The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump.

IMPORTANCE: The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.

Coelonin protects against PM2 .5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

One of the important monitoring indicators of the air pollution is atmospheric fine particulate matter (PM2.5 ), which can induce lung inflammation after inhalation. Coelonin can alleviate PM2.5 -induced macrophage damage through anti-inflammation. However, its molecular mechanism remains unclear. We hypothesized that macrophage damage may involve the release of inflammatory cytokines, activation of inflammatory pathways, and pyrosis induced by inflammasome. In this study, we evaluated the anti-inflammation activity of coelonin in PM2.5 -induced macrophage and its mechanism of action. Nitric oxide (NO) and reactive oxygen species (ROS) production were measured by NO Assay kit and dichlorofluorescein-diacetate (DCFH-DA), and apoptosis were measured by Flow cytometry and TUNEL staining. The concentration of inflammatory cytokines production was measured with cytometric bead arrays and ELISA kits. The activation of NF-κB signaling pathway and NLRP3 inflammasome were measured by immunofluorescence, quantitative reverse transcription-polymerase chain reaction and western blot. As expected, coelonin pretreatment reduced NO production significantly as well as alleviated cell damage by decreasing ROS and apoptosis. It decreased generation of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in PM2.5 -induced RAW264.7 and J774A.1 cells. Moreover, coelonin markedly inhibited upregulating the expression of toll-like receptor (TLR)4 and cyclo-oxygenase (COX)-2, blocked activation of p-nuclear factor-kappa B (NF-κB) signaling pathway, and suppressed expression of NLRP3 inflammasome, ASC, GSDMD, IL-18 and IL-1ß. In conclusion, the results showed that coelonin could protect against PM2.5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

Hinokitiol Selectively Enhances the Antibacterial Activity of Tetracyclines against Staphylococcus aureus.

The increasing prevalence of antibiotic resistance causes an urgent need for alternative agents to combat drug-resistant bacterial pathogens. Plant-derived compounds are promising candidates for the treatment of infections caused by antibiotic-resistant bacteria. Hinokitiol (ß-thujaplicin), a natural tropolone derivative found in the heartwood of cupressaceous plants, has been widely used in oral and skin care products as an antimicrobial agent. The aim of this work was to study the synergy potential of hinokitiol with antibiotics against Staphylococcus aureus, which is an extremely successful opportunistic pathogen capable of causing nosocomial and community-acquired infections worldwide. The MIC was determined by the broth microdilution method, and the effect of combinations was evaluated through fractional inhibitory concentration indices (FICI). The mechanism behind this synergy was also investigated by using fluorescence spectroscopy and high-performance liquid chromatography (HPLC). The MICs of hinokitiol alone against most S. aureus strains were 32 µg/mL. Selectively synergistic activities (FICIs of ≤0.5) were observed for combinations of this phytochemical with tetracyclines against all tested strains of S. aureus. Importantly, hinokitiol at 1 µg/mL completely or partially reversed tetracycline resistance in staphylococcal isolates. The increased accumulation of tetracycline inside S. aureus in the presence of hinokitiol was observed. In addition, hinokitiol promoted the uptake of ethidium bromide (EB) in bacterial cells without membrane depolarization, suggesting that it may be an efflux pump inhibitor. IMPORTANCE The disease caused by S. aureus is a public health issue due to the continuing emergence of drug-resistant strains, particularly methicillin-resistant S. aureus (MRSA). Tetracyclines, one of the old classes of antimicrobials, have been used for the treatment of infections caused by S. aureus. However, the increased resistance to tetracyclines together with their toxicity have limited their use in the clinic. Here, we demonstrated that the combination of hinokitiol and tetracyclines displayed synergistic antibacterial activity against S. aureus, including tetracycline-resistant strains and MRSA, offering a potential alternative approach for the treatment of infections caused by this bacterium.

Protocatechuic aldehyde prevents ischemic injury by attenuating brain microvascular endothelial cell pyroptosis via lncRNA Xist.

BACKGROUND: Pyroptosis is a pro-inflammatory cell death characterized by the formation of inflammasomes. Abnormal inflammation in brain microvascular endothelial cells (BMECs) has been correlated with ischemic stroke. Protocatechuic aldehyde (PCA) is a hydrophilic phenolic acid derived from the traditional Chinese herb Salvia miltiorrhiza with significant anti-inflammatory effects. However, the mechanism of PCA on BMEC pyroptosis under ischemic injury has been largely unexplored. PURPOSE: We aimed to study the effects and mechanism of PCA on BMEC pyroptosis under ischemic injury. METHODS: Sprague-Dawley (SD) rats were injected through the tail vein with different concentrations of PCA after transient middle cerebral artery occlusion (MCAO) was performed. The protective effects of PCA in SD rats were examined via neurological scores, infarct volume evaluation, and anti-pyroptosis effects using immunofluorescence staining and western blot. Rat BMECs (rBMECs) were treated with different concentrations of PCA after oxygen and glucose deprivation (OGD). The ability of PCA to protect rBMECs was examined via cell viability, anti-oxidative activity, and anti-pyroptosis effects as determined by qRT-PCR and western blot. Additionally, the role of lncRNA Xist in anti-pyroptosis responses of PCA-treated rBMECs was validated with lncRNA Xist siRNA. RESULTS: We found that treatment with MCAO and OGD increased the expression of NOD-like receptor protein 3, gasdermin D, Caspase-1, interleukin-1ß, and NIMA-related kinase 7, which was reversed by treatment with PCA or MCC950 (a pyroptosis inhibitor). In addition, PCA reduced the cerebral infarct volume in MCAO rats and promoted cell survival and proliferation in OGD/reperfusion-treated rBMECs. PCA enhanced the antioxidant activity and mitochondrial membrane potential in rBMECs. PCA also enhanced lncRNA Xist expression, and when the expression of lncRNA Xist was silenced, PCA could not alleviate pyroptosis well in rBMECs. CONCLUSION: Protocatechuic aldehyde prevents ischemic injury by attenuating rBMEC pyroptosis via lncRNA Xist.

Polysaccharides from Tetrastigma hemsleyanum Diels et Gilg: optimum extraction, monosaccharide compositions, and antioxidant activity.

The optimization of extraction of Tetrastigma hemsleyanum Diels et Gilg polysaccharides (THP) using ultrasonic with enzyme method and its monosaccharide compositions and antioxidant activity were investigated in this work. Single-factor experiments and response surface methodology (RSM) were performed to optimize conditions for extraction, and the independent variables were (XA) dosage of cellulase, (XB) extraction time, (XC) ultrasonic power, and (XD) ratio of water to the material. The extraction rate of THP was increased effectively under the optimum conditions, and the maximum (4.692 ± 0.059%) was well-matched the predicted value from RSM. THP was consisted of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, and arabinose, while glucose was the dominant (26.749 ± 0.634%). According to the total antioxidant capacity assay with the FRAP method, DPPH, and hydroxyl radical scavenging assay, THP showed strong antioxidant activity with a dose-dependent behavior. The results indicated that THP has the potential to be a novel antioxidant and could expand its application in food and medicine.

Protective effects of flavonoids from the leaves of Carya cathayensis Sarg. against H2O2-induced oxidative damage and apoptosis in vitro.

Hydrogen peroxide (H2O2) can induce apoptosis by releasing reactive oxygen species (ROS) and reactive nitrogen species, which cause mitochondrial damage. The present study aimed to investigate the protective effects of flavonoids from the leaves of Carya cathayensis Sarg. against H2O2-induced oxidative damage and apoptosis in vitro. The bioactivity of total flavonoids (TFs) and five monomeric flavonoids [cardamonin (Car), pinostrobin chalcone, wogonin, chrysin and pinocembrin] from the leaves of Carya cathayensis Sarg. (LCCS) were tested to prevent oxidative damage to rat aortic endothelial cells (RAECs) induced by H2O2. Oxidated superoxide dismutase, glutathione peroxidase, malondialdehyde, lactate dehydrogenase and ROS were analyzed to evaluate the antioxidant activity. Gene and protein expression patterns were assessed using reverse transcription-quantitative PCR and western blotting, respectively. The results indicated that TFs and Car inhibited H2O2-induced cytotoxicity and apoptosis of RAECs. Additionally, they regulated the level of oxidase and inhibited the production of ROS. Overall, the TFs extracted from LCCS could potentially be developed as effective candidate drugs to prevent oxidative stress in the future; moreover, they could also provide a direction in investigations for preventing antioxidant activity through the ROS pathway.

The Antibacterial Properties of 4, 8, 4', 8'-Tetramethoxy (1,1'-biphenanthrene) -2,7,2',7'-Tetrol from Fibrous Roots of Bletilla striata.

Biphenanthrene compound, 4, 8, 4', 8'-tetramethoxy (1, 1'-biphenanthrene)-2, 7, 2', 7'-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.

Polysaccharide Isolated From Tetrastigma hemsleyanum Activates TLR4 in Macrophage Cell Lines and Enhances Immune Responses in OVA-Immunized and LLC-Bearing Mouse Models.

Tetrastigma hemsleyanum Diels et Gilg is a valuable Chinese medicinal herb with a long history of clinical application. Our previous study isolated and characterized a purified polysaccharide from the aerial part of Tetrastigma hemsleyanum (SYQP) and found it having antipyretic and antitumor effects in mice. A preliminary mechanistic study suggests these effects may be related to the binding of toll-like receptor (TLR4). The objective of this study is to further explore the detailed stimulating characteristics of SYQP on TLR4 signaling pathway and its in vivo immune regulating effect. We use HEK-BLUE hTLR4, mouse and human macrophage cell lines, as research tools. In vitro results show SYQP activated HEK-BLUE hTLR4 instead of HEK-BLUE Null cells. The secretion and the mRNA expression of cytokines related to TLR4 signaling significantly increased after SYQP treatment in both PMA-induced THP-1 and RAW264.7 macrophage cell lines. The TLR4 antagonist TAK-242 can almost completely abolish this activation. Furthermore, molecules such as IRAK1, NF-κB, MAPKs, and IRF3 in both the MyD88 and TRIF branches were all activated without pathway selection. In vivo results show SYQP enhanced antigen-specific spleen lymphocyte proliferation and serum IgG levels in OVA-immunized C57BL/6 mice. Orally administered 200 mg/kg SYQP induced obvious tumor regression, spleen weight increase, and the upregulation of the mRNA expression of TLR4-related cytokines in Lewis lung carcinoma-bearing mice. These results indicate SYQP can act as both a human and mouse TLR4 agonist and enhance immune responses in mice (p < 0.05). This study provides a basis for the development and utilization of SYQP as a new type of TLR4 agonist in the future.

Role of Militarine in PM2.5-Induced BV-2 Cell Damage.

A growing number of studies have shown that air fine particulate matter (PM2.5) pollution is closely associated with neuroinflammation in humans. Militarine, a glucosyloxybenzyl 2-isobutylmalate compound isolated from Bletilla striata, has been found to exert significant neuroprotective effects. However, the anti-inflammatory, antioxidant and antiapoptotic effects of militarine on PM2.5-stimulated BV-2 microglial cells have not been reported. This study aimed to investigate the protective effects of militarine against PM2.5-induced cytotoxicity and its mechanism in BV-2 microglial cells. Our results revealed that pretreatment with 0.31-1.25 µg/mL militarine reversed the morphological changes caused by PM2.5 and decreased proinflammatory cytokine generation and gene expression in PM2.5-treated BV-2 cells. In particular, tumor necrosis factor-α and interleukin-6 expression was inhibited in a dose-dependent manner. Notably, militarine markedly inhibited the upregulation of Toll-like receptor 4, Toll-like receptor 2, and cyclo-oxygenase-2 expression at both the mRNA and protein levels and reduced NF-κB pathway-associated protein expression. Immunofluorescence analysis showed that militarine suppressed NF-κB activity through inhibiting p65 nuclear translocation. Our data suggested that militarine alleviated neuroinflammation in BV-2 microglial cells, possibly by inhibiting the expression of neuroinflammatory cytokines through the TLR/NF-κB signaling pathway. Additionally, militarine significantly reduced PM2.5-mediated reactive oxygen species (ROS) generation and cell apoptosis and restored the mitochondrial membrane potential (MMP; ΔΨm). Collectively, these findings demonstrate that militarine played a protective role against PM2.5-induced damage in BV-2 cells by exerting anti-inflammatory, antioxidant, and antiapoptotic effects.

New Insights Into the Antibacterial Mechanism of Cryptotanshinone, a Representative Diterpenoid Quinone From Salvia miltiorrhiza Bunge.

The rapid rise of antibiotic resistance causes an urgent need for new antimicrobial agents with unique and different mechanisms of action. The respiratory chain is one such target involved in the redox balance and energy metabolism. As a natural quinone compound isolated from the root of Salvia miltiorrhiza Bunge, cryptotanshinone (CT) has been previously demonstrated against a wide range of Gram-positive bacteria including multidrug-resistant pathogens. Although superoxide radicals induced by CT are proposed to play an important role in the antibacterial effect of this agent, its mechanism of action is still unclear. In this study, we have shown that CT is a bacteriostatic agent rather than a bactericidal agent. Metabolome analysis suggested that CT might act as an antibacterial agent targeting the cell membrane. CT did not cause severe damage to the bacterial membrane but rapidly dissipated membrane potential, implying that this compound could be a respiratory chain inhibitor. Oxygen consumption analysis in staphylococcal membrane vesicles implied that CT acted as respiratory chain inhibitor probably by targeting type II NADH:quinone dehydrogenase (NDH-2). Molecular docking study suggested that the compound would competitively inhibit the binding of quinone to NDH-2. Consistent with the hypothesis, the antimicrobial activity of CT was blocked by menaquinone, and the combination of CT with thioridazine but not 2-n-heptyl-4-hydroxyquinoline-N-oxide exerted synergistic activity against Staphylococcus aureus. Additionally, combinations of CT with other inhibitors targeting different components of the bacterial respiratory chain exhibit potent synergistic activities against S. aureus, suggesting a promising role in combination therapies.

Molecular docking simulation and in vitro studies on estrogenic activities of flavonoids from leaves of Carya cathayensis Sarg.

The main purpose of this study was to evaluate the estrogenic properties of total flavonoids (TFs) and five flavonoid monomers (cardamonin (Car), pinostrobin chalcone (PC), wogonin (Wo), chrysin (Chr) and Pinocembrin (PI)) from leaves of Carya cathayensis Sarg (LCC). TFs from LCC were isolated and determined using HPLC. The 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were performed to assess the effects of flavonoids on cell proliferation and cell cycle, respectively. The molecular docking technique was applied to investigate binding conformations of the monomers from LCC to the estrogen receptor ERα and ERß. Gene and protein expression patterns were assessed using quantitative real-time PCR (qRT-PCR) and western blot, respectively. The results showed that TFs, Car, PC, Wo and Chr promoted proliferation of MCF-7 cells and cell transition from the G1 to S phase, and inhabitation of MCF-7 cell proliferation was observed after the treatment of PI. Molecular docking studies confirmed ERs as molecular targets for the monomers. TFs, Car, PC, Wo and Chr from LCC promoted gene expression of ERα, ERß, progesterone receptor (PR) and pS2. Our collective results demonstrated that TFs and monomers from LCC may exert ER agonist activity through competitively bind to ER, inducing ER upregulation and active ER to estrogen response element (ERE)- independent gene regulation. As an abundant natural product, LCC may provide a novel medicinal source for treatment of diseases caused by estrogen deficiency.

Effective fraction of Bletilla striata reduces the inflammatory cytokine production induced by water and organic extracts of airborne fine particulate matter (PM2.5) in vitro.

BACKGROUND: Bletilla striata is a traditional Chinese medicine used to treat hemorrhage, scald, gastric ulcer, pulmonary diseases and inflammations. In this study, we investigated bioactivity of the effective fraction of B. striata (EFB) in reducing the inflammatory cytokine production induced by water or organic extracts of PM2.5. METHODS: PM2.5 extracts were collected and analyzed by chromatographic system and inductively coupled plasma mass spectrometer. Cell viability was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, and cell supernatant was analyzed by flow cytometry, ELISA, and qRT-PCR in cultured mouse macrophage cell line RAW264.7 treated with EFB and PM2.5 extracts. Expressions of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway were measured by Western blot. RESULTS: PM2.5 composition is complex and the toxicity of PM2.5 extracts were not noticeable. The treatment of EFB at a wide dose-range of 0-40 µg/mL did not cause significant change of RAW264.7 cell proliferation. EFB pretreatment decreased the inflammatory cytokines in the macrophage. Further analysis showed that EFB significantly attenuated PM2.5-induced proinflammatory protein expression and downregulated the levels of phosphorylated NF-κBp65, inhibitor of kappa B (IκB)-α, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38. CONCLUSIONS: Our study demonstrated the potential effectiveness of B. striata extracts for treating PM2.5-triggered pulmonary inflammation.

[Comparison of contents of three active ingredients in Bletilla striata from different sources].

In order to understand the difference of contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ in 60 different sources of Bletilla striata planted under the same conditions. UPLC method was used and the analysis was performed on a Waters ACQUITY UPLC BEH C18 column( 2. 1 mm×100 mm,1. 7 µm),eluted with acetonitril-0. 1% formic acid solution by gradient. The flow rate was 0. 208 m L·min-1,the detection wavelength was 270 nm,the column temperature was 35 ℃ and the injection volume was 4µL. Under the above chromatographic conditions,the three components can be separated well with good linearity in the range of 0. 156-5. 000 mg·L-1. The average contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ were( 0. 116 ± 0. 071) %,( 0. 386 ±0. 185) % and( 0. 086±0. 034) %,respectively. After planting for two years under the same conditions,there was no significant difference in chemical composition among different sources and varieties,but the contents of the three components had some regional differences,which indicated that the western region was higher than the eastern region,while the contents of coelonin and batatasin Ⅲ in B.sinensis were slightly higher than those in B. striata. The chromatographic method above is simple,stable and reproducible,and can be used for quantitative analysis of three components. The content analysis of different sources of B. striata can provide reference for future B. striata breeding and quality control.

[Effect of pinostrobin chalcone on adipogenic differentiation of mesenchymal stem cell C3H10T1/2].

Chalcones is a flavonoid wildly presented in many herbs. It has the effect to inhibit cells adipogenic differentiation. In order to study the effect of pinostrobin chalcone extracted and isolated from leaves of hickoryes on the adipogenic differentiation of murine embryonic mesenchymal stem cell (C3H10T1/2), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] method was used to detect the cell proliferation; adipogenic differentiation was characterized by oil red O staining and isopropanol extraction; the triglyceride content was detected by GAP-PAP enzyme method; and the C3H10T1/2 cell differentiation into adipocytes was also examined by the mRNA and protein expression of PPARγ, C/EBPα and FABP4 by RT-PCR and Western blot respectively. Results indicated that pinostrobin chalcone almost had no effect on cell proliferation activity when the concentration was less than or equal to 50 µmol•L⁻¹; the oil red O staining, isopropanol extraction and GAP-PAP enzyme method showed that pinostrobin chalcone significantly decreased the C3H10T1/2 adipogenic differentiation and triglyceride content in the cytoplasm of adipocytes; the RT-PCR and Western blot analysis showed that pinostrobin chalcone can down-regulate the mRNA and protein levels of FABP4, PPARγ and C/EBPα in C3H10T1/2 cells(P<0.05 or P<0.01). The experiment results suggest that pinostrobin chalcone can inhibit C3H10T1/2 adipogenic differentiation.

The anti-Staphylococcus aureus activity of the phenanthrene fraction from fibrous roots of Bletilla striata.

BACKGROUND: Bletillae Rhizoma, the tuber of Bletilla striata, has been used in Chinese traditional medicine to treat infectious diseases. Chemical studies indicated that phenanthrene was one of the most important components of the herb, with a broad spectrum of antibiotic activity against Gram-positive bacteria. The objective of this study was to further characterize the antibacterial activity of the phenanthrene fraction from the fibrous root of the pseudobulb of B. striata. METHODS: The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbs was isolated using polyamide column chromatography. The antibacterial activity of the fraction was evaluated in vitro using a 96-well microtiter plate and microbroth dilution method. The cytotoxicity of EF60 against mammalian cells was tested by hemolysis and MTT assays. RESULTS: EF60 was obtained using alcohol extraction and polyamide column chromatography, with a yield of 14.9 g per 1 kg of the fibrous roots of B. striata. In vitro tests indicated that EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 µg/mL. Minimum bactericidal concentration tests demonstrated that EF60 was bactericidal against S. aureus 3304 and ATCC 29213 and was bacteriostatic against S. aureus 3211, ATCC 25923, and ATCC 43300. Consistently, the time-kill assay indicated that EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively. Further studies indicated that EF60 (160 µg/mL) showed no cytotoxicity against human erythrocytes, and was minimally toxic to Human Umbilical Vein Endothelial Cells with an IC50 of 75 µg/mL. CONCLUSIONS: Our studies indicated that EF60 is worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA.

[Protective Effect of Total Flavonoids from Carya cathayensis Leaf on Cultured H9C2 Cardiomyocytes During Hypoxia / Reoxygenation Injury].

Objective: To study the protective effect of total flavonoids from Carya cathayensis leaf on cultured H9C2 cardimyocytes during hypoxia / reoxygenation( H / R) injury. Methods: Co Cl2 was used to induce the H / R injury model in H9C2 cells at different concentrations,different H / R time. Total flavonoids from Carya cathayensis leaf was added into culture medium with final concentration of2. 5,5 and 10 µg / m L before H / R. Lactate dehydrogenase( LDH), Malondialdehyde( MDA) content, Superoxidedimutase( SOD) activity in the bathing medium were assayed for the evaluation of myocardial cell injury. Myocardial cell viability was detected by the MTS assay kit. Apoptotic changes in H9C2 cells were observed by using Hoechst-PI staining and flow cytometry analysis. The expression of HIF-1αprotein was detected by Western blot. Results: Using 1 200 µmol / L Co Cl2 to hypoxia the cells for 18 h and then reoxygenation for 2 h were the best condition of H / R injury model. Compared with model group,the cells treated with total flavonoids from Carya cathayensis leaf could decrease the activity of LDH and the content of MDA, while increased the activity of SOD. Moreover, they could regulate the expression of HIF-1α protein to normal level. Conclusion: It is suggested that total flavonoids from Carya cathayensis leaf has a protective effect on H9C2 cardiomyocyte during H / R injury. The mechanism may be relate to enhancing the capability of the cell clearing the oxygen free radial, decreasing the production of lipid peroxidation and reducing apoptosis.

[Antibacterial Activity of Chemical Constituents Isolated from Fibrous Roots of Bletilla striata].

Objective: To investigate the chemical constituents isolated from the fibrous roots of Bletilla striata, and to research their antibacterial activities. Methods: The native products were isolated and purified by silica gel, Sephadex LH-20 column chromatography and preparative HPLC. Their structures were elucidated on the basis of various spectroscopic analysis, and their antibacterial activities were tested by microbroth dilution method in a 96-well microtiter plate. Results: Seven compounds were isolated from the ethanol extract of the fibrous roots of Bletilla striata, and identified as p-hydroxybenzaldehyde( 1),2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene( 2),4,5-dihydroxy-2-methoxy-9,10-dihydrohenanthrpene( 3),2-dihydroxy-4,7-dimethoxyphenan-threne( 4), militarine( 5), dactylorhin A( 6) and gastrodin( 7). Among them, compounds 2 ~ 4 showed moderate antibacterial activities against several Gram-positive bacterial strains( MIC 8 ~ 128 µg / m L),such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Bacillus subtilis. Conclusion: The fibrous roots and tubers of Bletilla striata contain similar compounds, including glucosyloxybenzyl 2-isobutylmalates,and phenanthrene compounds, which showed antimicrobial activities against Gram-positive bacterial strains. And compounds 3,4 are isolated from Bletilla genus for the first time.

[Research on the Anti-Pulmonary Fibrosis Effect of the Bletilla striata Polysaccharide in Rat Silicosis Model].

Objective: To investigate the anti-pulmonary fibrosis effect and the possible molecular mechanism of the Bletilla striata polysaccharide. Methods: Polysaccharide was prepared by water reflux extraction plus ethanol precipitation method, and following deproteinization process by Sevage method. Rat silicosis model was established by invasive intratracheal instillation method. The effect and molecular mechanism of the polysaccharide was evaluated by lung indexes, lung pathological change, serum levels of SOD,MDA,NF-κB,IL-1ß,PDGF,TGF-ß1,TNF-α,HYP were detected, and the contents of CD3~+,CD4~+,CD8~+T lymph cells and CD4~+/ CD8~+ratio were detected by flow cytometry. Results: Both low( 100 mg / kg) and high( 400 mg / kg) dosage polysaccharide treatment could remarkably elevate the serum SOD level and reduce the MDA,NO level, and effectively reverse the CD4~+/ CD8~+ratio comparing with the model group( P < 0. 01). Except the TNF-α level was significantly lower in the high dosage treatment group, there was no other effect in inflammatory cytokines and HYP content in serum. HE pathological section confirmed that the Bletilla striata polysaccharide treatment group can not effectively prevent lung fibrosis. Conclusion: The Bletilla striata polysaccharide has remarkable regulation effect on antioxidation system and immune system, but can not effectively prevent lung fibrosis, more effort should be made to study the active antipulmonary fibrosis components of Bletilla striata.

[Chrysin inhibits adipogenic differentiation of mouse embryonic fibroblasts].

Chrysin is an active flavonoid wildly presented in many herbs. It has the effect to reduce serum lipid. To investigate the effect of chrysin on the adipogenic differentiation of mouse embryonic fibroblasts, methyl thiazolyl tetrazolium (MTT) and crystal violet were used to detect the cytotoxic effect of chrysin on Immortalized mouse embryonic fibroblasts (iMEFs). Propidium iodide (PI) staining combined with flow cytometry (FCM) was employed to detect the effects of different concentrations of chrysin on iMEFs cell cycle. The effect of chrysin on adipogenic differentiation ability of iMEFs was determined by oil red O staining. Semi-quantitative PCR was employed to detect the effect of chrysin on mRNA transcriptional levels of adipogenic differentiation markers, including perilipin 2, adiponectin (adipoq), Fabp4, LPL, MCP-1 and adipogenic differentiation key transcription factor peroxisome proliferators-actiated receptor-gamma 2(PPAR-γ2). Results indicated that chrysin had certain cytotoxic effect for iMEFs in a dose-dependent manner, and the IC50 was identified nearly to 30 µmol•L⁻¹. FCM analysis showed that chrysin could affect the cell-cycle distribution of iMEFs, increasing the ratio of cells in G1 phase. Adipogenic differentiation inducing experiment showed that 30 µmol•L⁻¹ chrysin significantly reduced lipid drops accumulation induced by insulin and dexamethasone. In addition, the mRNA transcriptional levels of PPAR-γ2 and LPL were significantly decreased and mRNA levels of fabp 4, MCP-1, adipoq were also affected after chrysin treatment. The experiment results suggest that chrysin attenuates the adipogenic differentiation capacity of mesenchymal stem cells.

Cardamonin Regulates miR-21 Expression and Suppresses Angiogenesis Induced by Vascular Endothelial Growth Factor.

Cardamonin has promising potential in cancer prevention and therapy by interacting with proteins and modifying the expressions and activities, including factors of cell survival, proliferation, and angiogenesis. In our precious study, we have demonstrated that cardamonin suppressed vascular endothelial growth factor- (VEGF-) induced angiogenesis as evaluated in the mouse aortic ring assay. It is also known that microRNAs (miRNAs) play important roles in angiogenesis. Herein, we hypothesized whether antiangiogenesis effect of cardamonin in human umbilical vein endothelial cells (HUVECs) triggered by VEGF was associated with miRNAs. We found that cardamonin reduced the miR-21 expression induced by VEGF in HUVECs. Treatment with miR-21 mimics abolished the effects of cardamonin on VEGF-induced cell proliferation, migration, and angiogenesis in HUVECs. However, treatment with miR-21 inhibitors presented the opposite effects, indicating the vital role of miR-21 in this process. Our study provides a new insight of the preliminary mechanism of anti-VEGF-induced angiogenesis by cardamonin in HUVECs.