Microbiota-derived short chain fatty acids in fermented Kidachi Aloe promote antimicrobial, anticancer, and immunomodulatory activities.

BACKGROUND: Fermented Aloe leaf juice is a commonly used food supplement in Japan. In a previous study, fermentation of A. arborescence juice was performed and the presence of short-chain fatty acids (SCFAs) was confirmed and quantified. Samples were collected before and after the fermentation process to be subjected, in the present study, to DNA extraction, 16S rRNA gene (V3-V4 regions) amplification, and sequencing by the next-generation Illumina MiSeq sequencer. Our work aims to analyze the sequences to assess the bacterial diversity in the juice before and after fermentation, identify the beneficial microbes responsible for the production of SCFAs, and evaluate some of the biological activities of the fermented juice. RESULTS: Data revealed the richness and diversity of the bacterial community in the fermented juice compared to the unfermented control. Relative abundance of bacterial phyla showed that the majority of the microbial community in the test samples corresponded to Pseudomonadota (unfermented; 10.4%, fermented; 76.36%), followed by Bacillota (unfermented; 4.71%, fermented; 17.13%) and then Bacteroidota (unfermented; 0.57%, fermented; 1.64%). For the fermented sample, 84% of Bacillota were lactobacilli. A hierarchically clustered heatmap revealed that Lactobacillus was the most abundant genus in both samples suggesting its involvement in the production of SCFAs. To assess potential health benefits, the anticancer efficacy of the fermented product of A. arborescens was investigated against colorectal cancer (IC50 = 3.5 µg/ml) and liver cancer (IC50 = 6.367 µg/ml) compared to the normal peripheral blood mononuclear cells (PBMCs). Flow cytometric analysis of the cell cycle pattern revealed remarkable population arrest in G0 and G1, however, the highest percentages were mainly in the G1 phase for Hep-G2 (40.1%) and HCT-116 (53.2%) cell lines. This effect was accompanied by early apoptotic profiles of HCT-116 (36.9%) and late apoptosis for Hep-G2 (17.3%). Furthermore, immunomodulatory properties demonstrated a significantly (p < 0.001) reduced percentage of induced TNF-α while enhancing IFN-γ dramatically. For antimicrobial activities, marked broad-spectrum activities were recorded against some bacterial and fungal pathogens (17-37 mm inhibition zone diameter range). CONCLUSION: Therefore, this study affords the basis of bacterial community composition in fermented A. arborescens juice as well as its potential biological benefits.

ARHGAP1 Transported with Influenza Viral Genome Ensures Integrity of Viral Particle Surface through Efficient Budozone Formation.

Influenza viral particles are assembled at the plasma membrane concomitantly with Rab11a-mediated endocytic transport of viral ribonucleoprotein complexes (vRNPs). The mechanism of spatiotemporal regulation of viral budozone formation and its regulatory molecules on the endocytic vesicles remain unclear. Here, we performed a proximity-based proteomics approach for Rab11a and found that ARHGAP1, a Rho GTPase-activating protein, is transported through the Rab11a-mediated apical transport of vRNP. ARHGAP1 stabilized actin filaments in infected cells for the lateral clustering of hemagglutinin (HA) molecules, a viral surface membrane protein, to the budozone. Disruption of the HA clustering results in the production of virions with low HA content, and such virions were less resistant to protease and had enhanced antigenicity, presumably because reduced clustering of viral membrane proteins exposes hidden surfaces. Collectively, these results demonstrate that Rab11a-mediated endocytic transport of ARHGAP1 with vRNPs stimulates budozone formation to ensure the integrity of virion surface required for viral survival. IMPORTANCE The endocytic transport of the influenza viral genome triggers the clustering of viral membrane proteins at the plasma membrane to form the viral budozone. However, host factors that promote viral budozone formation in concert with viral genome transport have not been identified. Here, we found that ARHGAP1, a negative regulator of the Rho family protein, is transported with the viral genome and stabilizes actin filaments to promote budozone formation. We have shown that ARHGAP1-mediated efficient formation of viral budozone was crucial for the clustering of viral HA protein to the progeny viral particles. The clustering of HA proteins on the virions is responsible for the structural integrity of the viral particles, which promotes viral stability and viral immune evasion. This study highlights the molecular mechanism that works in concert with viral genome packaging to ensure the structural integrity of viral particles.

A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM).

The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.

Divergence of structural strategies for homophilic E-cadherin binding among bilaterians.

Homophilic binding of E-cadherins through their ectodomains is fundamental to epithelial cell-cell adhesion. Despite this, E-cadherin ectodomains have evolved differently in the vertebrate and insect lineages. Of the five rod-like, tandemly aligned extracellular cadherin domains of vertebrate E-cadherin, the tip extracellular cadherin domain plays a pivotal role in binding interactions. Comparatively, the six consecutive N-terminal extracellular cadherin domains of Drosophila E-cadherin, DE-cadherin (also known as Shotgun), can mediate adhesion; however, the underlying mechanism is unknown. Here, we report atomic force microscopy imaging of DE-cadherin extracellular cadherin domains. We identified a tightly folded globular structure formed by the four N-terminal-most extracellular cadherin domains stabilized by the subsequent two extracellular cadherin domains. Analysis of hybrid cadherins from different insects indicated that the E-cadherin globular portion is associated with determining homophilic binding specificity. The second to fourth extracellular cadherin domains were identified as the minimal portion capable of mediating exclusive homophilic binding specificity. Our findings suggest that the N-terminal-most four extracellular cadherin domains of insect E-cadherin are functionally comparable with the N-terminal-most single extracellular cadherin domain of vertebrate E-cadherin, but that their mechanisms might significantly differ. This work illuminates the divergence of structural strategies for E-cadherin homophilic binding among bilaterians.

An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy.

An improved unroofing method enabled the cantilever of an atomic force microscope (AFM) to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in phosphate-buffered saline (PBS) at a higher resolution than conventional electron microscopy. All of the actin filaments clearly exhibited a short periodicity of approximately 5-6 nm, which was derived from globular actins linked to each other to form filaments, as well as a long helical periodicity. The polarity of the actin filaments appeared to be determined by the shape of the periodic striations. Microtubules were identified based on their thickness. Clathrin coats and caveolae were observed on the cytoplasmic surface of cell membranes. The area containing clathrin molecules and their terminal domains was directly visualized. Characteristic ridge structures located at the surface of the caveolae were observed at high resolution, similar to those observed with electron microscopy (EM). Overall, unroofing allowed intracellular AFM imaging in a liquid environment with a level of quality equivalent or superior to that of EM. Thus, AFMs are anticipated to provide cutting-edge findings in cell biology and histology.

Direct observation of the actin filament by tip-scan atomic force microscopy.

Actin filaments, the actin-myosin complex and the actin-tropomyosin complex were observed by a tip-scan atomic force microscope (AFM), which was recently developed by Olympus as the AFM part of a correlative microscope. This newly developed AFM uses cantilevers of similar size as stage-scan AFMs to improve substantially the spatial and temporal resolution. Such an approach has previously never been possible by a tip-scan system, in which a cantilever moves in the x, y and z directions. We evaluated the performance of this developed tip-scan AFM by observing the molecular structure of actin filaments and the actin-tropomyosin complex. In the image of the actin filament, the molecular interval of the actin subunits (∼5.5 nm) was clearly observed as stripes. From the shape of the stripes, the polarity of the actin filament was directly determined and the results were consistent with the polarity determined by myosin binding. In the image of the actin-tropomyosin complex, each tropomyosin molecule (∼2 nm in diameter) on the actin filament was directly observed without averaging images of different molecules. Each tropomyosin molecule on the actin filament has never been directly observed by AFM or electron microscopy. Thus, our developed tip-scan AFM offers significant potential in observing purified proteins and cellular structures at nanometer resolution. Current results represent an important step in the development of a new correlative microscope to observe nm-order structures at an acceptable frame rate (∼10 s/frame) by AFM at the position indicated by the fluorescent dye observed under a light microscope.

Photoirradiation wavelength dependence of cycloreversion quantum yields of diarylethenes.

The cycloreversion quantum yields of compounds 1b and 2b increased upon irradiation with light at shorter wavelengths. The irradiation wavelength dependence is ascribed to the energy barrier on the 2A potential energy surface in the excited electronic state.

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

Antifibrotic effect of aloe vera in viral infection-induced hepatic periportal fibrosis.

AIM: To investigate the anti-oxidative and anti-fibrotic effects of aloe vera in patients with liver fibrosis. METHODS: Aloe vera high molecular weight fractions (AHM) were processed by patented hyper-dry system in combination of freeze-dry technique with microwave and far infrared-ray radiation. Fifteen healthy volunteers as the control group and 40 patients were included. The patients were randomly subdivided into two equal groups: the conventional group was treated with placebo (starch), and AHM group was treated with 0.15 gm/d AHM, both for 12 consecutive weeks. The patients were investigated before and after treatment. Serum activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), hyaluronic acid (HA), transforming growth factor-ß (TGF-ß) and matrixmetalloproteinase-2 (MMP-2) were determined. The reduced glutathione (GSH) and malondialdehyde (MDA) levels in liver were assayed and the expression of hepatic α-smooth muscle actin (α-SMA) was identified by immunohistochemistry. RESULTS: At the start of the study, the hematoxylin and eosin staining revealed fibro-proliferated bile ductules, thick fibrous septa and dense inflammatory cellular infiltration in the patients before treatment. The use of AHM for 12 wk significantly ameliorated the fibrosis, inhibited the inflammation, and resulted in minimal infiltration and minimal fibrosis compared to the conventional group. The enzyme activities of the liver (ALT, AST and ALP) were attenuated after treatment in both groups, and the decrease in the AHM group was more significant as compared with the conventional group. Similar to the AST, the MDA levels were significantly higher before treatment, and were attenuated after treatment in both groups. In contrast, the hepatic glutathione content in the patients were decreased significantly in the AHM group compared to the controls. The serum levels of the fibrosis markers (HA, TGF-ß and MMP-2) were also reduced significantly after treatment. The expression of α-SMA was modified in patients before and after treatment as compared with the normal controls. In the conventional group, there was only thin and incomplete parenchymal α-SMA positive septum joining the thickened centrilobular veins, while in the AHM group, few α-SMA positive cells were present in sinusoid and lobule after treatment. CONCLUSION: Oral supplementation with AHM could be helpful in alleviating the fibrosis and inflammation of hepatic fibrosis patients.

Improvement of some physicochemical properties of arundic acid, (R)-(-)-2-propyloctanonic acid, by complexation with hydrophilic cyclodextrins.

Arundic acid, (R)-(-)-2-propyloctanonic acid, is a novel neurological agent for intractable neurodegenerative diseases. However, arundic acid, an oily drug, has low aqueous solubility and severe bitter/irritating tastes. Consequently, these physicochemical properties of arundic acid need to be improved to develop its pharmaceutical preparations. In the present study, we evaluated whether parent cyclodextrins (CyDs) and 2-hydroxypropylated CyDs (HP-CyDs) can interact with arundic acid, and have powderization, solubilization and taste-masking properties. Of various CyDs, HP-ß-CyD had the most potent solubilizing effect for arundic acid. UV and (1)H NMR spectroscopic studies demonstrated that arundic acid formed inclusion complexes with CyDs at a molar ratio of 1:1 in solution. The complexation with CyDs changed an oily form of arundic acid to a solid form. The gustatory sensation studies indicate that of various CyDs, HP-ß-CyD and γ-CyD showed the most significant taste-masking effects in solution and powders, respectively. HP-ß-CyD significantly reduced the response of the electric potential caused by the adsorption of arundic acid to the taste sensor. These results suggest that hydrophilic CyDs have potential as multifunctional excipients for preparing solutions and powders containing arundic acid.

Possible hypoglycemic effect of Aloe vera L. high molecular weight fractions on type 2 diabetic patients.

Aloe vera L. high molecular weight fractions (AHM) containing less than 10 ppm of barbaloin and polysaccharide (MW: 1000 kDa) with glycoprotein, verectin (MW: 29 kDa), were prepared by patented hyper-dry system in combination of freeze-dry technique with microwave and far infrared radiation. AHM produced significant decrease in blood glucose level sustained for 6 weeks of the start of the study. Significant decrease in triglycerides was only observed 4 weeks after treatment and continued thereafter. No deterious effects on kidney and liver functions were apparent. Treatment of diabetic patients with AHM may relief vascular complications probably via activation of immunosystem.

Effects of tanshinone VI on phosphorylation of ERK and Akt in isolated cardiomyocytes and cardiac fibroblasts.

Effects of tanshinone VI, a diterpene from Tan-Shen, on humoral factor-induced phosphorylation of ERK and Akt during hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts isolated from neonatal rats were examined. Treatment of cultured cardiomyocytes with 10 nM insulin-like growth factor-1 (IGF-1) or 10 nM endothelin-1 resulted in an increase in leucine incorporation into acid-insoluble fraction. Treatment of cultured cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II increased incorporation of proline. IGF-1 increased phosphorylated extracellular signal-regulated kinase (pERK) and protein kinase B (pAkt) of cardiomyocytes, whereas endothelin-1 increased pERK, but not pAkt. Treatment of cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II also increased pERK, whereas pAkt was increased by treatment with IGF-1 alone. When the cardiomyocytes were incubated in the presence of 10 microM tanshinone VI, IGF-1- and endothelin-1-induced increases in pERK, but not pAkt, were partially attenuated. Treatment of cardiac fibroblasts with 10 microM tanshinone VI also attenuated IGF-1-induced increases in pERK and pAkt. Tanshinone VI also partially attenuated angiotensin II-induced increase in proline incorporation into cardiac fibroblasts. PD98059, an inhibitor for phosphorylation of extracellular signal-regulated kinase (ERK), but not wortmannin, that for protein kinase B phosphorylation, attenuated an increase in leucine incorporation into cardiomyocytes in the presence of either IGF-1 or endothelin-1. These results suggest that tanshinone VI is a possible agent that can attenuate the humoral factor-induced hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts via an attenuation of ERK phosphorylation in these cells.

Accumulation of bisphenol A in hemodialysis patients.

Bisphenol A [BPA, 2,2-bis(4-hydoxyphenyl)propane], an industrial chemical used in the production of polycarbonate, epoxide resin, and polyarylate, is considered to be an endocrine-disrupting chemical. BPA may be present in some hollow-fiber dialyzers used in hemodialysis. In this study, we tested the amounts of BPA eluted from various hollow fibers. Furthermore, we measured the BPA concentration in the sera of 22 renal disease predialysis patients, as well as 15 patients who were receiving hemodialysis, to see if there is BPA accumulation in these patients. The elution test of BPA showed that a much larger amount of BPA was eluted from polysulfone (PS), and polyester-polymeralloy hollow fibers. Among renal disease patients who had not undergone hemodialysis, the serum BPA concentration increased as the renal function deteriorated, showing a significant negative association. In a crossover test between PS and cellulose (Ce) dialyzers, the predialysis serum BPA concentration of PS dialyzer users decreased after changing to a Ce dialyzer, and the serum BPA increased again after switching back to PS dialyzers. In patients who were using PS dialyzers, the BPA level significantly increased after a dialysis session. However, in the Ce dialyzer users, the BPA level decreased. Since accumulation of BPA could affect the endocrine or metabolic system of the human body, it is important to perform further investigations on dialysis patients.

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL.

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.

Monoamine oxidase inhibitors from Gentiana lutea.

Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4''-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1'-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.

Effects of tanshinone VI on insulin-like growth factor-1-induced hypertrophy of isolated cardiomyocytes from neonatal rats.

The effects of tanshinone VI (Tan), a diterpene extracted from Salvia miltiorrhiza, on insulin-like growth factor-1 (IGF-1)-induced hypertrophy of cardiomyocytes were examined. Cultured cardiomyocytes were isolated from neonatal rat hearts. The incorporation of [(3)H]-leucine into the trichloroacetic acid (TCA)-insoluble fraction was measured as a marker of protein synthesis, which revealed cardiomyocyte hypertrophy. Various concentrations of IGF-1, ranging from 0.1 nM to 10 nM, increased [(3)H]-leucine incorporation into the TCA-insoluble fraction of cardiomyocytes in a dose-dependent manner. IGF-1 induced an increase in phosphorylated extracellular signal-regulated kinase 1/2 (ERK), but did not change ERK protein content in cardiomyocytes. When the cells were incubated in the presence of Tan (0.1 muM to 10 muM), [(3)H]-leucine incorporation into IGF-1-untreated cells was unaltered. When the cells were incubated with 10 muM Tan, IGF-1-induced increases in [(3)H]-leucine incorporation into the TCA-insoluble fraction and phosphorylated ERK were attenuated. These results suggest that Tan is a possible agent for the suppression of IGF-1-induced hypertrophy of cardiomyocytes via an attenuation of ERK activation.

A flavone from Manilkara indica as a specific inhibitor against aldose reductase in vitro.

Isoaffinetin (5,7,3',4',5'-pentahydroxyflavone-6-C-glucoside) was isolated from Manilkara indica as a potent inhibitor of lens aldose reductase by bioassay-directed fractionation. This C-glucosyl flavone showed specific inhibition against aldose reductases (rat lens, porcine lens and recombinant human) with no inhibition against aldehyde reductase and NADH oxidase. Kinetic analysis showed that isoaffinetin exhibited uncompetitive inhibition against both dl-glyceraldehyde and NADPH. A structure-activity relationship study revealed that the increasing number of hydroxy groups in the B-ring contributes to the increase in aldose reductase inhibition by C-glucosyl flavones.

[Anti-inflammatory constituents, aloesin and aloemannan in Aloe species and effects of tanshinon VI in Salvia miltiorrhiza on heart].

Cinnamoyl, p-coumaroyl, feruloyl, caffeoyl aloesin, and related compounds were isolated from Aloe species. The antiinflammatory and antioxidative activities of these compounds were examined based on the structure-activity relationship. It was suggested that the bioactivities may link to acyl ester groups in aloesin, together with those of aloesin-related compounds. However, investigations using the contact hypersensitivity response indicated a preventive effect of aloesin on the UV-B-induced immune suppression. Furthermore, aloesin inhibited tyrosine hydroxylase and dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase from normal human melanocyte cell lysates. These results show that aloesin prevents not only UV-B-induced immune suppression, but also could be a positive pigment-altering agent for cosmetic application. In preclinical study, aloe extract was investigated using phagocytosis and nitroblue tetrazolium chloride (NBT) reduction in adult bronchial asthma, and high molecular-weight materials, such as polysaccharide and glycoprotein fractions, were identified as active ingredients. The neutral polysaccharides, aloemannan and acemannan showed antitumor, antiinflammatory and immunosuppressive activities, and glycoprotein fractions with bradykinindegrading and cell proliferation-stimulating activities were identified from the nondialysate fraction of the gel part of Aloe species. Verectin fractionated from Aloe vera gel was examined biochemically and immunochemically, and verectin antibody was used in the appraisal of commercial Aloe vera gel products. It was reported that aloesin stimulates the proliferation of cultured human hepatoma SK-Hep 1 cells. Thus aloesin, related compounds, and high molecular-weight materials, such as aloemannan and verectin, may act in concert to exert therapeutic properties for wounds, burns and inflammation. The biodisposition of fluoresceinylisothiocyanate (FITC)--labeled aloemannan (FITC-AM) with the homogenate from some organs in mice was demonstrated, and FITC-AM was metabolized to a smaller molecule (MW 3000) by the large intestinal microflora in feces. The modified aloe polysaccharide (MW: 80000) with cellulase under restricted conditions, immunologically stimulated the recovery of UV-B-induced tissue in jury. Thus the modified polysaccharides of aloemannan, together with acemannan (MW: about 600000), are expected to participate in biological activity following oral administration. The effects of tanshinone VI, a diterpenoid isolated from Salvia miltiorrhiza, on the heart are reviewed. First, the effects on the posthypoxic recovery of contractile function of perfused rat hearts were examined. Hypoxia/reoxygenation induced a release of purine nucleosides and bases (ATP metabolites) and resulted in little recovery of contractile force of reoxygenated hearts. Pretreatment of the perfused heart with 42 nM tanshinone VI under hypoxic conditions attenuated the release of ATP metabolites during hypoxia/reoxygenation. Treatment with tanshinone VI enhanced the posthypoxic recovery of myocardial contractility. These results show that tanshinone VI may protect the heart against hypoxia/reoxygenation injury and improve the posthypoxic cardiac function. Second, the effects of tanshinone VI on in vitro myocardial remodeling were examined. Cardiomyocytes and cardiac fibroblasts were isolated from neonatal rat hearts, and simultaneously prepared insulin-like growth factor-1 (IGF-1) induced the hypertrophy of cardiomyocytes. IGF-1 increased the collagen synthesis of cardiac fibroblasts, that is, in vitro fibrosis. The hypertrophy of cardiomyocytes was attenuated in the presence of tanshinone VI in the culture medium. The fibrosis of cardiac fibroblasts was decreased by treatment with tanshinone VI. When tanshinone VI was added to cardiac fibroblast-conditioned medium, the medium-mediated hypertrophy of cardiomyocytes was also attenuated. These results show that tanshinone VI may attenuate in vitro cardiac remodeling. The series of studies has shown that tanshinone VI protects the myocardium against hypoxia/reoxygenation injury and attenuates progression of in vitro myocardial remodeling, suggesting that tanshinone VI is a possible agent for the treatment of cardiac disease with contractile failure.

Simultaneous determination of glycyrrhizin metabolites formed by the incubation of glycyrrhizin with rat feces by semi-micro high-performance liquid chromatography.

A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the simultaneous determination of 3alpha-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid together with glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide formed by incubation of glycyrrhizin with rat feces. The analysis was accomplished within 25 min with a TSKgel ODS-80TsQA (150 x 2.0 mm i.d.) column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 ml.min(-1), a thermostatic oven at 25 degrees C, and detection at 254 nm. The detection limits of these compounds were 0.2 pmol per injection (5 microl). The metabolites of glycyrrhizin, by anaerobic or aerobic incubation with rat fecal suspension over 48 h, were determined. Glycyrrhizin was almost completely converted to metabolite glycyrrhetic acid, and metabolites 3alpha-hydroxyglycyrrhetic acid and 3-dehydroglycyrrhetic acid in negligible amounts in anaerobic conditions. However, the metabolic time courses of 3-dehydroglycyrrhetic acid when incubated in aerobic conditions revealed that it apparently continued increasing during the whole incubation period.

Effects of tanshinone VI on the hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts of neonatal rats.

The possible effects of tanshinone VI (tsh), a diterpene from the root of Tan-Shen (Salvia miltiorrhiza, Bunge (Labiatae)) on hypertrophy and fibrosis in cultured neonatal rat cardiac myocytes and fibroblasts were examined. Tsh had no significant effect on protein synthesis, which was evaluated by [3H]-leucine incorporation into the acid insoluble fraction in the cells, in the absence of stimulatory factors in cardiac myocytes. The amount of protein produced in cardiac myocytes was increased by 10(-8) M endothelin-1 (ET-1), 10(-6) M phenylephrine (PE), or 10(-8) M insulin-like growth factor-1 (IGF-1), suggesting that hypertrophy of cardiac myocytes in vitro was induced by these factors. The ET-1-, PE-, or IGF-1-induced increase in protein synthesis was attenuated by treatment with 10(-5) M tsh. Treatment with 10(-5) M tsh significantly decreased the synthesis of collagen by cardiac fibroblasts, which was evaluated by [3H]-proline incorpolation into acid-insoluble fraction of the fiblobrasts, in the absence of stimulatory factors for the production. Fetal bovine serum (FBS) or IGF-1 increased collagen synthesis in a concentration-dependent manner. The increase at 5% FBS or 10(-8) M IGF-1 was inhibited by 10(-5) M tsh. Fibroblast-conditioned medium (FB-CM) increased protein synthesis in cardiac myocytes in a concentration-dependent manner (10; - 100 %). Tsh attenuated the FB-CM-induced increase in protein synthesis by cardiac myocytes. These results show that tsh may attenuate the humoral factor-induced hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts. The findings suggest that tsh may improve the development of cardiac remodeling under pathophysiological conditions. Abbreviations. ANP:atrial natriuretic peptide DMEM:Dulbecco-modified Eagle's medium ET-1:endothelin-1 FB-CM:fibroblast-conditioned medium FBS:fetal bovine serum IGF-1:insulin-like growth factor-1 PE:phenylephrine tsh:tanshinone VI