rose as time passed (P<0.05). We also observed increased mRNA expression of TLR-4, Bax (P<0.05) and deceased expression of Bcl-2 (P<0.05). Compared with the model group, the CS preincubated NRK-52E cells showed apparent tolerance to ischemia-reperfusion injury, which manifestated lower apoptosis ratio (P<0.05), decreased expression of mRNA of TLR-4, Bax and increased expression of Bcl-2 (All Ps<0.05). CONCLUSION: The number of apoptosis cells and the expression of TLR-4 mRNA increased with ischemia-reperfusion injury of NRK-52E in vitro. CS can prevent the NRK-52E cells from ischemia-reperfusion injury by downregulating TLR-4 gene.

Symphytum officinale augments osteogenesis in human bone marrow-derived mesenchymal stem cells in vitro as they differentiate into osteoblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Mesenchymal stem cells (MSCs) are multipotent stem cells possessing regenerative potential. Symphytum officinale (SO) is a medicinal plant and in homoeopathic literature, believed to accelerate bone healing. AIM OF THE STUDY: This study aimed to determine if homoeopathic doses of SO could augment osteogenesis in MSCs as they differentiate into osteoblasts in vitro. MATERIALS AND METHODS: Bone marrow samples were obtained from patients who underwent bone grafting procedures (n = 15). MSCs were isolated, expanded and characterized by flow cytometry (CD90, CD105). Cytotoxicity of SO was evaluated by MTT assay. Osteogenic differentiation was induced in MSCs with ß-glycerophosphate, ascorbic acid and dexamethasone over 2 weeks. Different homoeopathic doses of SO (MT, 3C, 6C, 12C and 30C) were added to the basic differentiation medium (BDM) and efficiency of MSCs differentiating into osteoblasts were measured by evaluating expression of Osteocalcin using flow cytometry, and alkaline phosphatase activity using ELISA. Gene expression analyses for osteoblast markers (Runx-2, Osteopontin and Osteocalcin) were evaluated in differentiated osteoblasts using qPCR. RESULTS: Flow cytometry (CD90, CD105) detected MSCs isolated from bone marrow (93-98%). MTT assay showed that the selected doses of SO did not induce any cytotoxicity in MSCs (24 hours). The efficiency of osteogenic differentiation (2 weeks) for different doses of Symphytum officinale was determined by flow cytometry (n = 10) for osteoblast marker, Osteocalcin, and most doses of Symphytum officinale enhanced osteogenesis. Interestingly, gene expression analysis for Runx-2 (n = 10), Osteopontin (n = 10), Osteocalcin (n = 10) and alkaline phosphatase activity (n = 8) also showed increased osteogenesis with the addition of Symphytum officinale to BDM, specially mother tincture. CONCLUSIONS: Our findings suggest that homoeopathic dose (specially mother tincture) of Symphytum officinale has the potential to enhance osteogenesis.

Site selectivity of competitive antagonists for the mouse adult muscle nicotinic acetylcholine receptor.

The muscle-type nicotinic acetylcholine receptor has two nonidentical binding sites for ligands. The selectivity of acetylcholine and the competitive antagonists (+)-tubocurarine and metocurine for adult mouse receptors is known. Here, we examine the site selectivity for four other competitive antagonists: cisatracurium, pancuronium, vecuronium, and rocuronium. We rapidly applied acetylcholine to outside-out patches from transfected BOSC23 cells and measured macroscopic currents. We have reported the IC(50) of the antagonists individually in prior publications. Here, we determined inhibition by pairs of competitive antagonists. At least one antagonist was present at a concentration producing > or =67% receptor inhibition. Metocurine shifted the apparent IC(50) of (+)-tubocurarine in quantitative agreement with complete competitive antagonism. The same was observed for pancuronium competing with vecuronium. However, pancuronium and vecuronium each shifted the apparent IC(50) of (+)-tubocurarine less than expected for complete competition but more than expected for independent binding. The situation was similar for cisatracurium and (+)-tubocurarine or metocurine. Cisatracurium did not shift the apparent IC(50) of pancuronium or vecuronium, indicating independent binding of these two pairs. The data were fit to a two-site, two-antagonist model to determine the antagonist binding constants for each site, L(alphaepsilon) and L(alphadelta). We found L(alphaepsilon)/L(alphadelta) = 0.22 (range, 0.14-0.34), 20 (9-29), 21 (4-36), and 1.5 (0.3-2.9) for cisatracurium, pancuronium, vecuronium, and rocuronium, respectively. The wide range of L(alphaepsilon)/L(alphadelta) for some antagonists may reflect experimental uncertainties in the low affinity site, relatively poor selectivity (rocuronium), or possibly that the binding of an antagonist at one site affects the affinity of the second site.

The kinetics of competitive antagonism of nicotinic acetylcholine receptors at physiological temperature.

Detailed information about the ligand-binding site of nicotinic acetylcholine receptors has emerged from structural and mutagenesis experiments. However, these approaches provide only static images of ligand-receptor interactions. Kinetic measurements of changes in protein function are needed to develop a more dynamic picture. Previously, we measured association and dissociation rate constants for competitive inhibition of current through embryonic muscle acetylcholine receptor channels at 25 degrees C. Little is known about competitive antagonism at physiological temperatures. Here, we performed measurements at 37 degrees C and used thermodynamics to estimate the energetics of antagonism. We used rapid solution exchange protocols to determine equilibrium and kinetics of inhibition of acetylcholine-activated currents in outside-out patches by (+)-tubocurarine, pancuronium and cisatracurium. Kinetic rates as high as 600 s(-1) were resolved by this technique. Binding was primarily enthalpy driven. The 12 degrees C increase in temperature decreased equilibrium antagonist binding by 1.7- to 1.9-fold. In contrast, association and dissociation rate constants increased 1.9- to 6.0-fold. Activation energies for dissociation were 90 +/- 6, 106 +/- 8 and 116 +/- 10 kJ mol(-1) for cisatracurium, (+)-tubocurarine and pancuronium, respectively. The corresponding apparent activation energies for association were 38 +/- 6, 85 +/- 6 and 107 +/- 13 kJ mol(-1). The higher activation energy for association of (+)-tubocurarine and pancuronium compared with cisatracurium is notable. This may arise from either a more superficial binding site for the large antagonist cisatracurium compared to the other ligands, or from a change in receptor conformation upon binding of (+)-tubocurarine and pancuronium but not cisatracurium. Differences in ligand desolvation and ligand conformation are not likely to be important.

A signaling-selective, nanomolar potent allosteric low molecular weight agonist for the human luteinizing hormone receptor.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) activate the LH receptor/cyclic AMP (cAMP) signaling pathway to induce ovulation. As an alternative to parenterally administered hCG to treat anovulatory infertility, orally active low molecular weight (LMW) LHR agonists have been developed at Organon. In this paper, we present the mechanism of action of a prototypic, nanomolar potent and almost full LHR agonist, Org 43553. Org 43553 interacts with the endodomain of the LHR, whereas LH acts via the N-terminal exodomain. LH stimulates the cAMP pathway with an EC50 of 35 pM, but this stimulation is not antagonized by simultaneous incubation with Org 43553. At nanomolar concentrations, LH also stimulates phospholipase C (PLC), but Org 43553 is hardly able to do so. In contrast, Org 43553 inhibits LH-induced PLC (IC50 approximately 10 nM). While Org 43553 stimulates dissociation of [125I]hCG from the LHR and reduces [125I]hCG binding, LH reduces specific [3H]Org 43553 binding. We conclude that Org 43553 is a signaling-selective, allosteric LHR agonist. We hypothesize that Org 43553 and LH induce a similar LHR conformation necessary for activating adenylyl cyclase, which initiates most, if not all, physiological responses of LH.

Homeopathic potencies of Arnica montana L. change gene expression in a Tamm-Horsfall protein-1 cell line in vitro model: the role of ethanol as a possible confounder and statistical bias.

Marzotto et al. showed that homeopathic preparations of Arnica montana L. acted directly on gene expression of Tamm-Horsfall protein-1 (THP-1) monocyte/macrophage cell lines activated with phorbol12-myristate13-acetate and interleukin-4 (IL-4). A. montana homeopathic dilutions are used in complementary and alternative medicine to treat inflammation disorders and post-traumatic events as well as for wound repair. The French Pharmacopoeia of these remedies uses 0.3% ethanol in each centesimal dilution. In this paper, we discuss how ethanol-containing A. montana homeopathic centesimal dilutions can change gene expression in IL-4-treated monocyte/macrophage THP-1. We assessed the role of ethanol in the Arnica homeopathic dilutions containing this alcohol by investigating its action on gene expression of THP-1 cell. Evidence would strongly suggest that the presence of ethanol in these remedies might play a fundamental role in the dilutions ability to affect gene expression, particularly for doses from 5c to 15c. Where, rather than playing a major role in the mesoscopic structure of water, the ethanol might have a chemical-physical role in the induction of THP-1 gene expression, apoptosis, and deoxyribonucleic acid function. This evidence generates a debate about the suggestion that the use of a binary-mixed solvent in homeopathic chemistry, used by Hahnemann since 1810, may be fundamental to explain the activity of homeopathy on cell models.

[The medicinal serum of yi-shen ruan-jian san antagonized the effect of aristolochic acid on human proximal tubular epithelial cells in vitro].

OBJECTIVE: To evaluate whether the medicinal serum of Yi-shen Ruan-jian san can antagonize the fibrogenic effect of human proximal tubular epithelial cell line (HKC) activated by aristolochic acid (AA) in vitro. METHOD: The HKC was incubated in the media containing 40 mg x L(-1) aristolochic acid sodium salt (AA-Na) with or without 10% concentration of Yi-shen Ruan-jian san medicinal serum. Then the cell proliferation and cytotoxicity of HKC were determined by MTF and lactate dehydrogenase (LDH) release assay respectively, the antigen expression of cytokeratin and alpha-smooth muscle actin on HKC was detected by immunocytochemistry, the mRNA expression of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I Collagen (Col I) of HKC was measured by RT-PCR, and their protein expression was measured by ELISA or Western blot. RESULT: No cytotoxic effect was found in HKC after stimulation of AA-Na with or without the medicinal serum of Yi-shen Ruan-jian san (P > 0.05). No epithelial-myofibroblast transdifferentiation was found in HKC after AA-Na stimulation. The mRNA and protein expression of TGF-beta1, CTGF, PAI-1 and TIMP-1 of HKC was significantly upregulated by AA-Na (P < 0.05). The above-mentioned enhanced mRNA and protein expression, except for PAI-1, was significantly downregulated by the medicinal serum of Yi-shen Ruan-jian san, compared with the control (normal rat serum in the same concentration) (P < 0.05). CONCLUSION: The fibrogenic effects of HKC activated by AA are antagonized by Yi-shen Ruan-jian san, through downregulating the expression of promoting excellular matrix (ECM) synthesis factors (TGF-beta1, CTGF) and inhibiting ECM degradation factor (TIMP-1).

Progesterone stimulates Krox-20 gene expression in Schwann cells.

The gene of the zinc finger transcription factor Krox-20 (Egr-2) is expressed in Schwann cells and plays an important role in myelination of peripheral nerves. We have shown that progesterone promotes myelination in the regenerating sciatic nerve and in cocultures of Schwann cells and sensory neurones. To determine whether progesterone regulates Krox-20 expression, we measured its effects on Krox-20 mRNA levels in the MSC80 mouse Schwann cell line by semi-quantitative RT-PCR. Although low levels of Krox-20 mRNA are detectable in MSC80 cells cultured in defined medium, treatment with 10(-6) M progesterone induces a rapid (15 min) and transient increase in the levels of Krox-20 mRNA. Lower doses of progesterone (10(-9), 10(-8) and 10(-7) M) are also effective in increasing Krox-20 mRNA. Other steroids including testosterone, dexamethasone, and estradiol are ineffective when added to the culture medium at 10(-6) M for 1 h. The induction of Krox-20 mRNA was also observed with the selective progesterone agonist Organon 2058 and was abolished by treating the MSC80 Schwann cells with the progesterone antagonist RU486, indicating that progesterone induces Krox-20 mRNA expression by binding to its intracellular receptor. The induction of Krox-20 by progesterone was also demonstrated in primary cultures of Schwann cells isolated from neonatal rat sciatic nerves, at the mRNA level by RT-PCR and at the protein level by immunohistochemistry. As Krox-20 is a necessary step for the initiation of myelin formation in peripheral nerves, its stimulation by progesterone suggests an important signalling function for this steroid in myelination.

The effect of homeopathic plant extract solutions on the cell proliferation of human cutaneous fibroblasts in vitro.

BACKGROUND: According to homotoxicology illness is defined as an overload of the connective tissue matrix with toxic substances, the homotoxins. In order to support elimination of these homotoxins, complex homeopathic medicines were developed. Fibroblasts are the local cells of this matrix and produce and modulate the composition of the extracellular matrix (ECM) in every organ. OBJECTIVE: In this study, the effect of 17 potentiated plant extracts, which are components of some antihomotoxic remedies, on the cell proliferation of human cutaneous fibroblasts (F54) was analyzed as an indication of their influence on the ECM. MATERIAL AND METHODS: Cell proliferation of the F54 cells was measured by a colorimetric XTT-based assay kit. RESULTS: Six of the tested agents had no effect on the cell proliferation of fibroblasts; 11 plant extracts had a dose-dependent inhibitory effect on cell proliferation. CONCLUSIONS: As the modulation of the ECM is dependent on the activity of the fibroblasts, these results indicate that the plant extracts may modulate the composition of the ECM via the inhibitory effect on fibroblasts cell growth.

[Growth capability of epithelial cell line of human poorly differentiated nasopharyngeal carcinoma and its response to Chinese medicinal herbs and marine drugs].

By 3H-TdR incorporation, dye exclusion and cell colony-forming tests, the capability of short-term in vitro growth of the epithelial cell line of human poorly differentiated nasopharyngeal carcinoma (CNE-2Z) was assayed. At the same time, its response to 54 kinds of Chinese medicinal herbs and marine drugs was studied. The results showed that the 3H-TdR incorporation rate of cells was 1.8 +/- 0.02%, reproduction rate was 60.9 +/- 13.0% and colony-forming rate, 40.8 +/- 3.5%. As to the ratios of the three cell growth indexes and response to medicines, the Chinese medicinal herbs and marine drugs causing the reduction of colony-forming and cell survival ratios were predominant (64.8% and 40.7%). The results indicate that the majority of drugs possess the cytotoxic and inhibitory effect on cell reproduction to different degrees. The composite cell response to every kind of drug could be divided into 6 types: descending, ascending, peaked, valley-like, depressed and stable. The depressing type drugs might inhibit or arrest the cell growth of nasopharyngeal carcinoma and are worthy of further study.